This study investigates genetic diversity in three species of Ephemeroptera, one eurytopic and therefore widespread (Afroptilum sudafricanum) and two stenotopic and thus endemic (Demoreptus natalensis and Demoreptus capensis) species, all of which co-occur in the southern Great Escarpment, South Africa. Mitochondrial DNA was analysed to compare the genetic diversity between the habitat generalist and the two habitat specialists. Afroptilum sudafricanum showed no indication of population genetic structure due to geographic location, while both Demoreptus species revealed clear genetic differentiation between geographic localities and catchments, evident from phylogenetic analyses and high FST values from AMOVA. In addition, the phylogenetic analyses indicate some deeper haplotype divergences within A. sudafricanum and Demoreptus that merit taxonomic attention. These results give important insight into evolutionary processes occurring through habitat specialisation and population isolation. Further research and sampling across a wider geographic setting that includes both major mountain blocks of the Escarpment and lowland non-Escarpment sites will allow for refined understanding of biodiversity and associated habitat preferences, and illuminate comparative inferences into gene flow and cryptic speciation.http://zookeys.pensoft.netam2021Plant Production and Soil Scienc
