DNA barcoding to confirm morphological traits and determine relative abundance of burrowing mayfly species in western Lake Erie

► Burrowing mayflies (Hexagenia spp.) have recently recovered in Lake Erie. ► Only adult male H. limbata and H. rigida show obvious differences in morphology. ► DNA barcoding of the mitochondrial COI gene distinguished these two species. ► We describe pigmentation patterns that separate these species at all life stages. ► DNA barcoding confirmed these morphological traits to be distinctive.