Aquatic larvae of many Rhithrogena mayflies (Ephemeroptera) inhabit sensitive Alpine environments. A number of species
are on the IUCN Red List and many recognized species have restricted distributions and are of conservation interest. Despite
their ecological and conservation importance, ambiguous morphological differences among closely related species suggest
that the current taxonomy may not accurately reflect the evolutionary diversity of the group. Here we examined the species
status of nearly 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included
22 type localities, general mixed Yule-coalescent (GMYC) model analysis of one standard mtDNA marker and one newly
developed nDNA marker, and morphological identification where possible. Using sequences from 533 individuals from 144
sampling localities, we observed significant clustering of the mitochondrial (cox1) marker into 31 GMYC species. Twenty-one
of these could be identified based on the presence of topotypes (expertly identified specimens from the species' type
locality) or unambiguous morphology. These results strongly suggest the presence of both cryptic diversity and taxonomic
oversplitting in Rhithrogena. Significant clustering was not detected with protein-coding nuclear PEPCK, although nine
GMYC species were congruent with well supported terminal clusters of nDNA. Lack of greater congruence in the two data
sets may be the result of incomplete sorting of ancestral polymorphism. Bayesian phylogenetic analyses of both gene
regions recovered four of the six recognized Rhithrogena species groups in our samples as monophyletic. Future
development of more nuclear markers would facilitate multi-locus analysis of unresolved, closely related species pairs. The
DNA taxonomy developed here lays the groundwork for a future revision of the important but cryptic Rhithrogena genus in
Europe.