We examined the systematics of a ubiquitously distributed southern African freshwater crab, Potamonautes sidneyi s.l. species complex. Specimens were subjected to DNA sequence analyses of two mitochondrial loci (16S rRNA + COI). We applied three species delimitations methods (ASAP, bGMYC and bPTP) to test their utility in delineating species boundaries in Potamonautes and three additional Afrotropical genera (Liberonautes, Nesonautes and Seychellum). The combined mtDNA dataset retrieved five clades. Clade 1 comprised of P. barbarai, clade 2 comprised of specimens from the interior of the Great Karoo Basin, sister to P. sidneyi s.s. in clade 3. Clade 4 was confined to Eswatini and the Mpumalanga Province of South Africa, and sister to clade 5 that comprised P. danielsi. The three species delimitation methods either over- or underestimated the number of species. Phylogenetically, specimens from the Great Karoo Basin (clade 2) were equidistant to P. sidneyi s.s. and P. perlatus, while the Eswatini and Mpumalanga specimens (clade 4) were sister to P. danielsi. Clades 2 and 4 are herein described as P. karooensis sp. nov. and P. valles sp. nov., respectively.
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