The present study used molecular and morphological approaches to investigate hidden diversity within the Hippopotamyrus ansorgii species complex in southern Africa. Phylogenetic reconstructions and three species delimitation methods based on two mitochondrial markers (cytochrome b and cytochrome oxidase I) and one nuclear marker (S7) revealed 12 Molecular Operational Taxonomic Units (MOTUs), with two of them representing two recently described species, Hippopotamyrus longilateralis and Hippopotamyrus szaboi. The highest diversity occurred in the Kwanza River system, which contained five MOTUs, and the Upper Zambezi River system that had two MOTUs. Five other river systems contained a single MOTU each. A major impediment to the review of this complex has been the uncertainty surrounding the type locality of the specimens that were used for the description of H. ansorgii. The present study has, through a careful examination of published literature and synthesis of information on the travel activities of Dr. William Ansorge who collected the specimens, identified the Kwanza River system as the most plausible source of the syntypes. The resolution of the type locality of H. ansorgii facilitates future work on the review of this complex which is critical for providing reliable biodiversity estimates, identifying effective conservation management strategies and understanding the evolutionary history and biogeographic patterns of the fishes of this region.

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