In the present work, a comparative morphological and molecular study of Nosopsyllus barbarus (Jordan & Rothschild 1912) and Nosopsyllus fasciatus (Bosc, d’Antic 1800) isolated from rodents from different geographical regions (Spain, Morocco, Belgium, France and Portugal) has been carried out. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and partial 18S rRNA, and partial cytochrome c oxidase 1 (cox1) and cytochrome b (cytb) mtDNA sequences were determined to clarify the taxonomic status of these two species and to assess intraspecific variation and inter-specific sequence differences. In addition, a phylogenetic analysis with other species of fleas using Bayesian inference was performed. We have found morphological differences between N. barbarus and N. fasciatus that did not correspond with molecular differences. Furthermore, any of the five molecular markers used in this study was able to discriminate between the two species. Thus, based on the phylogenetic and molecular study of three nuclear markers (18S, ITS1, ITS2) and two mitochondrial markers (cox1 and cytb), as well as the concatenated dataset of both species, we concluded that morphological characters traditionally used to discriminate between the two species do not provide solid arguments for considering these two “morphospecies” as two different species. Therefore, we propose N. barbarus should be considered as a junior synonym of N. fasciatus.
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