Zaprionus is an important drosophilid genus in the Afrotropical region. Here, two new species, Z. burlai n. sp. and Z. tsacasi n. sp., are described from Tanzania and São Tomé, respectively. The two species show incomplete reproductive isolation with Z. tuberculatus Malloch and Z. sepsoides Duda, respectively, with intercrosses producing fertile females but sterile males. The latter two have long been considered sibling species and together with three other species (Z. mascariensis Tsacas & David, Z. kolodkinae Chassagnard & Tsacas, and Z. verruca Chassagnard & McEvey) form the tuberculatus subgroup. The phylogenetic relationships of these seven species of the subgroup were revised in light of mitochondrial (COII) gene sequences and wing morphometrics. Mitochondrial DNA firmly distinguished most of the species, except for a triad of Z. tuberculatus, Z. verruca, and Z. burlai. Wing morphometrics was able to distinguish between closely related species and also indicated the altitudinal origin of each species. Most species can be identified through internal anatomy of the reproductive system (testis and seminal receptacle lengths), and the discovery of the new species with incomplete reproductive isolation may help in understanding the genetic basis of this variation through interspecific hybridization. The molecular phylogeny reconfirmed the Malagasy origin of the subgroup during the Late Pliocene. Colonization of Africa probably involved two independent events during the Pleistocene.
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