Resolution of the identity of three species of Diplostomum (Digenea: Diplostomidae) parasitising freshwater fishes in South Africa, combining molecular and morphological evidence

Reliable data on the diversity of the genus Diplostomum (Digenea: Diplostomidae) parasitising freshwater fishes in South Africa, as well as in Africa, is almost non-existent. Most of the morphology-based identifications of species within this genus reported from Africa require critical revision. The aim of the present study was to determine the diversity of Diplostomum metacercariae in South African fishes applying molecular and traditional morphological techniques. To achieve this aim, a total of 216 fishes belonging to 21 species collected in the Rivers Phongolo, Riet, Usuthu and Mooi in three provinces of South Africa were examined. Metacercariae of Diplostomum were recovered from the eye lenses of 38 fishes belonging to five species of the families Anguillidae, Cichilidae and Mochokidae, with an overall low prevalence of infection (18%). Metacercariae were subjected to morphological study and molecular sequencing of the partial mithochondrial cox1 and ribosomal 28S rDNA genes as well as of ribosomal ITS1-5.8S-ITS2 region. Morphological and phylogenetic analyses revealed the presence of three species which matched those previously reported from Nigeria, Iraq and China, therefore those from Tilapia sparrmanii and Synodontis zambezensis were named Diplostomum sp.; those from Anguilla labiata, Oreochromis mossambicus and S. zambezensis were named Diplostomum sp. 14; and those from Pseudocrenilabrus philander were named Diplostomum sp. 16. Geographic distribution of several species of Diplostomum appeared to be wider than expected. Morphological description and novel sequence data generated during this study will contribute to the elucidation of the life cycles of Diplostomum sp., Diplostomum sp. 14 and Diplostomum sp. 16 and advance further research of diplostomids in Africa. Keywords: Trematoda, Metacercariae, Freshwater fish, Morphology, DNA, South Africa