Morphological and Molecular Characterization of and Phenotypes from Co-endemic Localities in Mpumalanga and KwaZulu-Natal Provinces of South Africa

Fasciolosis is a food- and water-borne disease caused by digenean trematode species, and . They are widely distributed and infect a wide range of definitive hosts, causing enormous economic loss due to reduced productivity in domestic ruminants. The two species have been previously reported to be co-endemic in KwaZulu-Natal and Mpumalanga provinces of South Africa. Hybridization between the two species has been reported elsewhere. Despite the overlap of the two species in two provinces, there has been no attempt to determine the presence of the intermediate forms or hybrids. Therefore, this study aimed at morphological and molecular characterization of spp. collected from cattle slaughtered at abattoirs located in the two provinces of South Africa, where two species are endemic. A total of 71 fluke specimens were collected cattle from abattoirs in Enhlazeni and Nelspruit in Mpumalanga province and Pietermaritzburg in KwaZulu-Natal province of South Africa, and Zimbabwe (Bulawayo abattoir). specimen collected from Zimbabwe where it has been confirmed as the only species occurring and this was used as control in the morphological and molecular assessment of the collected specimens. Of the 71 specimens collected, 37 were classified as , 12 as and 22 as using morphological characters. Of these species, 11 of 37  and 6 of 22 were found to be aspermic or having very scanty sperm. Fifteen flukes which were spermatic were all identified morphologically as whilst 5 flukes which were aspermic were identified morphologically as . Molecular analysis of the same 15 spermatic specimens confirmed the presence of ( = 9) and ( = 6) using the CO1 marker and as ( = 4), ( = 7) and sp. ( = 1) for the same specimens using the ITS-1/5.8S/ITS-2 marker. The remaining 4 aspermic flukes (one did not resolve) were all identified morphologically as and molecular analysis confirmed them as ( = 4) by both CO1 and ITS-1/5.8S/ITS-2. Phylogenetic analysis based on both CO1 and ITS-1/5.8S/ITS-2 showed that species formed a moderately supported monophyletic clade with . Their ancestral history was further confirmed by haplotype network, which formed novel haplotypes that corresponded with the structure of the phylogenetic tree. Results from this study showed that morphological characters alone have limitations in identifying and in areas where the two species occur, although both methods confirmed the presence of occurring in Zimbabwe, in KwaZulu-Natal, and both species occurring in Mpumalanga province. Therefore, the use of morphological techniques, complemented by molecular techniques are recommended, especially in endemic areas where the two species are co-endemic.