Echinostomatids from South African freshwater limpets: Phylogenetic analyses and diagnostic morphological features for cercariae of Petasiger

Species of the family Echinostomatidae use diverse gastropod taxa as first intermediate hosts. However, identification of echinostomatid larvae often proves difficult because of incomplete information on their life cycles and lack of molecular data that can link larvae to the corresponding known adults. Here, echinostomatids that were isolated from freshwater limpets in South Africa were described using light and scanning electron microscopy, and ribosomal (28S, ITS, and 18S) and mitochondrial (cox1) DNA sequences. The analyses revealed three species: Petasiger radiatus, Petasiger sp., and Echinostomatidae gen. sp. Considering the close morphological resemblance between cercariae of Petasiger spp., the current species were compared with data from literature. The results showed that cercarial size is generally unsuitable for species discrimination. The numbers of flame cells and refractile granules in the excretory system, and penetration gland cell patterns, may indicate, but do not prove species identity. Although papillary patterns were distinct between species, papillae were clearly discernible only using scanning electron microscopy and are known for only a few species. Phylogenetic reconstruction indicated that 28S rDNA sequences of Petasiger on GenBank are for P. exaeretus, P. phalacrocoracis, P. radiatus, and six unnamed species. Furthermore, the results revealed that multiple ITS rDNA and cox1 sequences labelled as Stephanoprora amurensis and P. phalacrocoracis on GenBank, are from isolates whose identities are questionable. Echinostomatidae gen. sp. could not be assigned to any currently known genus. Expansion of the genetic database of the family Echinostomatidae is necessary for the delineation of putative species and elucidation of intergeneric relationships.