Evolutionary affinities and morphological characterization of the enigmatic Zonocotyle bicaecata (Trematoda: Paramphistomoidea: Zonocotylidae) from the Upper Paraná River basin.

Neotropical fish amphistomes represent a highly diverse group within the Paramphistomoidea, with wide distribution across major South American hydrological drainages. However, the limited molecular characterization of these taxa has impeded a comprehensive assessment of their evolutionary relationships and the systematic relevance of morphological features in classification schemes. Our study, based on the critical evaluation of the type material of both nominal species of Zonocotyle (type genus of the monotypic Zonocotylidae), and newly collected specimens of Zonocotyle bicaecata from Steindachnerina insculpta (Curimatidae) in the Upper Paraná River basin, Brazil, presents a morphological reappraisal of Z. bicaecata and provides molecular data (28S rDNA, ITS1-5.8S-ITS2 region, and COI mtDNA) to assess its phylogenetic relationships. Our phylogenetic analyses confirm this species belongs to the Paramphistomoidea. The most comprehensive analyses (based on 28S and COI) further indicate a close relationship with other fish amphistomes from the Neotropical region. Additionally, we emphasized the necessity for a new classification within Paramphistomoidea and briefly discussed the host range of Zonocotyle among curimatid fish hosts.