The first molecular phylogenetic analysis of Balanorchis anastrophus (Paramphistomoidea: Balanorchiidae), a rumen fluke found in cattle in South Brazil.

The rumen fluke Balanorchis anastrophus Fischoeder, 1901 is a paramphistome found in introduced domestic and native wild ruminants in South America, and the sole member of the family Balanorchiidae. No DNA sequences have been obtained from B. anastrophus, and little is known about the life cycle, distribution, pathology and epidemiology of this species. In this study, paramphistomes found in the rumen of slaughtered cattle from Rio Grande, Southern Brazil, in 2022 were subjected to morphological and molecular analyses. Stained whole and manually cut worms were subjected to morphological and morphometric analyses by light microscopy, which allowed the identification of B. anastrophus. In the molecular analysis, the first genetic data for the species were generated by Sanger [28S (1162 bp), ITS2 (480 bp) and cox1 (764 bp)] and Next Generation [mt genome (14,259 bp, 22 protein coding genes); rDNA operon (7214 bp)] sequencing, which were used in phylogenetic analyses. In all phylogenetic analyses, B. anastrophus formed a distinct lineage from other paramphistomoid taxa with data available for comparison. Thus, at the current stage, Balanorchiidae is maintained as a distinct family of Paramphistomoidea. In most phylogenies, B. anastrophus was more closely related to New World paramphistomoids than to taxa from the Old World. This finding supports the South American origin of B. anastrophus and suggests that the infection of cattle is probably related to a parasite spillover from wild deer.