Multigene taxonomy of three freshwater ciliates from the Chishui River, with establishment of a new subfamily extracted from the intricate puzzle of oxytrichids (Protista: Ciliophora)

The systematics of hypotrich ciliates have long been problematic owing to their high polymorphism and morphological complexity. In this study, the taxonomy of three freshwater hypotrichs, Apoamphisiella tihanyiensis, Aponotohymena australis and Rubrioxytricha haematoplasma, with the first two species representing the first records of their occurrence in China, was investigated through combined morphological, morphogenetic, and multi-gene-based phylogenetic analyses, incorporating 16 newly obtained sequences. Apoamphisiella and Aponotohymena form a monophyletic group with genus Paraurostyla, all of which were classified historically under the controversial family Oxytrichidae. Given their shared morphological features, especially three rows of caudal cirri and transverse cirri arranged in an oblique pseudorow, Paraurostylinae subfam. nov. is proposed to accommodate these genera. The first morphogenetic data for Apoamphisiella tihanyiensis corroborated developmental uniformity within the genus. Notably, our analyses revealed significant incongruence in the ITS–5.8S tree compared with 18S and 28S results, probably attributable to chimeric or misassigned sequences in previous datasets. Including or excluding this ITS–5.8S sequence from the concatenated dataset clearly demonstrates how a single problematic marker can distort terminal branch topology and disrupt the monophyly of Apoamphisiella. This result highlights the importance of using complete ribosomal operon sequences from single cells or clonal populations.