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Phylogenetic analyses of ciliated protozoa have been increasingly relied on multigene information, which was revealed to provide more robust interpretations than single-gene information. Previous studies showed that Amphisiellidae was an extremely divergent group within the order Stichotrichida, with species widely dispersed throughout the stichotrichid assemblage, while Trachelostylidae, excluding gonostomatid species, is a monophyletic group within the order Sporadotrichida. In the present study, we provide 38 new sequences of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes to infer phylogenetic relationships among all taxa available in Amphisiellidae and Trachelostylidae. The results indicate that: (1) Amphisiellidae is polyphyletic, with Amphisiella , Hemiamphisiella , Orthoamphisiella , Uroleptoides , and Urospinula distributing in different clades; (2) Amphisiella is separated into two clades in phylogenetic trees, corroborated by difference in cortical granule distribution. Thus, cortical granule pattern and distribution may be strong diagnostic features to divide Amphisiella species into two subgenera; (3) the monophyly of Trachelostylidae sensu Berger (2008) is strongly supported, suggesting it is a well-defined family; (4) Gonostomatidae is confirmed to be a valid family. [ABSTRACT FROM AUTHOR]