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Free‐living litostomatean ciliates, prominent microeukaryote predators commonly encountered in freshwater and marine habitats, play vital roles in maintaining energy flow and nutrient cycles. Nevertheless, understanding their biodiversity and phylogenetic relationships remains challenging due to insufficient morphological information and molecular data. As a new contribution to this group, three haptorian ciliates, including two new species (Actinobolina bivacuolata sp. nov. and Papillorhabdos foissneri sp. nov.) and the insufficiently described type species, Actinobolina radians, were isolated from wetlands around Lake Weishan, China and investigated by a combination of living morphology, stained preparations, and 18S rRNA gene sequence data. An illustrated key of the valid species within the two genera is provided. In addition, we reveal the phylogenetic positions of these two genera for the first time. Although they differ in all key morphologic characters such as general appearance (ellipsoidal with numerous tentacles vs. cylindrical), extrusomes (stored in tentacles vs. anchored to pellicle), circumoral kinety (present vs. absent), composition of somatic kineties (kinetosome clusters vs. monokinetids), and number of dorsal brush rows (1 vs. 4), they both cluster in a fully supported clade in the phylogenetic tree, which indicates that the biodiversity and additional molecular markers of this group need further exploration. [ABSTRACT FROM AUTHOR]

[Display omitted] • 53 new SSU, ITS1-5.8S rDNA-ITS2, and LSU rDNA sequences of spirotrich ciliates. • Novistrombidium asserts an ancestral ciliary pattern in Oligotrichia. • Division of subgenera Novistrombidium and Parallelostrombidium is fully supported. • Cyrtostrombidiidae, Pelagostrombidiidae and Tontoniidae evolved from Strombidiidae. Marine planktonic ciliates are largely oligotrichs and choreotrichs, which are two subclasses of the class Spirotrichea. The current phylogenetic assignments of oligotrichs and choreotrichs are inconsistent with previous results based on morphological features, probably hindered by the limited information from a single gene locus. Here we provide 53 new sequences from small subunit ribosomal RNA (SSU rDNA), ITS1-5.8S rDNA-ITS2, and large subunit ribosomal RNA (LSU rDNA) gene loci in 25 oligotrich and choreotrich species. We also predict RNA secondary structures for the ITS2 regions in 55 species, 48 species of which are reported for the first time. Based on these novel data, we make a more comprehensive phylogenetic reconstruction, revealing consistency between morphological taxonomy and an updated phylogenetic system for oligotrichs and choreotrichs. With the addition of data from ciliature patterns and genes, the phylogenetic analysis of the subclass Oligotrichia suggests three evolutionary trajectories, among which: 1) Novistrombidium asserts an ancestral ciliary pattern in Oligotrichia; 2) the subgenera division of Novistrombidium and Parallelostrombidium are fully supported; 3) the three families (Tontoniidae, Pelagostrombidiidae and Cyrtostrombidiidae) all evolved from the most diverse family Strombidiidae, which explains why strombidiids consistently form polyphyletic clades. In the subclass Choreotrichia, Strombidinopsis likely possesses an ancestral position to other choreotrichs, and both phylogenetic analysis and RNA secondary structure prediction support the hypothesis that tintinnids may have evolved from Strombidinopsis. The results presented here offer an updated hypothesis for the evolutionary history of oligotrichs and choreotrichs based on new evidence obtained by expanding sampling of molecular information across multiple gene loci. [ABSTRACT FROM AUTHOR]