Many ciliated protists prey on other large microbial organisms, including other protists and microscopic metazoans. The ciliate class Litostomatea unites both predatory and endosymbiotic species. The evolution of predation ability in ciliates remains poorly understood, in part, due to a lack of genomic data. To fill this gap, we acquired the transcriptome profiles of six predatory litostomateans using single-cell sequencing technology and investigated their transcriptomic features. Our results show that: (1) in contrast to non-predatory ciliates, the predatory litostomateans have expanded gene families associated with transmembrane activity and reactive oxidative stress response pathways, potentially as a result of cellular behaviors such as fast contraction and extension; (2) the expansion of the calcium-activated BK potassium channel gene family, which hypothetically regulates cell contractility, is an ancient evolutionary event for the class Litostomatea, suggesting a rewired metabolism associated with the hunting behavior of predatory ciliates; and (3) three whole genome duplication (WGD) events have been detected in litostomateans, with genes associated with biosynthetic processes, transmembrane activity, and calcium-activated potassium channel activity being retained during the WGD events. In addition, we explored the evolutionary relationships among 17 ciliate species, including eight litostomateans, and provided a rich foundational dataset for future in-depth phylogenomic studies of Litostomatea. Our comprehensive analyses suggest that the rewired cellular metabolism via expanded gene families and WGD events might be the potential genetic basis for the predation ability of raptorial ciliates.

Recent high-throughput sequencing endeavors have yielded multigene/protein phylogenies that confidently resolve several inter- and intra-class relationships within the phylum Ciliophora. We leverage the massive sequencing efforts from the Marine Microbial Eukaryote Transcriptome Sequencing Project, other SRA submissions, and available genome data with our own sequencing efforts to determine the phylogenetic position of Mesodinium and to generate the most taxonomically rich phylogenomic ciliate tree to date. Regardless of the data mining strategy, the multiprotein data set, or the molecular models of evolution employed, we consistently recovered the same well-supported relationships among ciliate classes, confirming many of the higher-level relationships previously identified. Mesodinium always formed a monophyletic group with members of the Litostomatea, with mixotrophic species of Mesodinium-M. rubrum, M. major, and M. chamaeleon-being more closely related to each other than to the heterotrophic member, M. pulex. The well-supported position of Mesodinium as sister to other litostomes contrasts with previous molecular analyses including those from phylogenomic studies that exploited the same transcriptomic databases. These topological discrepancies illustrate the need for caution when mining mixed-species transcriptomes and indicate that identifying ciliate sequences among prey contamination-particularly for Mesodinium species where expression from stolen prey nuclei appears to dominate-requires thorough and iterative vetting with phylogenies that incorporate sequences from a large outgroup of prey.

Triadinium was created to include Triadinium caudatum. Further, four other species were included, T. minimum, T. galea, T. elongatum, and T. magnum, all sharing a characteristic helmet-shaped body. Wolska and Grain argued that the inclusion of T. minimum and T. galea into Triadinium was done based on superficial morphological aspects, and established two new genera to accommodate these species: Circodinium and Gassovskiella. Although the phylogenetic relationships within Entodiniomorphida have been investigated by multiple authors, none of them discussed the evolutionary relationship of helmet-shaped entodiniomorphids. We performed molecular phylogenetics and revisited old literature digging for morphological data to explain our results. According to our analyses, the helmet-shaped body is homoplastic and may have evolved from at least three different entodiniomorphid ancestors. Circodinium minimum is phylogenetically related to members of Blepharocorythidae, T. caudatum emerged within Spirodiniidae and G. galea within Polydiniellidae. This phylogenetic hypothesis is partially supported by information on infraciliature and ultrastructure of C. minimum and T. caudatum. However, such morphological information is not available for polydiniellids. In order to shed some light into the evolution of the helmet-shaped ciliates, future works should focus to collect information on the infraciliature and the ultrastructure of Polydiniella mysorea and of other Triadinium species.

The morphology, ontogenesis, conjugation, and phylogenetic position of Metopus boletus nov. spec. were studied using live observation, various silver impregnation methods, scanning electron microscopy, morphometry, and the 18S rRNA gene sequence. The new species is outstanding in having a mushroom-like appearance; a globular to broadly ellipsoid macronucleus in anterior body half; 5-10 elongated caudal cilia; 4-6 dikinetids curved rightwards in the anterior portion of the first postoral kinety; and an adoral zone composed of an average of 28 small polykinetids. Ontogenesis of M. boletus follows the metopid mode and the species-specific vegetative morphology is obtained after division. Its conjugation is temporary, isogamic and the partners unite ventral-to-dorsal, forming strongly arched to almost rod-like pairs, which indicates a heteropolar arrangement. There are only two maturation divisions and a single synkaryon division in exconjugants. The conjugation data corroborate a sister group relationship of the classes Armophorea and Litostomatea within the SAL (Spirotrichea + Armophorea + Litostomatea) supercluster in that the partners unite ventral-to-dorsal and the main body axes are antiparallel. On the other hand, the last common ancestor of the spirotricheans very likely had a ventral-to-ventral and homopolar conjugation mode with the main body axes oriented in parallel.

The validity of genus Eodinium has been historically disputed due to morphological similarities with Diplodinium (absence of skeletal plates as well as adoral and dorsal ciliary zones at the same body level). To address this issue, the 18S rDNA of four Eodinium posterovesiculatum morphotypes and four Diplodinium anisacanthum morphotypes were sequenced and phylogenetically analyzed. The different inference methods suggest the existence of a last common ancestor of Eodinium and Ostracodinium that is not shared with Diplodinium, strongly supporting the validity of genus Eodinium. Since skeletal plates are present in all members of genus Ostracodinium, the most parsimonious is a secondary loss of skeletal plates in E. posterovesiculatum. This work represents a breakthrough in the taxonomy and phylogeny of the family Ophryoscolecidae indicating that the skeletal plates may not reflect evolutionary divergence within this group of ciliates as traditionally proposed.

Two new pleurostomatid ciliates, Loxophyllum lembum sp. n., L. vesiculosum sp. n., and the poorly known L. perihoplophorum Buddenbrock, 1920, isolated from brackish waters in coastal regions of southern China, are described following observations of live cells and protargol-impregnated specimens. Loxophyllum lembum sp. n. is distinguished by a combination of characters including two macronuclear nodules, 6-9 contractile vacuoles along the ventral margin, 11-14 right and 6-8 left kineties and the presence of cortical granules. Loxophyllum vesiculosum sp. n. differs from its congeners mainly by the unique distribution of contractile vacuoles, several of which lie along the dorsal margin and one on the ventral margin, and 15-21 right and 6-8 left kineties. Loxophyllum perihoplophorum is characterized by its large cell size (350-450 μm long in vivo), 3-5 contractile vacuoles along the dorsal margin in the posterior region of the body, and 19-23 right and 7-9 left kineties. An improved diagnosis of L. perihoplophorum is provided. The SSU rDNA sequence of L. perihoplophorum is reported for the first time and its molecular phylogeny is analyzed. Maximum likelihood and Bayesian inference analyses of SSU rDNA sequence data recover the monophyly both of the order Pleurostomatida and of the genus Loxophyllum.