Hypotrich ciliates, a large group of mainly free-living protists with highly diverse cortical structures, are commonly found in a broad variety of habitats worldwide. Methodological difficulties and insufficient faunistic studies have limited our understanding of their true biodiversity and phylogeny. In this report, two new Strongylidium species from China are investigated using live observation, protargol staining and 18S rRNA gene sequencing, and the evolution of strongylidiid ciliates is investigated based on morphological, morphogenetic and molecular data. Recently, the genera Strongylidium Sterki, 1878 and Pseudouroleptus Hemberger, 1985 were assigned to the family Spirofilidae Gelei, 1929. Given that these genera differ morphologically and phylogenetically from the type genus of Spirofilidae, Hypotrichidium Ilowaisky, 1921, their familial classification should be reconsidered. Moreover, the highly characteristic formation pattern of the mixed left ventral cirral row and other morphological features shared by Strongylidium, Pseudouroleptus and Hemiamphisiella Foissner, 1988, together with their 18S rRNA gene sequences, suggest that the three genera form a group closely related to Dorsomarginalia Berger, 2006. The family Strongylidiidae Fauré-Fremiet, 1961 is reactivated for these three genera.

The classification of the family Psilotrichidae, a curious group of ciliated protists with unique morphological and ontogenetic features, is ambiguous and poorly understood particularly due to the lack of molecular data. Hence, the systematic relationship between this group and other taxa in the subclass Hypotrichia remains unresolved. In this paper the morphology and phylogenetics of species from two genera of Psilotrichida are studied to shed new light on the phylogeny and species diversity of this group of ciliates. The 18S rRNA gene sequences of species from two psilotrichid genera were obtained. In the phylogenetic trees, the available psilotrichid sequences are placed in a highly supported clade, justifying the establishment of the family Psilotrichidae. The morphology of two little-known species, packed with green algae, including a new species, Hemiholosticha kahli nov. spec., and Psilotrichides hawaiiensis Heber et al., 2018, is studied based on live observation, protargol impregnation, and scanning electron microscopy. Both species are easily recognized by their green coloration due to the intracellular algae, and a comprehensive discussion as to the possible roles of the intracellular algae is provided. The 18S rRNA gene phylogeny supports the morphological argument that Hemiholosticha, Psilotrichides and Urospinula belong to the same family, Psilotrichidae. However, the single-gene analysis, not surprisingly, does not resolve the deeper relationships of Psilotrichidae within the subclass Hypotrichia. Two little-known psilotrichid genera with green algae were collected from the same puddle on the island of Guam, indicating a high species diversity and broader geographic distribution of this group of ciliates than previously supposed. Phylogenetic inferences from transcriptomic and/or genomic data will likely be necessary to better define the systematic position and evolution of the family Psilotrichidae. Further studies are also needed to clarify the role of the intracellular eyespot-bearing algae in these ciliates.

Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups, leading to their systematics remaining unresolved. Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data. In this study, a new strongylidiid genus Heterouroleptus is established, mainly based on the novel mode of origin of the ventral cirral rows: left ventral cirral row (LVR) originates from frontal–ventral–transverse cirral anlagen (FVTA) III (anterior portion), IV (middle portion), and V (rear portion); right ventral cirral row comes from the entire FVTA VI. A new species, Heterouroleptus weishanensis gen. nov., sp. nov., is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense. Eight new sequences and nuclear gene markers (single-gene and multi-gene) are provided to analyze the phylogenetic relationships of strongylidiids, with the COI gene utilized to uncover further genetic information at species level and below. The results reveal that: (1) Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia; (2) Heterouroleptus gen. nov. forms a clade that is sister to all the other strongylidiids; (3) Hemiamphisiella Foissner, 1988 and Pseudouroleptus Hemberger, 1985 should not be synonyms, and both genera should be subdivided due to their variable morphological characteristics; (4) LVR originating from three anlagen is a plesiomorphy of Strongylidiidae. The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus, but also helps to improve the diagnosis of the family Strongylidiidae.