Macronuclear Nodules Research Articles

Morphology and morphogenesis of a soil urostylid ciliate, Paragastrostyla terricola (Foissner, 1988) Berger, 2006 (Ciliophora: Hypotrichia)

ABSTRACT The soil urostylid ciliate Paragastrostyla terricola, isolated from northern China, was studied for its morphology and morphogenesis using live observation and protargol impregnation. The main ontogenetic features of P. terricola are as follows: (a) in the proter, the anterior portion of the parental adoral zone of membranelles is retained, while only the posterior part is renewed; undulating membranes anlage undergo, like most hypotrichs, depolymerisation of old undulating membranes and differentiation of new structures; (b) usually eight frontoventral cirral anlagen are formed, the penultimate anlage produces the midventral row and the rightmost anlage produces three frontoterminal cirri; (c) marginal and dorsal kineties anlagen are formed intrakinetally; one caudal cirrus is formed at the posterior end of each dorsal kinety; (d) the macronuclear nodules fuse into a single mass during the process. http://www.zoobank.org/urn:lsid:zoobank.org:pub:A150EE18-03BF-46B4-9E5B-48273FBA2CFF

Morphology, morphogenesis, and molecular phylogeny of Holostichides (Holostichides) parachardezi nov. Spec. (Ciliophora, Hypotrichia, Urostylida), a new soil ciliate from China

A new urostylid ciliate, Holostichides (Holostichides) parachardezi, collected from a Chinese tropical city, was studied using morphological and molecular methods. Diagnostic features of the new species include: size 140–180 × 30–60 μm in vivo; 25–43 macronuclear nodules; colourless cortical granules; three to six frontoterminal cirri; a midventral complex composed of seven to eleven cirral pairs and one midventral row of five to eleven cirri; four bipolar dorsal kineties and six to nine caudal cirri in four groups; and a terrestrial habitat. Its ontogenesis follows the typical Holostichides mode in that (1) the proximal portion of the parental adoral zone of membranelles is partly renewed and the opisthe’s oral primordium originates very close to the midventral complex; (2) the last anlage n generates the frontoterminal cirral row, and the midventral complex is formed from anlage IV to n − 1; and (3) both marginal rows and dorsal kinety anlagen develop within the parental rows. The present SSU rDNA phylogenies corroborated the monophyly of the genus Holostichides.

Morphology, morphogenesis and molecular phylogeny of a new soil ciliate, Lamtostyla paravitiphila nov. spec. (Ciliophora, Hypotrichia)

The morphology and morphogenesis of Lamtostyla paravitiphila nov. spec., a novel soil hypotrichous ciliate collected from eastern China, were investigated based on live observations and protargol-stained specimens. The new species is morphologically characterized as follows: seven to twelve macronuclear nodules, cortical granules absent, 19–26 adoral membranelles, three or four frontoventral cirri, the amphisiellid median cirral row extends to about mid-body and composed of 12–18 cirri, two or three transverse cirri, 27–39 left and 30–41 right marginal cirri, three almost bipolar dorsal kineties. Morphogenetically, it is characterized by the initial formation of six frontal-ventral-transverse cirral anlagen as primary primordia. Notably, the amphisiellid median cirral row and the posterior frontoventral cirrus (or cirri) contribute to the development of the frontal-ventral-transverse cirral anlagen, while the buccal cirrus may not participate in this process. Phylogenetic analyses based on small subunit ribosomal DNA sequence data indicate that the Lamtostyla species with available molecular data do not form a monophyletic group.

A new contribution to the raptorial ciliate genus Lacrymaria (Protista: Ciliophora): a brief review and comprehensive descriptions of two new species from Changjiang Estuary.

Ciliates serve as excellent indicators for water quality monitoring. However, their utilization is hindered by various taxonomic confusions. The ciliate genus Lacrymaria Bory de Saint-Vincent, 1824 is commonly found in different aquatic habitats, but its taxonomy has been sparsely investigated using state-of-the-art methods. This study investigated two new Lacrymaria species from Nanhui Wetland, Shanghai, China, using living observation, protargol staining, and molecular phylogeny methods. Lacrymaria songi sp. nov. is 180-340 × 20-25 μm in size and possesses 12-16 somatic kineties, 1 terminal contractile vacuole, 2 macronuclear nodules, and 2 types of rod-shaped extrusomes. Lacrymaria dragescoi sp. nov. is distinguished from its congeners by its cell size of 210-400 × 25-35 μm, 14-17 somatic kineties, 1 terminal contractile vacuole, 1 macronucleus, and 2 types of rod-shaped extrusomes. Phylogenetic analyses based on SSU rRNA gene sequences indicate that Lacrymariidae is monophyletic but Lacrymaria is not. Additionally, a brief review of the genus Lacrymaria is provided in this study. We suggest that L. bulbosa Alekperov, 1984, L. lanceolata Kahl, 1930, and L. ovata Burkovsky, 1970 be removed from the genus and propose Phialina lanceolata nov. comb. and Phialina ovata nov. comb. for the latter two. ZooBank registration: Present work: urn:lsid:zoobank.org:pub:CDFB1EBD-80BD-4533-B391-CEE89F62EDC4 Lacrymaria songi sp. nov.: urn:lsid:zoobank.org:act:417E7C2D-DAEC-4711-90BB-64AB3CD2F7D5 Lacrymaria dragescoi sp. nov.: urn:lsid:zoobank.org:act:8778D6B0-1F2E-473C-BE19-3F685391A40D.

Morphology, morphogenesis and molecular phylogeny of a novel saline soil ciliate, Urosoma quadrinucleatum n. sp. (Ciliophora, Hypotrichia)

The morphology and morphogenesis of a new saline soil hypotrichous ciliate, Urosoma quadrinucleatum n. sp., collected from northwestern China, were studied based on live observations and protargol stained specimens. The new species is characterized as follows: size in vivo 90–130 × 20–30 μm; body outline elongate-elliptical with both ends broadly rounded; four macronuclear nodules; cortical granules present; paroral in front of endoral; usually 16 frontal-ventral-transverse cirri. Urosoma quadrinucleatum n. sp. has almost the same morphogenetic characteristics as its congeners U. gigantea and U. salmastra, and differs from other three congeners whose morphogenesis is known in the formation of the frontal-ventral-transverse cirral anlagen as well as the development of marginal and dorsal kineties anlagen. The sequence differences among U. quadrinucleatum n. sp. and other Urosoma species further support the validity of the present organism as novel species. Further, U. quadrinucleatum n. sp. clusters with U. salmastra in the phylogenetic analyses based on small subunit ribosomal RNA gene (SSU rDNA) sequence data.

Morphology and phylogenetic position of three anaerobic ciliates from the classes Odontostomatea and Muranotrichea (Ciliophora).

The diversity of the classes Odontostomatea and Muranotrichea, which contain solely obligate anaerobes, is poorly understood. We studied two populations of Mylestoma sp., one of Saprodinium dentatum (Odontostomatea), two of Muranothrix felix sp. nov., and one of Muranothrix sp. (Muranotrichea) employing live observation, protargol impregnation, scanning electron microscopy, and 18S rRNA gene sequencing. Conspecificity of Mylestoma sp., described here, with a previously described species of this genus cannot be excluded since no species have been studied with modern methods. Phylogenetically, the genus Mylestoma is closely related to the odontostomatid Discomorphella pedroeneasi, although the phylogenetic position of class Odontostomatea itself remains unresolved. The newly described muranotrichean species, Muranothrix felix sp. nov., is morphologically similar to M. gubernata but can be distinguished by its fewer macronuclear nodules and fewer adoral membranelles; moreover, it is clearly distinguished from M. gubernata by its 18S rRNA gene sequence. Another population, designated here as Muranothrix sp., most likely represents a separate species.

Description of a new oxytrichid ciliate, Oxytricha buxai n. sp. and redescription of O. quadricirrata Blatterer and Foissner, 1988 based on morphology and 18S rDNA analyses

The morphology of a new oxytrichid ciliate, Oxytricha buxai n. sp., isolated from a soil sample collected from the Buxa Tiger Reserve, West Bengal, India, was studied based on live observation and protargol impregnation. The new species is characterised by a body size of 85×35 µm in vivo, two macronuclear nodules with one or two micronuclei attached at variable positions, a few colourless cortical granules scattered throughout cortex, adoral zone of membranelles about 35% of body length with 26 membranelles on average, about 18 cirri in left and 16 cirri in right marginal row, right marginal row starts at the level of buccal vertex, usually 18 frontoventral transverse cirri, five dorsal kineties including one dorsomarginal row, three caudal cirri. Further, a redescription based on live and protargol-impregnated specimens of Oxytricha quadricirrata Blatterer and Foissner, 1988, isolated from a moss sample collected from the Kangra district, Himachal Pradesh, India, is provided. The Indian population of O. quadricirrata is similar in morphology to the type population. However, the dorsal side shows some variation, i.e., the presence of a second dorsomarginal row with one or two bristles and incomplete fragmentation of dorsal kinety 3 (vs single dorsomarginal row and complete fragmentation). The resting cyst is spherical and about 20 µm across, with a wrinkled surface. Morphogenesis is in typical Oxytricha pattern. Based on 18S rDNA, phylogenetic analyses show Oxytricha to be a polyphyletic genus. Further, O. quadricirrata clusters away from O. granulifera, thereby supporting the validity of the former.

Strongylidium koreanum n. sp. (Protozoa: Ciliophora), a new soil species from South Korea.

Strongylidium koreanum n. sp., a new soil ciliate from Jeju Island, South Korea, is described based on live observations, protargol impregnation, and molecular analysis of the 18S rRNA gene sequence. It is characterized by the following morphological features: cell outline more or less fusiform, posterior end broader than anterior end; grayish under low magnification; cortical granules absent; 23-32 adoral membranelles; three enlarged frontal cirri; buccal cirrus and postoral ventral cirrus present; 27-42 left and 15-28 right ventral cirri; 23-36 left and 30-46 right marginal cirri; three dorsal kineties; three caudal cirri; and two macronuclear nodules with two or three micronuclei. Phylogenetic analyses show that Strongylidium is monophyletic.

The description of a new brackish water ciliate species from China, Trachelostyla aestuarina n. sp., with a species key and biogeographic investigation for Trachelostyla (Ciliophora, Sporadotrichida)

The ciliated protists are a diverse group of microbial eukaryotes that play an important role in the functioning of microbial food webs. The genus Trachelostyla Borror, 1972, comprised a few species with a non-twisted distinctly cephalized shape. The overall taxonomy has been challenging for this group, and the understanding of their diversity and distribution is therefore relatively limited. To further investigate this group, we report here on two species from the East China Sea, Trachelostyla aestuarina n. sp. and Trachelostyla caudata Borror, 1972, including their in vivo morphology, infraciliature, and phylogenetic relationships based on 18S rRNA gene sequences. The physiological reorganization stages of the latter were also investigated. T. aestuarina is characterized by a combination of features including a bipartite cell shape, a size of 150–220 × 15–35 μm, scattered cortical granules, and multiple macronuclear nodules (37–55). T. caudata has only 10 cirri in the frontal region, fewer than its congeners; thus, we improved the definition of the genus. The global distribution of Trachelostyla was summarized based on the available literature and a key to the valid species provided. This work improves the overall knowledge of hypotrich biodiversity and provides underpinning for future researchers in global regions for environmental monitoring and resource investigations.

Ontogenesis and systematic position of a new hypotrichous ciliate, Chaetospira sinica sp. nov., with an improved diagnosis of the poorly defined family Chaetospiridae Jankowski, 1985 (Protozoa, Ciliophora, Hypotrichia).

Ciliates are unique single-celled organisms that play important roles in ecological, environmental, evolutionary, and ontogenetic research. In the present study, phylogenetic analyses based on 18S rRNA gene sequence data reveal that Chaetospira sinica sp. nov. clusters with Stichotricha aculeata with strong to full support (97% ML, 1.00 BI), but is not closely related to members of Spirofilidae Gelei, 1929 to which Chaetospira and Stichotricha have previously been assigned. Phylogenetic analyses, together with morphological and morphogenetic data from Chaetospira sinica sp. nov., support the validity of family Chaetospiridae Jankowski, 1985. Chaetospira and Stichotricha are here assigned to the family Chaetospiridae, the improved diagnosis of which is as follows: non-dorsomarginalian Hypotrichia with flask-shaped body; oral region extending along narrow anterior neck region; lorica usually present; two ventral and two marginal cirral rows, all distinctly spiraled or obliquely curved; pretransverse and transverse cirri absent. The basic morphogenetic features in C. sinica sp. nov. can be summarized as: (1) the oral primordium for the opisthe develops de novo and the parental adoral zone is completely retained by the proter; (2) all ventral cirral anlagen and marginal anlagen developed intrakinetally; (3) three dorsal kineties anlagen formed intrakinetally in each daughter cell; and (4) macronuclear nodules fuse into a single mass. Exconjugant cells were also isolated and their morphologic and molecular data are provided.

Morphology and molecular phylogeny of Keronopsis koreana n. sp. (Ciliophora, Hypotricha), a new soil species from South Korea

The morphology and molecular phylogeny of a new soil ciliate, Keronopsis koreana n. sp., which was discovered in soil from Jindo-gun, South Korea, were investigated. The new species is characterized by the following morphological features: cell more or less ellipsoid or ovoid, both ends broadly rounded; cortical granules irregularly distributed, colorless, 1 µm across; 35–54 adoral membranelles; 12–23 cirri in frontal corona; two frontal and two frontoventral cirral rows; two or three buccal cirri; 23–38 left and 22–37 right marginal cirri; three bipolar dorsal kineties; and two macronuclear nodules and three to five micronuclei. Phylogenetic analyses show that Keronopsis koreana n. sp. is placed within a clade containing K. helluo (type species of Keronopsis) and species belonging to Paraholosticha, Uroleptoides, Parabistichella, Orthoamphisiella, Lamtostyla, and Bistichella in non-dorsomarginalian hypotrichs. Also, the sequences belonging to the Keronopsidae did not cluster together. In addition, a new combination is provided: Parabistichella tasmaniensis (Blatterer and Foissner, 1988) n. comb. (original combination: Keronopsis tasmaniensis).

Morphology, morphogenesis and phylogeny of a new soil ciliate, Bistichella sinensis n. sp., and morphology of two oxytrichids (Ciliophora, Hypotrichia)

The living morphology, infraciliature, morphogenesis and phylogenetic position of a new soil ciliate, Bistichella sinensis n. sp. collected from Northwest of China, were investigated. The new species is characterized by an elongate-oval body, two macronuclear nodules, colourless cortical granules, three frontal and three or four buccal cirri, three frontal rows with 7–10 cirri in total, and usually-two frontoventral rows with the left one usually terminating at 90% down length of body. The main morphogenetic features of the novel species are as follows: (1) the posterior part of the parental adoral zone is renewed; (2) frontal-ventral cirral anlagen III to V each forms a frontal row, and anlagen VI to n each produces a frontoventral row; (3) marginal rows and dorsal kineties develop intrakinetally. Furthermore, the phylogenetic analyses based on small subunit ribosomal DNA reveal close relationship between Bistichella sinensis n. sp., Parabistichella, Uroleptoides, Lamtostyla, Keronopsis, Paraholosticha and Orthoamphisiella. The in vivo morphology and the infraciliature of the Chinese populations of Monomicrocaryon euglenivorum fimbricirratum and Laurentiella strenua are basically identical to previous descriptions. Improved diagnoses for M. euglenivorum and its two subspecies, as well as a redescription of a Chinese population of L. strenua are supplied.

Morphology and morphogenesis of the hypotrich ciliate Parentocirrus hortualis Voβ, 1997, with notes on the phylogeny of Parentocirrus (Ciliophora, Hypotrichia)

The morphology, morphogenesis and molecular phylogeny of the hypotrich ciliate Parentocirrus hortualis, isolated from soil in northwestern China, was studied using live observation and protargol staining. The Chinese population closely matches the German type population although some features such as the number of macronuclear nodules (4–8 vs 5–16) and transverse cirri (4 or 5 vs 2–4) differ considerably. These differences can probably be explained, at least in part, by environmental adaptability. We also compared the ontogenetic processes in the two populations and did not find any significant differences. Phylogenetic analyses based on SSU rDNA gene sequence data showed that Parentocirrus hortualis clusters with Gastrostyla steinii and Parentocirrus sp., which together form a clade with another species of Gastrostyla within the Stylonychinae. These findings suggest that Parentocirrus and Gastrostyla are closely related.

Two hypotrichs (Ciliophora, Hypotricha) from China: morphology and SSU rDNA sequence of Holosticha aestuarina nov. spec. and H. muuiensis Kim et al., 2017

The morphology, four ontogenetic stages, and the phylogenetic relationships based on the small subunit RNA-gene of Holosticha aestuarina nov. spec. from the Yangtze River Estuary (Shanghai) were investigated. The new species differs from the congeners by the following combination of features: body size 160–196 × 27–36 μm in vivo, a vesicular structure containing one or two bean-shaped opaque particles, 24–32 adoral membranelles, 16–34 midventral pairs, 17–26 left marginal cirri, 22–32 right marginal cirri, six or seven transverse cirri, four dorsal kineties, and usually two macronuclear nodules. The anlage for the left marginal row of the proter very likely originates de novo (anterior portion) and from parental cirri (posterior portion). Further, we provide a morphological description of a Chinese population of H. muuiensis Kim et al., 2017, originally discovered in South Korea, indicating that it is confined to the Eastern Asia region. Its SSU rDNA sequence is identical with that of the type population and forms a clade with the available Holosticha sequences. By contrast, H. aestuarina nov. spec. is sister to the Holosticha + Uncinata clade, indicating that Uncinata is a subgenus of Holosticha.

Morphology, Morphogenesis and Molecular Phylogeny of a New Soil Ciliate, Holostichides eastensis nov. spec. (Ciliophora, Hypotrichia)

A new bakuellid ciliate, Holostichides eastensis nov. spec. isolated from China, is investigated in terms of its morphology, ontogenesis, and molecular phylogenesis. It is characterized by size 150-180 µm × 45-60 µm in vivo; 27-46 macronuclear nodules; cortical granules dark brown, spherical, about 0.5 µm across, clustered in groups and then arranged longitudinally; three frontal cirri with one to four smaller cirri below the middle one, a buccal cirrus, nine to 15 frontoterminal cirri, and midventral complex composed of five to eight cirral pairs and two long midventral rows. The main events during binary fission are as follows: (1) in the proter, the undulating membrane anlage is formed from both the dedifferentiation of old undulating membranes and the basal bodies developing de novo, and the proximal part of the parental adoral zone of membranelles is renewed by a field of basal bodies that originates de novo; (2) in the opisthe, the oral primordium is formed intrakinetally; (3) besides the middle frontal cirrus and buccal cirrus, the FVT-anlagen II produces the extra one to four cirri. Phylogenetic analyses based on SSU rDNA sequence data suggest a close relationship between Holostichides eastensis nov. spec. and its congeners, indicating that the genus Holostichides is monophyletic.

Taxonomy and Phylogeny of Two Spirotrichean Ciliates (Alveolata, Protista, and Ciliophora) From a Plateau Saline–Alkaline Lake in Tibet, China, Including Description of a New Species

There are few researches concerning ciliates from “extreme” environments such as plateau salt lakes; nevertheless, such a faunistic survey is of great significance for understanding the diversity and biogeography of these microeukaryotes. In this study, two ciliates, namely, Hemiurosomoida tibetensis sp. nov. and Euplotes euryhalinusValbonesi and Luporini, 1990, isolated from Kyêbxang Co, a Tibet Plateau saline-alkaline lake, China, were investigated based on observations of live and silver-stained specimens, and 18S rDNA sequences were analyzed. The new species can be characterized by having a size of 60–95 × 20–40 μm in vivo, an elongate elliptical body, colorless cortical granules mainly grouped around cirri and dorsal bristles, about 21 adoral membranelles, 20 cirri on each marginal row, and two macronuclear nodules and two micronuclei. The Chinese population of E. euryhalinus corresponds well with the original one. Phylogenetic analyses show that Hemiurosomoida is non-monophyletic, H. tibetensis sp. nov. has a closer relationship with H. longa within this genus, and that Euplotes euryhalinus presents a wide geographic distribution. This study first reveals the ciliature, morphogenesis, and rRNA gene sequence data for H. tibetensis sp. nov. and the Chinese population of E. euryhalinus, which thus increases our knowledge about the diversity of ciliates at high altitudes.

Multi-Gene Phylogeny of the Ciliate Genus Trachelostyla (Ciliophora, Hypotrichia), With Integrative Description of Two Species, Trachelostyla multinucleata Spec. nov. and T. pediculiformis (Cohn, 1866).

Many hypotrich genera, including Trachelostyla, are taxonomically challenging and in a need of integrative revision. Using morphological data, molecular phylogenetic analyses, and internal transcribed spacer 2 (ITS2) secondary structures, we attempt to cast more light on species relationships within the genus Trachelostyla. The present multifaceted approach reveals that (1) a large-sized species with numerous macronuclear nodules, isolated from sandy littoral sediments in southern China, is new to science and is endowed here with a name, T. multinucleata spec. nov.; (2) two other Chinese populations previously identified as T. pediculiformis represent undescribed species; and (3) multigene phylogeny is more robust than single-gene trees, recovering the monophyly of the genus Trachelostyla with high bootstrap frequency. Additionally, ITS2 secondary structures and the presence of compensatory base changes in helices A and B indicate the presence of four distinct taxa within the molecularly studied members of the genus Trachelostyla. Molecular data are more suitable for delimitation of Trachelostyla species than morphological characters as interspecific pairwise genetic distances of small subunit (18S) rDNA, ITS1-5.8S-ITS2, and large subunit (28S) rDNA sequences do not overlap, whereas ranges of multiple morphometric features might transcend species boundaries.

Morphogenesis and molecular phylogeny of a freshwater ciliate, Oxytricha multilineata n. sp. (Ciliophora, Hypotrichia)

The morphology, morphogenesis, and molecular phylogeny of a new oxytrichid ciliate, Oxytricha multilineata n. sp. isolated from a tributary of the YuYao River, Ningbo, China, were investigated. Oxytricha multilineata n. sp. is characterized as follows: body size about 85–110 × 30–40 µm in vivo; 18 frontoventral-transverse cirri; paroral distinctly bipartite; seven dorsal kineties with cilia about 18 μm long; two macronuclear nodules and two micronuclei. The main events during morphogenesis follow a typical Oxytricha pattern with two exceptions: (1) only one anlage is formed initially within parental dorsal kineties 1–3, each of which then divides and is distributed into the two filial products, that is, the dorsal kineties anlagen are formed in primary mode; (2) dorsal kineties 3 and 4 are formed by incomplete fragmentation. Phylogenetic analyses based on SSU rDNA sequence data reveal that O. multilineata n. sp. clusters with Oxytricha lithofera, Heterourosomoida lanceolata and Quadristicha setigera, and species of the genus Oxytricha did not form a monophyletic group, however nodal support is not sufficiently high to recover a robust phylogeny of the Oxytrichidae.

Taxonomic and phylogenetic studies on two new freshwater Amphileptus species (Ciliophora, Pleurostomatida), from Lake Weishan, northern China

The morphology and phylogeny of two new species of pleurostomatid ciliates, Amphilepus weishanensis sp. nov. and A. parapleurosigma sp. nov., collected from Lake Weishan in northern China, were studied using live observation, protargol staining and phylogenetic analyses based on small subunit ribosomal DNA (SSU rDNA) sequence data. In both species, extrusomes form an apical group and contractile vacuoles are distributed along both the dorsal and the ventral margin. Amphileptus weishanensis sp. nov. is characterized by its body size (560–780 × 60–100 μm in vivo) and the possession of five left and 56–61 right somatic kineties, filiform extrusomes and 3–9 macronuclear nodules. Amphileptus parapleurosigma sp. nov. is characterized by possessing 4–6 left and 19–24 right somatic kineties and clavate extrusomes. The SSU rDNA sequences differ among ten similar species by 3–60 bp. Phylogenetic analyses based on the SSU rDNA sequences indicate that the family Amphileptidae is monophyletic while Amphileptus is paraphyletic with Pseudoamphileptus nesting within this genus.

Taxonomy and systematics of a new pleurostomatid ciliate, Pseudolitonotus spirelis gen. et sp. n. (Protozoa, Ciliophora, Haptoria).

Recent studies have revealed a high diversity of pleurostomatid ciliates in brackish habitats. Here, a novel species, Pseudolitonotus spirelis gen. et sp. n., isolated from a mangrove wetland of southern China, was investigated based on living observation, protargol staining, and molecular analyses. The new genus Pseudolitonotus gen. n. is characterized by the last left somatic kinety (LKn) being shortened and none of the right somatic kineties extending to the anterior end of the cell, thus distinguishing it from all known pleurostomatid genera. The type species, Pseudolitonotus spirelis sp. n., is characterized by the possession of two macronuclear nodules, 11-15 right and 7-9 left kineties, a single contractile vacuole subterminally located, extrusomes evenly spaced along the entire ventral margin and some forming an "apical group", two types of cortical granules, and the bottom of the oral slit invariably being twisted. Litonotus gracilis (Pan et al. Eur J Protistol 51:494-506, 2015) is believed to be another member of this new genus as its LKn and right somatic kineties are all shortened. Hence, a new combination, Pseudolitonotus gracilis (Pan et al., 2015) comb. n., is suggested and its diagnosis is improved. Molecular phylogenetic analyses based on SSU rDNA sequence data reveal that Pseudolitonotus gen. n. is monophyletic and groups with Apolitonotus (Pan et al. J Eukaryot Microbiol 67:252-262, 2020) of the family Protolitonotidae (Wu et al. Zool Scr 46:245-253, 2017). However, the familial assignment of this new genus is uncertain based on current data.