Protargol Staining Research Articles

Morphology, Morphogenesis and Molecular Phylogeny of Two Freshwater Ciliates (Alveolata, Ciliophora), with Description of Pseudosincirra binaria sp. nov. and Redefinition of Pseudosincirra and Perisincirra

Ciliated microeukaryotes are insufficiently investigated despite their ubiquity and ecological significance. The morphology and morphogenesis of a new Stichotrichida species, Pseudosincirra binaria sp. nov., and the known Perisincirra paucicirrata Foissner et al., 2002, are here studied using live observations and protargol staining methods. The new species is characterized by having one buccal, one parabuccal and three frontal cirri, one frontoventral row extending to the posterior half of the cell, three left and two right marginal rows and three dorsal kineties with the left kinety conspicuously bipartite, along with one caudal cirrus at the rear end of each kinety. During morphogenesis, there exist five frontal-ventral cirral anlagen with anlagen IV and V forming a frontoventral row in the proter, and four cirral anlagen with only anlage IV generating a frontoventral row in the opisthe. The anlagen for marginal rows and dorsal kineties develop intrakinetally. The new population of Perisincirra paucicirrata corresponds well with other isolates regarding morphology and cell development. Phylogenetic analyses based on small subunit ribosomal gene sequence data revealed that both Perisincirra and Pseudosincirra are deeply clustered in the clade consisting of species from the genera Deviata and Heterodeviata, supporting the placement of both genera into the family Deviatidae Foissner, 2016.

Taxonomy and molecular phylogeny of genus Plagiocampa (Ciliophora, Prostomatea), with redescriptions of two poorly known species

Prostomateans, as common inhabitants in diverse aquatic environments, are among the simplest ciliate lineages, and serve as trophic links in food webs. However, only a few members are well-known and thoroughly studied, and the diversity of this group remains elusive. The unique genus Plagiocampa has a long history of research, but few studies have been performed using up-to-date methods. In the present work, Plagiocampa longis Kahl, 1927 and Plagiocampa minima Kahl, 1927, collected from Chinese coastal habitats, were investigated based on microscopical observation, protargol staining, and SSU rRNA gene sequencing. Their ciliature and morphometric data as well as gene sequences are documented. Phylogenetic analyses revealed that the family Plagiocampidae is likely monophyletic and has a closer relationship with parasitic Cryptocaryon.

Coleps shanghaiensis n. sp. challenges the validity of the genus Levicoleps (Ciliophora, Prostomatida)

Coleps is a common genus of pelagic ciliates in freshwater and brackish water habitats. Classification and phylogeny of Coleps species are, however, still full of confusion. In this study, we investigated Coleps shanghaiensis n. sp., collected from a river in Shanghai, China, by living observation, protargol staining, and molecular methods. Coleps shanghaiensis is about 70–90 µm × 35–55 µm in size, has a barrel-shaped body with three posterior spines, and possesses 21–24 ciliary rows, each composed of two perioral dikinetids and 19–22 monokinetids, and six caudal cilia. In SSU rRNA gene phylogenies, C. shanghaiensis fell within the clade of subspecies of Levicoleps biwae, which questions the validity of the genus Levicoleps. Furthermore, the biogeography of the genus Coleps is discussed.

Morphology and molecular phylogeny of a Chinese population of Rubrioxytricha guamensis Kumar et al., 2018 (Ciliophora: Hypotrichia)

ABSTRACT So far, Rubrioxytricha guamensis Kumar et al. 2018 is known only from the type locality, namely Guam, USA. Recently, another isolate was recorded from a freshwater habitat in China that is 3500 km from the type locality. Its morphology and molecular phylogeny were investigated using live observation, protargol staining and ribosomal gene sequencing methods. The morphology of the present isolate of R. guamensis corresponds well with that of the type population despite a broad range of morphometric data in the Chinese population. Conspecificity of the two populations is supported by 99.9% 18S rRNA gene sequence similarity. Phylogenetic analysis reveals that these two populations of R. guamensis group together and cluster within the genus. The present study contributes to understanding the wide distribution of the species and helps to circumscribe the species. http://www.zoobank.org/urn:lsid:zoobank.org:pub:8D742552-78DA-4698-A316-BDAE0D887E92

Integrative data of a novel ciliate (Alveolata, Ciliophora) propose the establishment of Heterodeviata nantongensis nov. sp.

BackgroundAs unicellular eukaryotes, ciliates are an indispensable component of micro-ecosystems that play the role of intermediate nutrition link between bacteria or algae and meiofauna. Recent faunistic studies have revealed many new taxa of hypotrich ciliates, indicating their diversity is greater than previously thought. Here we document an undescribed form isolated from an artificial brackish water pond in East China. Examination of its morphology, ontogenesis and molecular phylogeny suggests that it represents a new species.ResultsThe morphology and morphogenesis of the new brackish-water deviatid ciliate, Heterodeviata nantongensis nov. sp., isolated from Nantong, China, were investigated using live observations and protargol staining. The diagnostic traits of the new species include three frontal cirri, one buccal cirrus, one or two parabuccal cirri, an inconspicuous frontoventral cirral row of four to six frontoventral cirri derived from two anlagen, three left and two right marginal rows, two dorsal kineties, dorsal kinety 1 with 9–14 dikinetids and dorsal kinety 2 with only two dikinetids, and one to three caudal cirri at the rear end of dorsal kinety 1. Its main morphogenetic features are: (i) the old oral apparatus is completely inherited by the proter except undulating membranes, which are reorganized in situ; (ii) anlagen for marginal rows and the left dorsal kinety develop intrakinetally in both proter and opisthe; (iii) dorsal kinety 2 is generated dorsomarginally; (iv) five cirral anlagen are formed in both proter and opisthe; (v) in the proter, anlagen I and II very likely originate from the parental undulating membranes and the buccal cirrus, respectively, anlage III from anterior parabuccal cirrus, anlage IV originates from the parental frontoventral cirri and anlage V from the innermost parental right marginal row; and (vi) anlagen I–IV of the opisthe are all generated from oral primordium, anlage V from the innermost parental right marginal row. Phylogenetic analyses based on SSU rRNA gene sequence data were performed to determine the systematic position of the new taxon.ConclusionsThe study on the morphology, and ontogenesis of a new brackish-water taxon increases the overall knowledge about the biodiversity of this ciliate group. It also adds to the genetic data available and further provides a reliable reference for environmental monitoring and resource investigations.

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.

FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates.

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

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.

Taxonomy and molecular phylogeny of two new Blepharisma species (Ciliophora, Heterotrichea) from northeastern China

The morphology and ciliary pattern of two new heterotrich ciliates, Blepharisma orientale n. sp. and B. sinicum n. sp., collected from Sanjiang Plain Wetland, northeastern China, were investigated using living observation and protargol staining. Blepharisma orientale n. sp. is characterized by the following combination of characteristics: limnetic; body in vivo 280–380 × 120–190 μm, slender to elongate, cortical granules dark pink, ventral margin somewhat sigmoid; buccal field about 55–60% of body length; 30–39 somatic kineties; 70–76 adoral membranelles, each comprising two long rows of basal bodies; paroral membrane consists of dikinetids with the posterior third obliquely arranged, both basal bodies in each dikinetid are ciliated. Blepharisma sinicum n. sp. is recognized by the combination of the following characters: limnetic; body 190–260 × 100–170 μm in size in vivo, irregularly sigmoid and slightly bilaterally flattened, dorsal margin curved; cortical granules pale pink in color; buccal field about 60–75% of body length; 25–29 somatic kineties; 53–82 adoral membranelles; paroral membrane consisting entirely of dikinetids. Phylogenetic analyses based on small subunit rRNA (SSU rRNA) gene sequence data recovered the two new species as sister taxa, both the sister group of B. penardi.

New Contribution to the Diversity of the Anaerobic Genus Metopus (Ciliophora, Armophorea), With Descriptions of Three New Marine Species

Armophorean ciliates constitute an important component of microeukaryotic community in anaerobic or hypoxic environments. Yet, their diversity remains poorly known due to under-sampling or the scarcity of knowledge. In this study, three metopid ciliates, i.e., Metopus paraes sp. n., Metopus spiculatus sp. n., and Metopus parapellitus sp. n., collected from coastal sediments in Qingdao and Rizhao, China, were investigated using live observation, protargol staining, and molecular phylogenetic methods. M. paraes sp. n. can be distinguished by its long caudal cilia. M. spiculatus sp. n. resembles M. vestitus in many ways, but differs mainly in having a beak-like preoral dome end and a conspicuous tail. The most remarkable features of M. parapellitus sp. n. include an ovate body shape, caudal cilia located at the rear end and right posterior body, and an adoral zone that never extends onto the dorsal surface. Sequence divergences supported the species identification of these three species. Phylogenetic analyses confirmed that the Metopus is not monophyletic, and first revealed that all marine species of Metopus form a well-supported clade. The clustering of these marine forms with M. es (type species) is not rejected by the AU test, which infers that the marine clade represents the genus Metopus together with M. es.

Taxonomy and SSU rRNA gene-based phylogeny of two new Euplotes species from China: E. chongmingensis n. sp. and E. paramieti n. sp. (Protista, Ciliophora)

BackgroundThe genus Euplotes Ehrenberg, 1830, one of the most complicated and confused taxa, contains about 160 nominal species. It was once proposed to be divided into four genera, two of which were proved to be non-monophyletic. At least 19 new species have been discovered in the past decade, implying that there is a large undiscovered diversity of this genus.ResultsThe morphology of two new freshwater euplotid ciliates, Euplotes chongmingensis n. sp. and E. paramieti n. sp., isolated from Shanghai, China, were investigated using live observations, protargol staining, and Chatton-Lwoff silver staining method. Euplotes chongmingensis is characterized by its small size (40–50 × 25–35 μm), about 24 adoral membranelles, 10 frontoventral cirri, two marginal and two caudal cirri, eight dorsolateral kineties with 11–16 dikinetids in the mid-dorsolateral kinety and a double type of silverline system. Euplotes paramieti n. sp. is 180–220 × 110–155 μm in vivo and strongly resembles E. amieti but having a difference of 57 bp in their SSU rRNA gene sequences. Phylogenetic analyses based on SSU rRNA gene sequence data were used to determine the systematic positions of these new taxa.ConclusionsThe description of two new freshwater taxa and their SSU rRNA gene sequences improve knowledge of biodiversity and enrich the database of euplotids. Furthermore, it offers a reliable reference for environmental monitoring and resource investigations.

Taxonomy and phylogeny of the freshwater tintinnid Tintinnopsis tubuformis Chiang, 1956 (Ciliophora, Oligotrichea) and a proposed synonymization of T. longa nom. corr. Chiang, 1956.

Tintinnid ciliates are traditionally identified by their loricae; however, increasing evidence indicates that some lorica features (e.g. its length, spiraled structures) are not reliable. The vast majority of tintinnids inhabit the marine pelagial; merely, about thirty species live in freshwater. In the present study, two morphotypes with similar lorica shapes and opening diameters but deviating lorica lengths were isolated from freshwater samples collected at different water temperatures near Chongming Island in the Yangtze Estuary, China. The specimens were studied in vivo and after protargol staining, and their phylogenetic placement was inferred from three ribosomal RNA markers; further, cell division was investigated in the short morphotype. Based on the original descriptions, the longer morphotype is identified as Tintinnopsis longa nom. corr. Chiang, 1956, and the shorter one as Tintinnopsis tubuformis Chiang, 1956. Despite distinct differences in the lorica lengths, the identity of the three molecular markers in both morphotypes suggests conspecificity, which is supported by overlapping ranges in the lorica opening diameters and the length-independent features of the somatic ciliary pattern (e.g. number of kineties). Hence, we synonymized T. longa nom. corr. with T. tubuformis and neotypified the later species.

Morphology, cell division, and phylogeny of Notohymena antarctica Foissner, 1996 and Engelmanniella mobilis (Engelmann, 1862) Foissner, 1982 (Ciliophora, Hypotrichia)

The morphogenesis of two hypotrich ciliates, Notohymena antarctica Foissner, 1996 and Engelmanniella mobilis (Engelmann, 1862) Foissner, 1982, were investigated using protargol staining. The main events of the oxytrichid Notohymena antarctica during binary fission are as follows: (1) the oral primordium of the opisthe is formed apokinetally left of transverse cirrus II/1; (2) six streaks of frontal-ventral-transverse cirral anlagen are segmented in the ordinary pattern 1: 3: 3: 3: 4: 4 from left to right, which form three frontal, four frontoventral, one buccal, three ventral, two pretransverse ventral and five transverse cirri; (3) marginal anlagen are formed intrakinetally and the right marginal anlagen develop much earlier than those on the left. Phylogenetic analyses based on SSU rDNA sequence data indicate that Notohymena is monophyletic. A remarkable morphogenetic feature in E. mobilis is the mode of formation of the right marginal rows. In this paper, we clarified that the right marginal anlagen of E. mobilis are formed and develop to the right of the parental outer right marginal row, and that the grandparental (inner) right marginal row and great-grand parental (the outermost) left marginal row are resorbed. It is the first time to report a population of E. mobilis with comprehensive information, i.e. the morphology, ontogeny and molecular sequence.

Morphological, Molecular, and Growth Characteristics of a Cryptic Species, Strombidium parasulcatum n. sp. (Alveolata: Ciliophora: Oligotrichida).

A new marine planktonic ciliate from Taiwan, Strombidium parasulcatum sp. n., is described based on live observations, protargol staining, and molecular data. Its morphological characters are similar to those of Strombidium sulcatum Claparède and Lachmann, 1859 sensu Song et al., 2000 but differ from S. sulcatum sensu Fauré-Fremiet, 1912 and sensu Fauré-Fremiet and Ganier, 1970 by several morphological characters. The 18S rRNA gene sequences of the two forms display 76 base pair differences (about 5%), indicating that they should be considered separate species. The highest observed specific growth rates of S. parasulcatum in culture were 1.79 day–1 over 1 day and 1.52 day–1 over 2 days, both starting at day 5. Bacteria appear to be an important food resource for the cultivation of this medium-sized oligotrich ciliate. This and other recent studies suggest that cryptic species may be common in the genus Strombidium, and an integrative approach including morphological, ecological, and molecular data should be used to address this question.

New considerations of the systematics of the family Holophryidae (Protozoa, Ciliophora, Prostomatea) with a description of Holophrya paradiscolor sp. nov. and a redescription of Pelagothrix plancticola

The diversity of the family Holophryidae is relatively poorly known due to a lack of studies using modern methods, the paucity of taxonomically informative characters for species circumscription, and the morphological similarity among holophryids. In the present study, two holophryid ciliates, namely Pelagothrix plancticola and Holophrya paradiscolor sp. nov., isolated from two freshwater ponds in Qingdao, China, are reported. The morphology of both species was described based on living observation and protargol staining, and their molecular phylogeny was analysed using SSU rRNA gene sequence data. Pelagothrix plancticola has never been redescribed since its establishment. Based on new data, an improved diagnosis of P. plancticola is supplied. Holophrya paradiscolor sp. nov. is distinguished by a combination of features including a single macronucleus, the absence of endosymbiotic green algae, and the presence of two types of extrusomes. Following the addition of two new sequences, the molecular phylogeny of the family Holophryidae was analysed. Finally, the information on the habitats and geographic distribution of holophryids was reviewed. http://zoobank.org/urn:lsid:zoobank.org:pub:3D2D3902-58BA-47D7-A5DE-FEA0A5163384

Redescription and phylogenetic position of the terrestrial ciliates Gastrostylides dorsicirratus and Heterourosomoida lanceolata (Hypotricha, Dorsomarginalia)

The morphology and infraciliature of two soil hypotrichous ciliates, Heterourosomoida lanceolata (Shibuya, 1930) Singh and Kamra, 2015 and Gastrostylides dorsicirratus (Foissner, 1982) Foissner, 2016, were investigated using live observation and protargol staining. The Chinese population of H. lanceolata differs slightly from other populations in the body size in vivo, the relative length of the adoral zone, the number of right and left marginal cirri, the total number of dorsal bristles, and the number of micronuclei. The Chinese population of G. dorsicirratus corresponds well with Austrian and Indian populations. We also document the morphogenetic processes during binary fission of G. dorsicirratus, including the formation of the frontoventral-transverse cirral anlagen in a primary pattern. In addition, phylogenetic analyses based on SSU rDNA sequence data reveal that the two populations of G. dorsicirratus for which data are available cluster together with full support and form a clade with Heterourosomoida sinica and two populations of H. lanceolata.

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.

Redescription, Morphogenesis, and Molecular Phylogeny of Pseudosincirra longicirrata nov. comb., With Establishment of a New Genus Pseudosincirra nov. gen. (Ciliophora, Hypotrichia).

The morphology and morphogenesis of Pseudosincirra longicirrata nov. gen. and nov. comb., isolated from southern China, were investigated with living observation and protargol staining. Our population is similar to the original population in living characteristics and ciliary patterns. The main determinable morphogenetic features of P. longicirrata nov. comb. are the presence of five frontoventral-transverse cirral anlagen (FVT-anlagen) and a dorsomarginal kinety anlage. According to the origin of FVT-anlagen IV and V in proter, it can be determined that P. longicirrata nov. comb. possesses two frontoventral rows and one right marginal row. Hence, a new genus, Pseudosincirra nov. gen., is proposed, and the diagnosis of P. longicirrata nov. comb. is improved. The new genus is diagnosed as follows: adoral zone of membranelles and undulating membranes is in a Gonostomum pattern; there are three enlarged frontal cirri, one buccal cirrus, and one parabuccal cirrus; postperistomial cirrus and transverse cirri are lacking; there are two more or less long frontoventral rows and one right and two or more left marginal rows; cirri within all rows very widely spaced; dorsal kinety pattern is of Urosomoida type, that is, three dorsal kineties and one dorsomarginal kinety; and caudal cirri are present. Phylogenetic analyses based on the small subunit ribosomal (SSU rDNA) sequence data indicate that P. longicirrata nov. comb. clusters with Deviata and Perisincirra. It is considered that Pseudosincirra nov. gen. and Perisincirra paucicirrata should be assigned to the family Deviatidae; fine cirri, and cirri within all rows being relatively widely spaced, should be considered as plesiomorphies of Deviatidae; and Deviatidae is closely related to Dorsomarginalia or Strongylidium–Hemiamphisiella–Pseudouroleptus.

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.

Morphology and molecular phylogeny of the anaerobic freshwater ciliate Urostomides spinosus nov. spec. (Ciliophora, Armophorea, Metopida) from China

The morphology and molecular phylogeny of a new metopid ciliate, Urostomides spinosus nov. spec., discovered in a freshwater ditch in Qingdao, China, were investigated using live observation, morphometry and protargol staining as well as molecular phylogenetic methods. Diagnostic features of the new species include a broadly obpyriform body carrying three posterior spines, eight somatic kineties, five preoral dome kineties with specialized row 3, adoral zone composed of about 28 membranelles, making a 270° turn around body axis. Phylogenetic analyses of the SSU rDNA sequence revealed that the genus Urostomides is monophyletic, but its interspecific relationships remained unresolved. Moreover, a closer relationship of the new species with the morphologically similar Urostomides campanula was not supported by the molecular data.