Two New Brackish Ciliates,Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China,with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa,Ciliophora, Pleurostomatida)

Two new brackish pleurostomatid ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. were collected from mangrove wetlands of southern China and their morphology and molecular phylogeny were studied. Amphileptus spiculatus sp. n. can be distinguished from congeners by the presence of 11–14 right and 6–8 left kineties, two macronuclear nodules and a conspicuous beak‐like anterior body end. Amphileptus bellus sp. n. is characterized by the presence of 2–4 macronuclear nodules, 31–35 right and 6 or 7 left kineties and two types of extrusomes. Phylogenetic analyses based on SSU rDNA sequences data indicate that the family Amphileptidae is paraphyletic.     

Morphogenesis and Molecular Phylogeny of the Soil Ciliate Holostichides chardezi (Ciliophora,Hypotrichia, Bakuellidae), with Redefinition of Holostichides Foissner, 1987 and Establishment of a New Genus Anteholostichides

Morphogenesis of the soil hypotrich ciliate, Holostichides chardezi Foissner, 1987, collected from southeastern China, was investigated using the protargol staining method. The main morphogenetic events follow a similar process with that of its congeners. Phylogenetic analyses based on the SSU rDNA sequence data indicate that Holostichides is nonmonophyletic; H. chardezi, the type species of Holostichides, clusters with H. heterotypicus, while H. terrae is distinctly separate from these species. H. terrae can be distinguished from H. chardezi (type species of Holostichides) and H. heterotypicus by undulating membranes relatively long and distinctly curved (vs. relatively short and straight), pharynx with (vs. without) rod‐shaped structure, and two (vs. more than two) frontoterminal cirri. Therefore, a new genus, Anteholostichides nov. gen., has been proposed for H. terrae. Further, the diagnosis of the genus Holostichides is improved.     

Morphology,Ontogeny, and Phylogeny of Two Brackish Urostylid Ciliates (Protist,Ciliophora, Hypotricha)

The diversity of hypotrichous ciliates has encouraged numerous researchers to use a combination of morphological, morphogenetic, and phylogenetic data to provide a better understanding of the evolutionary relationships within this complex group. In this study, we investigate the morphology and morphogenesis of Pseudourostyla subtropica sp. nov., isolated from mangrove wetland. The new species can be distinguished from its congeners by the huge body size, many more adoral membranelles and marginal cirral rows, and numerous macronuclear nodules. In addition, we provide a morphological characterization of a population of Pseudourostyla nova Wiackowski 1988 from an estuarine habitat. The main events during binary fission of P. subtropica sp. nov. and the Chinese population of P. nova are also revealed to be conservative. The morphological, ontogenetic, and phylogenetic analyses based on the SSU rDNA sequences corroborate the monophyly of Pseudourostyla Borror, 1972, which corresponds well with previous research. The phylogenetic analyses also show that Pseudourostyla and Hemicycliostyla Stokes, 1886, both of which are assigned to the family Pseudourostylidae based on morphological and morphogenetic data, in fact fall into separated clades. The approximately unbiased tests, however, do not reject the possibility that the family Pseudourostylidae is a monophyletic lineage.     

Morphology and Phylogeny of Two Novel Pleurostomatids (Ciliophora,Litostomatea), Establishing a New Genus

Pleurostomatida Schewiakoff, 1896 is a cosmopolitan order of ciliates. In the present study, we investigated two new pleurostomatid species, Apolitonotus lynni gen. et sp. nov. and Protolitonotus clampi sp. nov., with state‐of‐the‐art methods. Apolitonotus lynni lacks its oral extrusomes and its right kineties form an anterior semi‐suture near the dorsal margin. Based on these two features, the new genus Apolitonotus was established within the Protolitonotidae Wu et al., 2017. Protolitonotus clampi differs from its congeners by its size of 80–130 × 15–30 μm, 4–6 left, and 9–11 right kineties, extrusomes arranged along the oral slit, and two macronuclear nodules. Because Litonotus antarcticus possesses an anterior semi‐suture and oral extrusomes, it was transferred to the genus Protolitonotus, becoming P. antarctius comb. nov. (basionym Litonotus antarcticus Song and Wilbert, 2002). Phylogenetic analyses based on SSU rDNA sequences suggest a sister group relationship of P. clampi and the family Kentrophyllidae, and A. lynni is adelphotaxon to Litonotus gracilis, both within the order Pleurostomatida. Based on the new findings, an improved diagnosis for Protolitonotus was also provided.     

Morphology and Molecular Phylogeny of Two Poorly Known Species of Protocruzia (Ciliophora: Protocruziida)

The ciliate genus Protocruzia is a highly confused group, which was formerly placed in the class Heterotrichea or Karyorelictea, and is according to the most recent system tentatively assigned to the class Spirotrichea. In the present study, the morphology, ciliary pattern, and molecular phylogeny of two poorly known species, Protocruzia tuzeti Villeneuve‐Brachon, 1940, and Protocruzia granulosa Kahl, 1933, isolated from coastal waters of China, were investigated. Protocruzia tuzeti differs from its congeners mainly in possessing 6 adoral membranelles, 8–11 somatic kineties, and postoral dikinetids. Protocruzia granulosa is characterized by its extremely slender body, three postoral kineties, and 13 or 14 somatic kineties. The morphogenesis of P. granulosa is similar to that of P. tuzeti, especially in the parakinetal mode of stomatogenesis and the reorganization of the parental paroral membrane; however, more than one somatic kinety joins in the formation of the oral primordium in P. granulosa. Phylogenetic analyses based on small subunit ribosomal RNA gene revealed that six Protocruzia species form a fully supported clade that does not belong to any ciliate class; therefore, our data support the establishment of the class Protocruziea Gao et al. (Sci. Rep., 6, 2016, 24874).     

A Contribution to the Morphology and Phylogeny of Chlamydodon,with Three New Species from China (Ciliophora,Cyrtophoria)

Three new cyrtophorian ciliates isolated from coastal areas of China were described based on morphological and genetic data. The Chlamydodon mnemosyne‐like species Chlamydodon similis sp. n. differs from its congeners mainly by its number of somatic kineties. Chlamydodon oligochaetus sp. n. is distinguished from its congeners mainly by having fewer somatic kineties, and/or an elongated body shape. Chlamydodon crassidens sp. n. is characterized mainly by an inverted triangular body shape, a posteriorly interrupted cross‐striated band (5–6 μm wide), and a large cytostome. Moreover, we provided small‐subunit (SSU) rDNA sequences of C. similis sp. n. and C. oligochaetus sp. n. Maximum likelihood (ML) and Bayesian inference (BI) consistently placed C. similis sp. n. as a sister to C. paramnemosyne, but showed different branching position of C. oligochaetus sp. n., which may be due to a low taxon sampling in the Chlamydodontidae and/or an insufficient resolution of the marker gene at species level.     

Morphology and taxonomy of Pseudokeronopsis rubra (Ehrenberg, 1836) and Pseudokeronopsis parasongi sp. nov. (Ciliophora,Hypotrichia, Urostylida) from the Yellow Sea

Pseudokeronopsis rubra (Ehrenberg, 1836), type species of Pseudokeronopsis, is a widely reported but taxonomically confused species with most available sequences either misidentified or dubious. Based on the Yellow Sea population, of which the molecular data have been analyzed in a previous work, we redescribe P. rubra and try to clarify the identifications of the populations designated as P. rubra or those allocated to this species. Nonetheless, there is still a need of integrated investigation of the species combing both morphological and molecular data based on specimens from its type locality. Recent molecular analyses indicate a Yellow Sea population of Pseudokeronopsis (marked as P. cf. songi) likely represents a new species. Here we describe it as P. parasongi sp. nov. based on the specimens from the same population. The new species possesses a yellow-brownish body with both pigmented cortical granules and colorless blood cell-shaped structures underneath the body surface, invariably one contractile vacuole at 66% of body length and occasionally a second one at 25%, and the midventral complex terminating 2–23 μm ahead of 2–4 transverse cirri. It differs from closely related species by its yellow-brownish body color and separates from them in the ITS1-5.8S-ITS2 and COI genetic distances.     

Morphology,Ontogeny, and Molecular Phylogeny of Two Freshwater Species of Deviata (Ciliophora,Hypotrichia) from Southern China

The morphology and partial morphogenesis of two freshwater hypotrichous ciliates, Deviata brasiliensis Siqueira‐Castro et al., 2009 and Deviata rositae Küppers et al., 2007, isolated from southern China, were investigated using live observation and protargol staining. Our populations resemble the original ones in terms of their live characters and ciliary patterns. The main determinable morphogenetic features of D. brasiliensis basically correspond with those of the type population. However, the origin of anlage V for either proter or opisthe is ambiguous: whether anlage V for the proter originates from parental frontoventral row 2 (the same as in the original population) or parental frontoventral row 3 (the same as in Deviata abbrevescens) or even de novo is not clear; the anlage V for the opisthe is possibly derived from frontoventral row 3 and further migrates to frontoventral row 2, like that in D. abbrevescens. In addition, the SSU rRNA gene was first sequenced for both species. Molecular phylogenetic analyses suggest that the genus Deviata is non‐monophyletic and has a close relationship with Perisincirra paucicirrata.     

Molecular Phylogeny and Taxonomy of Two Novel Brackish Water Hypotrich Ciliates,with the Establishment of a New Genus,Antiokeronopsis gen. n. (Ciliophora,Hypotrichia)

Two novel brackish water urostyloid ciliates, Anteholosticha paramanca sp. n. and Antiokeronopsis flava gen. n., sp. n., isolated from the Shenzhen Mangrove Nature Protection Area on the coast of the South China Sea, were investigated using live observation and protargol impregnation techniques. Anteholosticha paramanca sp. n. is characterized by its spherical yellowish cortical granules arranged in lines, shortened midventral complex and three transverse cirri. Morphogenesis is similar to that in Anteholosticha manca. The new genus Antiokeronopsis is diagnosed by having a continuous adoral zone of membranelles, frontal cirri arranged in a bicorona, midventral complex composed of midventral pairs only, one marginal cirral row on each side, the presence of frontoterminal and transverse cirri, and the lack of buccal and caudal cirri. The type species A. flava sp. n. is characterized by its elongated body shape, brown to yellowish body color and two types of cortical granules. Small subunit ribosomal RNA gene sequence data justify the classification of both species. Phylogenetic analyses indicate that A. paramanca clusters with Bakuella subtropica within a clade that includes two other Anteholosticha species, while Antiokeronopsis groups within the core urostylids and is most closely related to the well‐known genera Pseudokeronopsis and Uroleptopsis.     

Two New Marine Ciliates,Caryotricha rariseta n. sp. and Discocephalus pararotatorius n. sp. (Ciliophora,Spirotrichea), with Phylogenetic Analyses Inferred from the Small Subunit rRNA Gene Sequences

The morphology, infraciliature, and molecular phylogeny of two novel spirotrich ciliates, Caryotricha rariseta n. sp. and Discocephalus pararotatorius n. sp., isolated from coastal waters of China, were investigated. Caryotricha rariseta n. sp. differs from its congeners mainly in possessing seven sparsely ciliated cirral rows that are conspicuously shortened posteriorly and three transverse cirri aligned in a row. Discocephalus pararotatorius n. sp. is characterized by the conspicuous spine‐like dorsal cilia, one extra (endoral?) membrane, and seven frontal, six ventral, and seven posterolateral marginal cirri. The small subunit rRNA gene was sequenced for both species. Complete SSU rRNA gene sequences of two Caryotricha spp. (including C. rariseta n. sp.) and two Discocephalus spp. (including D. pararotatorius n. sp.), along with those of 40 other ciliates, were used to determine their molecular phylogeny using maximum likelihood, neighbor joining and maximum parsimony analyses. The two Caryotricha species cluster with Kiitricha marina in the well‐supported Protohypotrichia clade that is basal to the main spirotrich assemblage. The two discocephalids form a clade that is sister to the Hypotrichia.     

Morphogenesis and Molecular Phylogeny of a Soil Ciliate Uroleptoides longiseries (Foissner,Agatha and Berger, 2002) Berger 2008 (Ciliophora,Hypotrichia)

Morphogenesis of the hypotrich ciliate Uroleptoides longiseries, isolated from sandy soil beside the Yellow River, Suide, Yulin, Shaanxi Province, China, was investigated using protargol staining. The main events during binary fission are as follows: (1) the long frontoventral row is formed by a single anlage; (2) five frontoventral‐transverse cirral anlagen are formed in primary mode; (3) only the posterior part of the parental adoral zone of the membranelles is renewed; and (4) the oral primordium of the opisthe is formed intrakinetally. This is the first detailed record of all stages of morphogenesis for Uroleptoides. We also provide the first record of the small subunit ribosomal DNA (SSU rDNA) sequences for U. longiseries. Phylogenetic analyses based on the SSU rDNA sequences data show that Uroleptoides longiseries clusters with U. magnigranulosa with moderate to high support which together form a clade with Orthoamphisiella breviseries. These three species share the morphogenetic feature of the long frontoventral row being formed by a single anlage.     

Morphology and Phylogeny of Two New Pleurostomatid Ciliates,Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp. (Protozoa,Ciliophora) from A Mangrove Wetland,South China

ABSTRACT. The morphology, infraciliature, and small subunit (SSU) rRNA gene sequences of two new pleurostomatid ciliates, Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp., isolated from a mangrove wetland near Shenzhen, South China, were investigated. Epiphyllum shenzhenense n. sp. is morphologically characterized by leaf‐shaped cell about 150 × 35 μm in vivo, usually with four contractile vacuoles, 20–29 right kineties and 10–26 left kineties, ca. four macronuclear nodules, and two types of extrusomes (i.e. short spindle‐shaped and long bar‐shaped). As a new species, L. spirellum n. sp. is distinguished from its congeners by its posterior dorsal margin twisted onto the left side, the distribution of extrusomes (evenly arranged along the oral slit, the posterior end, and clustered to 7–13 warts on dorsal margin), the subterminally positioned contractile vacuole, the number of kineties (8–10 on right side, 4–5 on left side), and its genetic distance from congeners. Phylogenetic trees based on the SSU rRNA gene sequence for both organisms were constructed, which indicate that Epiphyllum is a distinct genus and occupies a basal position in the Pleurostomatida clade; L. spirellum n. sp. falls well into the Loxophyllum clade, which has a close relationship with Litonotus and Spiroloxophyllum.     

Morphological Description and Molecular Phylogeny of Two Species of Levicoleps (Ciliophora,Prostomatida), L. taehwae nov. spec. and L. biwae jejuensis nov. subspec., collected in Korea

Two colepid ciliates, Levicoleps taehwae nov. spec. and L. biwae jejuensis nov. subspec., were collected from the brackish water of the Taehwa River and a small freshwater pond in Jeju Island, South Korea, respectively. Their living morphology, infraciliature, and small subunit (SSU) rRNA gene sequences were determined using standard methods. Barrel‐shaped L. taehwae nov. spec. is a small ciliate with an average size of 45 × 25 μm in vivo, about 15 ciliary rows each composed of 12 monokinetids and two perioral dikinetids, and two 20 μm‐long caudal cilia. The sequence length and GC content of the SSU rRNA gene are 1,669 bp, 44.5%. This novel species is similar in body size to Coleps hirtus, and has six armor tiers and hirtus‐type tier plates, and the same number of ciliary rows as C. hirtus; however, it can be distinguished from the latter by the absence of armor spines and its sequence similarity of SSU rRNA gene is about 92.8% which indicates that it is a distinct form. Levicoleps biwae jejuensis nov. subspec., is a medium colepid ciliate which has a barrel‐shaped body, about 22 somatic kineties and 16 transverse ciliary rows, three mini adoral organelles, and four 15 μm‐long caudal cilia. The sequence length and GC content of the SSU rRNA gene are 1,666 bp and 44.4%.     

Morphology and Molecular Phylogeny of Two New Brackish Water Ciliates of Bakuella (Ciliophora: Urostylida: Bakuellidae) from South Korea

Two new species of Bakuella, B. (B.) incheonensis n. sp. and B. (Pseudobakuella) litoralis n. sp., were discovered in brackish water near Aamdo Shore Park, Incheon, South Korea. We conducted a morphological study based on live observations and protargol‐impregnated specimens, and a molecular analysis using nuclear SSU rRNA gene sequences. Bakuella (B.) incheonensis is diagnosed by: body size 70–105 × 20–40 μm in vivo, 21–25 adoral membranelles, three or four frontoterminal cirri, midventral complex composed of 7–10 midventral pairs with one or two rows and terminating at about 62% of body length, 20–28 left and 25–32 right marginal cirri, 58–87 macronuclear nodules, and yellowish cortical granules. Bakuella (Pseudobakuella) litoralis is diagnosed by: body size 90–125 × 30–40 μm in vivo, 25–33 adoral membranelles, 3–5 buccal cirri, midventral complex composed 10–15 midventral pairs with one or two rows and terminated at 70% of body length, one or two pretransverse cirri, 3–6 transverse cirri, 26–39 left and 29–47 right marginal cirri, 49–84 macronuclear nodules, and two types of cortical granules. Molecular phylogeny using SSU rRNA gene sequences shows a nonmonophyletic relationship among Bakuella species and emphasizes the need for further morphogenetic studies of this genus and other related hypotrichs.     

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora,Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al‐Adaid Lagoon in Qatar

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al‐Adaid Lagoon in Qatar were investigated based on live observation as well as protargol‐ and silver nitrate‐impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50–65 × 33–40 μm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral‐transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans–Euplotes nobilii–Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.     

Morphological and Molecular Identification of a New Ciliate,Zoothamnium palmphlatum nov. spec. (Ciliophora,Peritrichia) from North China

A new peritrichous ciliate, Zoothamnium palmphlatum nov. spec., was collected from an estuary in Yantai, China. It was investigated, using both live observation and silver staining. The new species can be identified by a palm‐shaped colony consisting of highly developed and alternately arranged secondary branches, a double‐layered peristomial lip, and an infundibular polykinety 3 composed of two parallel kinetosomal rows. Phylogenetic analyses of the small subunit (SSU) rRNA gene sequence show that Z. palmphlatum clusters with other members of the family Zoothamniidae. Furthermore, the comparison of primary and secondary SSU rDNA structures indicates that Z. palmphlatum is distinctly different from its morphologically similar species (93.2–97.0% in sequence similarity) in combination of H10 and H31 regions. Although Z. palmphlatum shares highest sequence similarity with Zoothamnium mucedo (98.9%), the new species has distinctly different structures in the H11, H12, H18, and H31 regions compared to Z. mucedo, which indicates that sequence similarity may not determine the similarity of the secondary structure.     

Morphology and molecular phylogeny of two new soil ciliates,Hemiurosomoida warreni nov. spec. and Hemiurosoma clampi nov. spec. (Ciliophora,Hypotrichia) from Tibet

Two novel hypotrichous ciliates, Hemiurosomoida warreni nov. spec. and Hemiurosoma clampi nov. spec., isolated from soil in the Lhalu Wetland and Motuo Virgin Forest in Tibet, respectively, were investigated using live observation and protargol staining. Hemiurosomoida warreni nov. spec. strongly resembles the type species H. longa but can be distinguished by its body size in vivo (110–145 × 30–40 μm vs. 50–100 × 18–40 μm), number of adoral membranelles (25–38 vs. 15–22), and numbers of right (29–39 vs. 14–23) and left (26–35 vs. 13–23) marginal cirri, transverse cirri (3 vs. 4 or 5) and macronuclear nodules (4–8 vs. 2). Hemiurosoma clampi nov. spec. is characterized by its vermiform body shape, colourless cortical granules distributed in irregular rows, two macronuclear nodules, three frontal cirri, one buccal cirrus, four frontoventral cirri ranged in a line, two transverse cirri, lacking postoral ventral and pretransverse ventral cirri, and marginal rows that are not posteriorly confluent. Phylogenetic analyses based on SSU rDNA gene sequences suggest that Hemiurosomoida is not monophyletic. A close relationship is revealed between Hemiurosomoida warreni nov. spec., Parakahilella macrostoma, Hemiurosoma clampi nov. spec., and the type species Hemiurosoma terricola. As expected, all these species are classified within the “non-oxytrichid Dorsomarginalia”.     

Morphology and Phylogeny of Three Pleuronema Species (Ciliophora,Scuticociliatia) from Hangzhou Bay,China, with Description of Two New Species,P. binucleatum n. sp. and P. parawiackowskii n. sp.

The morphology and phylogeny of Pleuronema binucleatum n. sp., P. parawiackowskii n. sp., and P. marinum Dujardin 1841, collected from Hangzhou Bay estuary, China, were investigated using standard methods. Pleuronema binucleatum n. sp. can be identified by possessing about 90–120 × 35–50 μm cell size in vivo, reniform body outline, two macronuclei, six to eight preoral kineties, 32–41 somatic kineties, and posterior end of the anterior fragment of membranelle 2 (M2a) hook‐like. Pleuronema parawiackowskii n. sp. is characterized by the combination of the following characters: body size about 60–90 × 20–40 μm in vivo, elliptical in outline, four to eight preoral kineties, 20–29 somatic kineties, posterior portion of the M2a slightly curved but nonhooked, and single macronucleus sausage‐like. After comparison with other populations of P. marinum, it is suggested that many misidentifications exist in previous studies. And an improved diagnosis of P. marinum was supplied: cell about 95–180 μm long, elliptical in outline; 2–4 preoral kineties and 53–70 somatic kineties; both membranelle 1 and membranelle 3 three‐rowed; posterior end of the M2a straight; single contractile vacuole characteristically positioned near mid‐body. The small subunit rRNA genes of three forms were sequenced. Phylogenetic analyses indicate that the monophyly of the genus Pleuronema is still not supported.