Morphology and molecular phylogeny of a new brackish pleurostomatid ciliate,Loxophyllum paludosum sp. n. (Ciliophora,Litostomatea, Haptoria), from a mangrove wetland in China

A new ciliate species of the genus Loxophyllum Dujardin, 1841, Loxophyllum paludosum sp. n., is described from a mangrove wetland near Daya Bay in Guangdong Province, southern China, based on morphological and molecular analyses. The new species can be distinguished from its congeners by a combination of the following characters: (1) 12–14 right kineties and 4–6 left kineties; (2) two macronuclear nodules and one micronucleus; (3) a single contractile vacuole located terminally; (4) extrusomes bar-shaped, evenly spaced along entire ventral margin, and clustered to form 5–7 warts along dorsal margin; and (5) presence of three ridges on the left side of cell. The new species is divergent from its congeners from 0.4% to 6.7% (5–104 nucleotide sites) based on the small subunit (SSU) rRNA gene sequence data. The validity of the new species is also supported by molecular phylogenetic trees inferred from SSU rRNA gene sequences.     

The Morphology of Three Loxophyllum Species (Ciliophora,Pleurostomatida) from Southern China,L. lembum sp. n., L. vesiculosum sp. n. and L. perihoplophorum Buddenbrock, 1920, with Notes on the Molecular Phylogeny of Loxophyllum

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.     

Three New Loxophyllum Species (Ciliophora: Pleurostomatida) from China with a Brief Review of the Marine and Brackish Loxophyllum Species

Recent studies indicate that there is a high diversity of pleurostomatid ciliates in the coastal waters of China. Here, three new congeners of Loxophyllum, L. caudatum sp. n., L. rugosum sp. n., and L. chinense sp. n., are described following observations of live cells and protargol‐impregnated specimens. All three species usually have two macronuclear nodules and prominent warts along the dorsal margin formed by clustered extrusomes. In addition, L. caudatum sp. n. is characterized by its long conspicuous tail, dot‐like cortical granules, 4 or 5 left and 9 or 10 right kineties, and a single subterminal contractile vacuole. Loxophyllum rugosum sp. n. is distinguished by possessing three prominent ridges on the left side, 7–11 right and 5–7 left kineties. Loxophyllum chinense sp. n. is characterized by having several contractile vacuoles distributed along the ventral margin, 13–18 right and 6–8 left kineties. The small subunit ribosomal DNA (SSU rDNA) sequence similarities among six congeners range from 96.46% to 99.94%. Phylogenetic trees based on the SSU rDNA sequences indicate that all Loxophyllum spp. form a well‐supported monophyletic group. A brief review of the marine and brackish Loxophyllum species is supplied and one new combination, Litonotus multiplicatus (Kahl 1931) comb. n. (basionym Loxophyllum multiplicatum Kahl 1931), and one new name, Litonotus dragescoi nom. n. (basionym L. fasciolatus Dragesco 1966), are suggested.     

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.     

Morphology of Three Species of Amphileptus (Protozoa,Ciliophora, Pleurostomatida) from the South China Sea,with Note on Phylogeny of A. dragescoi sp. n.

Two new and one problematic species of pleurostomatids, Amphileptus dragescoi sp. n., A. wilberti sp. n., and A. marinus from coastal areas of the South China Sea, are described based on observations of live and protargol‐impregnated specimens. Amphileptus dragescoi is different from its congeners by the presence of an apical group of extrusomes and the possession of 12–15 right and five left somatic kineties, two macronuclear nodules, and a single terminally positioned contractile vacuole. Amphileptus wilberti is diagnosed by oval or pyriform body, 15–19 right and seven or eight left somatic kineties, extrusomes arranged only in anterior portion of oral slit, usually three ventrally located contractile vacuoles, and two macronuclear nodules. Amphileptus marinus (Kahl, 1931) Song et al., 2004 is redescribed and its diagnosis is improved. One isolate which was misidentified as A. marinus by Song et al. (2004) is believed to represent an unknown form, named here as Amphileptus songi sp. n. Phylogenetic analyses of the SSU rDNA sequences indicate that the genus Amphileptus is paraphyletic, but its monophyly is not rejected by statistical tree topology tests.     

Morphology and Phylogeny of Two Novel Ciliates,Arcanisutura chongmingensis n. gen., n. sp. and Naxella paralucida n. sp. from Shanghai,China

The morphologies of two novel ciliates, Arcanisutura chongmingensis n. gen., n. sp. and Naxella paralucida n. sp., collected from Shanghai, China, have been investigated using live observation and silver staining methods. Arcanisutura n. gen. can be easily distinguished from related genera by its inconspicuous, oblique anterior suture. Arcanisutura chongmingensis n. sp. is mainly recognized by its elongated body with a tail‐like posterior end, 25–33 somatic kineties, and 4–11 excretory pores. Naxella paralucida n. sp. can be distinguished from its congeners based on its two short nassulid organelles, fusiform trichocysts, 37–49 somatic kineties, and 16 nematodesmal rods. The small‐subunit (SSU) rRNA gene sequences of these two species are presented, revealing the phylogenetic positions of Arcanisutura and Naxella. Phylogenetic analyses show that Arcanisutura forms a sister clade to other synhymeniid genera, namely, Chilodontopsis, Orthodonella, and Zosterodasys; Naxella is most closely related to Nassula spp. and is located within the monophyletic clade of the family Nassulidae.     

Morphology and Molecular Phylogeny of a New Haptorian Ciliate,Chaenea mirabilis sp. n., with Implications for the Evolution of the Dorsal Brush in Haptorians (Ciliophora,Litostomatea)

We discovered a new haptorian ciliate, Chaenea mirabilis sp. n., in brackish water collected near the town of Busan, Korea. Its morphology was studied using standard taxonomical methods and its phylogenetic relationships were assessed by phylogenetic analysis of the 18S rRNA gene. Chaenea mirabilis is distinguished from all congeners by the combination of the following traits: (i) a narrowly bursiform to flask‐shaped, 60–100 μm long body; (ii) 11–21 doughnut‐shaped or sometimes horseshoe‐shaped macronuclear nodules; (iii) two types of extrusomes: type I is rod‐shaped and 6–8 μm long, while type II is narrowly to broadly teardrop‐shaped and only 1.5–2 μm long; (iv) highly refractive special granules tightly arranged between the first and second brush row, forming a conspicuous bulge; and (v) 12–13 somatic kineties. In the 18S rRNA gene phylogeny, C. mirabilis clustered with full support with other congeners. However, there was no statistical support for classification of Chaenea into the families Fuscheriidae, Acropisthiidae, or Trachelophyllidae, but a sister relationship with the Lacrymariidae could not be excluded. Therefore, we establish a new family, Chaeneidae, within the order Lacrymariida. This affiliation is strongly corroborated by the distinctly subapical dorsal brush bearing cilium‐like bristles.     

Morphology and Molecular Phylogeny of a New Marine Hypotrichous Ciliate,Hypotrichidium paraconicum n. sp. (Ciliophora,Hypotrichia)

A new saline hypotrich, Hypotrichidium paraconicum n. sp., found in coastal waters near Hong Kong, China, was investigated with emphasis on its living morphology, infraciliature, ontogenesis, and phylogenetic position. A body that is pyriform, with a posterior end that is twisted helically toward the left, a distinct tail and a dark colour, characterizes H. paraconicum n. sp. The buccal cavity is prominent and the cortical granules, which are scattered or sparsely distributed, are colourless, round, and about 1 μm in diam. The contractile vacuole is near the left middle of the cell and there are one or two frontal cirri, four meridional rows and five or six cirral rows which exhibit a left‐handed spiral and which are distributed in the posterior portion on both sides. Three dorsal kineties are distributed in the anterior portion of the dorsal side. In our molecular phylogenies, based on SSU rRNA gene sequences, the position of H. paraconicum n. sp. is rather poorly resolved, providing some indication of a relationship with Neokeronopsis aurea and Rubrioxytricha ferruginea, which fall into the assemblage of oxytrichids (s.l.).     

Morphological and Molecular Description of Three New Species of the Cyrtophorid Genus Chlamydodon (Ciliophora,Cyrtophoria)

This study investigates the morphology and molecular characteristics of three new cyrtophorid ciliates isolated from China seas: Chlamydodon salinus n. sp., Chlamydodon caudatus n. sp., and Chlamydodon paramnemosyne n. sp. Of these, C. salinus n. sp. differs from its congeners through a combination of body size, a cross‐striated band that is not continuous, the presence of 30–34 somatic kineties, 11–15 nematodesmal rods, and 13 contractile vacuoles. Chlamydodon caudatus n. sp., meanwhile, is characterized by having a conspicuous tail, a continuous cross‐striated band, 34–40 somatic kineties, about 15 contractile vacuoles, and 20–24 nematodesmal rods. Compared with other Chlamydodon species, the third new one, C. paramnemosyne n. sp., could be identified by its continuous cross‐striated band, 16–18 somatic kineties, 5 contractile vacuoles, and 9–12 nematodesmal rods. Based on the sequence of the small subunit (SSU) rRNA gene, the phylogeny of these three new species was analyzed, indicating that they all clustered with other congeners to form a monophyletic assemblage. Based on previous studies and the present work, a brief revision of the genus Chlamydodon is supplied, and a key to aid the identification of Chlamydodon species is given.     

Morphology,Morphogenesis, and Molecular Phylogeny of Anteholosticha multicirrata n. sp. (Ciliophora,Spirotrichea) with a Note on Morphogenesis of A. pulchra (Kahl, 1932) Berger, 2003

Two marine urostylid ciliates, Anteholosticha multicirrata n. sp. and Anteholosticha pulchra (Kahl, 1932) Berger, 2003, were collected from South Korea. These species were identified based on morphology, morphogenesis, and SSU rRNA gene sequence comparison. Anteholosticha multicirrata n. sp. is characterized by the following features: body size 90–125 × 30–45 μm in vivo, shape slender to ellipsoidal in outline, with yellow‐greenish cortical granules distributed along and between dorsal kineties and cirri; single contractile vacuole positioned on left at mid‐body; three frontal, five to seven frontoterminal, one buccal, one to two pretransverse and four to six transverse cirri; three complete dorsal kineties; one left and one right marginal cirral row; about 117 macronuclear nodules; and three to four micronuclei observed during morphogenesis. In addition, based on the observations of morphogenesis, we found that A. pulchra has pretransverse cirri, which were not described in detail in previous studies. Nuclear small subunit ribosomal RNA (SSU rRNA) gene was used to analyse their phylogenetic relationship, and the gene tree supports that the genus Anteholosticha is a highly polyphyletic group.     

Ontogeny and phylogeny of Metaurostylopsis cheni sp. n. (Protozoa,Ciliophora), with estimating the systematic position of Metaurostylopsis

Chen, X., Huang, J. & Song, W. (2010). Ontogeny and phylogeny of Metaurostylopsis cheni sp. n. (Protozoa, Ciliophora), with estimating the systematic position of Metaurostylopsis. —Zoologica Scripta, 40, 99–111. The ciliate genus Metaurostylopsis seems to be a highly divergent marine‐habiting group, of which neither systematic position nor the variation of their ontogeny has been critically checked. In the present work, the morphology and morphogenesis during asexual division of a new form, Metaurostylopsis cheni sp. n., isolated from the Yellow Sea, China, were investigated and comparison among known congeners was performed. The new species has two types of cortical granules, the larger ones of which are flattened and oval or circular in outline with a longitudinal groove, yellow–green in colour, and arranged along the cirral rows and dorsal kineties, whereas the smaller ones are colourless or grayish and sparsely distributed. The main morphogenetic features are: (i) the entire parental ciliature, including the old oral apparatus, is renewed, (ii) the oral primordium of the proter originates de novo and beneath the surface of the buccal cavity, that is, sub‐apokinetally, (iii) the anlagen of the marginal rows and of the dorsal kineties are formed intrakinetally and (iv) fusion of the macronuclear nodules results in an irregular mass with only few branches. The small subunit ribosomal RNA (SSU rRNA) gene of M. cheni was sequenced. Phylogenetic analysis based on SSU rRNA gene sequence data shows that M. cheni clusters with all other Metaurostylopsis spp. sequenced to date indicating that the genus is monophyletic and is probably closely related to the Apokeronopsis–Thigmokeronopsis‐group, within the order Urostylida.     

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.     

Trichopodiella faurei n. sp. (Ciliophora,Phyllopharyngea, Cyrtophoria): Morphological Description and Phylogenetic Analyses Based on SSU rRNA and Group I Intron Sequences

ABSTRACT. A new marine cyrtophorian ciliate Trichopodiella faurei n. sp., which belongs to the order Dysteriida, family Hartmannulidae, was investigated at the morphological and molecular levels. A combination of morphological features of the organism including the oval body shape, 2–3 contractile vacuoles, 22–28 nematodesmal rods in the cytopharyngeal basket, and 31–39 somatic kineties, distinguishes it from all other known congeners. In reconstructed small subunit (SSU) rRNA phylogenies, T. faurei groups with Isochona, a representative genus of the subclass Chonotrichia. The similarity of the infraciliature between hartmannulids and several chonotrichian examples also suggests that these taxa should be closely related. A new S943 intron belonging to group IC1 was identified in the SSU rRNA gene of this species. This intron is phylogenetically related to the S891 introns previously found in the suctorians Acineta sp. and Tokophrya lemnarum, and their internal guide sequences share four nucleotides, indicating that these introns were vertically inherited from a common phyllopharyngean ancestor and that reverse splicing might have been involved in the transposition.     

Morphogenesis and Molecular Phylogeny of a New Freshwater Ciliate,Notohymena apoaustralis n. sp. (Ciliophora,Oxytrichidae)

The live morphology, infraciliature, and morphogenesis of a new oxytrichid ciliate, Notohymena apoaustralis n. sp. collected from a freshwater pond in Qingdao (Tsingtao), China, were studied in vivo and after protargol impregnation. Notohymena apoaustralis n. sp. is characterized as follows: undulating membranes in Notohymena‐pattern; cortical granules yellow‐green, grouped around the marginal cirri and dorsal bristles, and in short irregular rows elsewhere in the cell; single contractile vacuole positioned at anterior 1/3 of the body length; two macronuclear nodules and one micronucleus; about 39 adoral membranelles; 18 frontoventral transverse cirri in typical Oxytricha‐pattern; one right and one left marginal row, almost confluent posteriorly; dorsal ciliature in typical Oxytricha‐pattern; 8–10 caudal cirri arranged in three rows, one each at the posterior end of dorsal kineties 1, 2, and 4, indistinguishable from marginal cirri in life. The morphogenetic process in N. apoaustralis n. sp. is consistent with that of the type species, Notohymena rubescens Blatterer and Foissner, 1988. Phylogenetic analyses based on small subunit rDNA sequence data suggest a sister relationship between N. apoaustralis n. sp. and Paraurostyla weissei, which cluster in a clade with Rubrioxytricha ferruginea.     

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.     

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.     

Morphology,morphogenesis and molecular phylogeny of a novel soil ciliate,Afrokahliella paramacrostoma n. sp. (Ciliophora,Hypotrichia)

A soil hypotrich ciliate, Afrokahliella paramacrostoma n. sp., was discovered in China. Its morphology, morphogenesis and molecular phylogeny were investigated using standard methods. The new species is characterized as follows: body about 140–180 × 60–70 μm in vivo, cortical granules absent, contractile vacuole positioned about 40% down length of body, 5–9 macronuclear nodules, 34–49 adoral membranelles, 3–5 buccal and 3–6 parabuccal cirri, usually two frontoventral rows, three or four left and two or three right marginal rows, three dorsal kineties and one dorsomarginal kinety; 1–3 and one or two caudal cirri located at the ends of dorsal kineties 1 and 2, respectively. The ontogenetic process is characterized by: (1) the marginal anlagen on each side develop in the outer right and the inner left marginal rows, respectively; (2) five frontoventral-transverse cirral anlagen, anlagen II–IV develop in secondary mode; (3) dorsal morphogenesis follows a typical Urosomoida-pattern, no parental dorsal kineties are retained; (4) caudal cirri are generated at the ends of dorsal kineties 1 and 2. Phylogenetic analyses based on SSU rDNA sequence data reveals that Afrokahliella paramacrostoma n. sp. is closely related to Parakahliella macrostoma and Hemiurosomoida longa.     

Morphology and Molecular Phylogeny of Apoterritricha lutea n. g., n. sp. (Ciliophora,Spirotrichea, Hypotrichia): A Putative Missing Link Connecting Cyrtohymena and Afrokeronopsis

A new hypotrichous ciliate, Apoterritricha lutea n. g., n. sp., was discovered in a sample of a terrestrial liverwort from Korea. Its morphology was studied using detailed in vivo observation and protargol impregnation. Its phylogenetic relationships were revealed by analyses of the 18S rRNA gene. This new taxon is characterized by a combination of the following traits: (i) ellipsoidal to narrowly ellipsoidal body with an average size of 230 × 85 μm; (ii) two macronuclear nodules and two to five micronuclei; (iii) golden yellow cortical granules, forming small groups along the microtubular appendages of cirri, adoral membranelles, and dorsal kineties; (iv) typically three frontal cirri, one buccal cirrus, four frontoventral cirri, seven midventral cirri, two pretransverse cirri, seven transverse cirri, ca. 38 left, and ca. 36 right marginal cirri; and (v) on average six dorsal kineties, three dorsomarginal kineties, and three caudal cirri. In molecular phylogenies, A. lutea clusters with strong support within a clade containing Afrokeronopsis aurea and several “typical” oxytrichids having golden yellow to brown cortical granules. In this light we propose a hypothesis that is not unambiguously rejected by the present phylogenetic analyses, which shows how the Afrokeronopsis‐like pattern could have evolved from a Rubrioxytricha‐like ancestor via an Apoterritricha‐like stage by cirri‐multiplication.