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 Small Subunit rDNA Phylogeny of Two New Marine Urostylid Ciliates,Caudiholosticha marina sp. nov. and Nothoholosticha flava sp. nov. (Ciliophora,Hypotrichia)

Two marine urostylid ciliates, Caudiholosticha marina sp. nov. and Nothoholosticha flava sp. nov., isolated from intertidal sediment in the Yellow Sea, are investigated using morphological and small subunit rDNA phylogenetic analyses. Caudiholosticha marina is 210?310 μm × 40?55 μm in vivo, and has 10?20 macronuclear nodules, 23?37 midventral cirral pairs extending to 5?8 transverse cirri, and two caudal cirri. It differs from congeners by its marine habitat, larger size, macronuclear arrangement pattern and high number of midventral pairs. Molecular phylogenetic analyses indicate a polyphyly of Caudiholosticha. Nothoholosticha flava is yellow to brownish and 240?320 μm × 40?60 μm sized, and has a bipartite adoral zone, six frontal cirri in atypical bicorona, usually four frontoterminal, one buccal and 5?7 transverse cirri and 28?54 midventral pairs. Phylogenetic analyses allocate N. flava as sister of N. fasciola, type of the genus. The two Nothoholosticha species differ distinctly by the presence/absence of frontoterminal cirri, a feature often used to define genera in the Hypotrichia. However, the SSU rDNA sequence similarity between these two species is 99.3%, which weakens the justification for separating the new isolate at genus level. The taxonomic significance of frontoterminal cirri is discussed based on morphological and molecular data.     

Morphology,Morphogenesis and Phylogenetic Position of the Soil Hypotrichous Ciliate,Parabistichella dieckmanni (Foissner, 1998) Foissner, 2016 (Ciliophora,Hypotrichia), with Notes on the Phylogeny of Parabistichella

The morphology, morphogenesis and molecular phylogeny of Parabistichella dieckmanni (Foissner, 1998) Foissner, 2016, isolated from north China, were investigated. The Chinese population was characterized as having five to seven frontal cirri in corona, four to seven buccal cirri arranged in a row, two to four short frontal rows and two long frontoventral rows, three to seven transverse cirri, four macronuclear nodules, three dorsal kineties following a Gonostomum-pattern, and caudal cirri absent. Morphogenetic research reveals that the main characteristics during binary fission are as follows: (1) the long left frontoventral row is formed by two or three anlagen; (2) the posterior part of the parental adoral zone of membranelles is renewed, and the oral primordium of the opisthe is formed intrakinetally; (3) FVT-anlagen I to VI (or V, VII) produce each a frontal cirrus to form the frontal corona; (4) development of dorsal kineties follows the Gonostomum-pattern. Phylogenetic analyses showed that P. dieckmanni does not group with other Parabistichella species. Therefore, the genus Parabistichella is polyphyletic. Additionally, Parabistichella variabilis n. comb. (basionym: Bistichella variabilis He & Xu, 2011) and Parabistichella cheni n. nom. (basionym: Parabistichella variabilis Jiang et al., 2013) were suggested.     

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.     

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.     

Molecular Phylogeny and Ontogeny of a New Ciliate Genus,Paracladotricha salina n. g., n. sp. (Ciliophora,Hypotrichia)

A hypotrichous ciliate, Paracladotricha salina n. g., n. sp., was discovered in hypersaline waters (salinity about 80‰) from Qingdao, China. Its morphology and some major ontogenetic stages were studied and the phylogenetic position was estimated using standard methods. Paracladotricha salina is characterized by a flexible, more or less slender body (size 50–120 × 20–35 μm), a gonostomatid oral apparatus, one short and two long frontoventral rows, four macronuclear nodules, almost completely reduced dorsal kineties 1–3, and a loss of several parts of the ciliature, namely, the slightly shortened ciliary row of the adoral membranelles, the paroral, and the buccal, the postoral and pretransverse ventral, the transverse, and the caudal cirri. The ontogenesis is rather simple: anlage II of both filial products and anlage III of the opisthe originate de novo, while anlagen IV and V are formed within the parental rows. This combination of features requires the establishment of a new genus, Paracladotricha, which is, according to the morphological data, closely related to Schmidingerothrix and Cladotricha. The small‐subunit rRNA gene was sequenced, indicating that P. salina is, as also demonstrated by the oral apparatus, a member of the gonostomatids. We provide a first, vague hypothesis about the phylogenetic relationships of the Gonostomatidae, Cladotrichidae, and Schmidingerotrichidae. However, since molecular data of the type species of these higher taxa are lacking, their validity and relationships remain obscure.     

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 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).     

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.     

Morphogenesis and molecular phylogeny of two soil ciliates Australocirrus australis (Foissner, 1995) Kumar and Foissner, 2015 and A. aspoecki (Foissner, 2004) Kumar and Foissner, 2015 (Ciliophora,Hypotrichia)

The morphogenesis and molecular phylogeny of two soil hypotrich ciliates, Australocirrus australis and A. aspoecki, collected from Xi’an, Shaanxi Province, China, were investigated using protargol preparation. The main features of morphogenesis of A. australis are as follows: (1) the parental adoral zone of membranelles is retained completely in the proter; (2) parental cirri and the undulating membranes are involved in the formation of six primordial streaks; (3) multiple fragmentation of the third dorsal kinety anlage; (4) more than three dorsomarginal kineties are formed. An early divider of A. aspoecki was also found, and its morphogenetic events resemble those of other Australocirrus species. Furthermore, the SSU rDNA sequence of the Shaanxi populations of A. australis and A. aspoecki was provided. The molecular phylogeny indicates that A. aspoecki clusters together with A. rubrus and two populations of A. australis with full support, which are separated from the clade comprising the two populations of A. shii.     

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.     

Morphological Characterizations of Four Species of Parallelostrombidium (Ciliophora,Oligotrichia), with a Note on the Phylogeny of the Genus

The morphology and phylogeny of four oligotrichid ciliates, Parallelostrombidium paraellipticum sp. n., P. dragescoi sp. n., P. jankowskii (Xu et al. 2009) comb. n., and P. kahli (Xu et al. 2009) comb. n., are described or redescribed based on live observation, protargol stained material, and SSU rRNA gene sequences. The new species P. paraellipticum sp. n. is characterized by its obovoidal cell shape, adoral zone composed of 17–21 collar, 9–11 buccal, and two thigmotactic membranelles, and extrusomes attached in one row along the girdle kinety. The new species P. dragescoi sp. n. is distinguished from its congeners by its obovoidal cell shape and a lack of thigmotactic membranelles. Based on ciliary patterns recognizable in the original slides, Omegastrombidium jankowskii Xu et al. 2009 and O. kahli Xu et al. 2009 should be transferred to the genus Parallelostrombidium Agatha 2004. Phylogenetic analyses based on SSU rRNA gene sequence data demonstrate that all four new sequences cluster with previously described congeners. The genus Parallelostrombidium is separated into two clusters, suggesting its non‐monophyly and probably corresponding to the two subgenera proposed by Agatha and Strüder‐Kypke (2014), as well as their morphological difference (cell dorsoventrally flattened vs. unflattened).     

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.     

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a “Well‐known” Euplotes Ciliate,Euplotes harpa Stein, 1859 (Ciliophora,Euplotida)

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and the previously well described Aspidisca lynceus (Müller, 1773 ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44–68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11–13 dorsal kineties with 17–25 dikinetids in the mid‐dorsal row, and dorsal silverline system of the double‐eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the “well‐known” Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this “well‐known” form in molecular databases requires further confirmation.     

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 a New Soil Ciliate,Sterkiella multicirrata sp. nov. (Ciliophora,Hypotrichia) from China

The morphology and morphogenesis of a new soil hypotrich ciliate, Sterkiella multicirrata sp. nov., was investigated using live observation and protargol staining. The new species is characterised by: body elliptical, 110–180 × 45–75 μm in vivo, an average of 35 adoral membranelles; usually 19 frontoventral‐transverse cirri, consisting of three frontal, five frontoventral, one buccal, four postoral ventral, two pretransverse, and four transverse cirri; four macronuclear nodules, and 2–5 micronuclei. S. multicirrata sp. nov. differs from its congeners mainly in the number of frontoventral‐transverse cirri and macronuclear nodules. Morphogenesis of the new species is similar to its congeners; the primary difference exists in the segmentation of the frontoventral‐transverse cirral anlagen, which is usually generated in a 1:2:3:3:5:5 pattern. Based on the small subunit ribosomal DNA sequence, the phylogenetic position of the new species is discussed.     

Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora,Metopida), with Identification of the Intracellular Bacteria

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18–21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21–29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion‐resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).     

Morphology and Phylogeny of Two Species of Loxodes (Ciliophora,Karyorelictea), with Description of a New Subspecies,Loxodes striatus orientalis subsp. n.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35–55 vs. 50–70) and a variable number of macronuclei (2–4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.