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.     

Morphology and Phylogeny of Four New Vorticella Species (Ciliophora: Peritrichia) from Coastal Waters of Southern China

Four new species of Vorticella, V. parachiangi sp. n., V. scapiformis sp. n., V. sphaeroidalis sp. n., and V. paralima sp. n., were isolated from coastal brackish waters of southern China. Their morphology, infraciliature, and silverline system were investigated based on observations of specimens both in vivo and following silver staining. Vorticella parachiangi sp. n. is distinguished by: a J‐shaped macronucleus; a single dorsally located contractile vacuole; a two‐rowed infundibular polykinetid 3, in which row 1 is shorter than row 2; 21–31 silverlines between peristome and aboral trochal band, 6–11 between aboral trochal band and scopula. Vorticella scapiformis sp. n. is characterized by its conspicuously thin and irregularly edged peristomial lip; a J‐shaped macronucleus; a single, ventrally located contractile vacuole; row 1 of the infundibular polykinetid 3 proximally shortened; 18–25 silverlines between peristome and aboral trochal band, 8–12 between aboral trochal band and scopula. Vorticella sphaeroidalis sp. n. can be identified by its small, sub‐spherical zooid; a C‐shaped macronucleus; a ventrally located contractile vacuole; an aboral trochal band adjacent to the scopula; 16–18 silverlines between persitome and aboral trochal band, two between aboral trochal band and scopula. Vorticella paralima sp. n. can be identified by its ovoidal zooid; a J‐shaped macronucleus; a dorsally positioned contractile vacuole; rows 1 and 2 of the infundibular polykinetid 3 proximally shortened; 26–35 silverlines from peristome to aboral trochal band, and 7–13 from aboral trochal band to scopula. The SSU rDNA genes of these four species were sequenced and their phylogeny was analyzed.     

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 Three Trachelocercid Ciliates,with Description of a New Species,Trachelocerca orientalis spec. nov. (Ciliophora,Karyorelictea)

Three trachelocercid ciliates, Trachelocerca orientalis spec. nov., Prototrachelocerca fasciolata (Sauerbrey, 1928) Foissner, 1996 and Tracheloraphis huangi Xu et al., 2011, isolated from marine coastal habitats at Qingdao, China, were taxonomically studied using observation in vivo and silver staining methods. The new species T. orientalis spec. nov. can be recognized by the combination of its size (600–1,200 μm in vivo), 15–21 somatic kineties and about 13 groups of macronuclear nodules forming a strand and the colorless globular cortical granules. Together with the sequence data of the small subunit ribosomal RNA (SSU rRNA) gene, the information of a new isolate of P. fasciolata and three populations of T. huangi is also documented based on the present work. According to the molecular data, the phylogeny of three species is estimated and the analyses show that they are all found within the trachelocercid assemblage though T. huangi does not cluster with its congeners but with Trachelocerca species. Nonetheless, the monophyly of Trachelocerca is not rejected by the approximately unbiased test (p = 0.345 > 0.05), while that of Tracheloraphis is not confirmed (p = 0.0002 < 0.05).     

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 Phylogeny of Three Pseudovorticella Species (Ciliophora: Peritrichia) from Brackish Waters of China

The biodiversity of peritrich ciliates from brackish biotopes is rarely investigated, especially members of the genus Pseudovorticella. Here, the morphology of three species of Pseudovorticella, i.e. P. cf. vestita (Stokes, 1883) Jankowski, 1976, P. spathulata sp. n., and P. qinghaiensis sp. n. isolated from brackish waters were studied. Pseudovorticella cf. vestita is characterized by inverted bell‐shaped cell; a J‐shaped macronucleus; a single contractile vacuole ventrally located; P3 three‐rowed; pellicle striated with highly developed pellicular vesicles; 18–22 transverse silverlines between peristome and aboral trochal band, and 9–13 between aboral trochal band and scopula. Pseudovorticella spathulata sp. n. differs from its congeners by the following combination of characters: elongate‐elliptical cell; a single contractile vacuole near ventral wall of infundibulum; a J‐shaped macronucleus; P3 three‐rowed; 24–34 silverlines between oral area and aboral trochal band and 6–10 between aboral trochal band and scopula. Pseudovorticella qinghaiensis sp. n. is characterized by: cell with an oval outline; a single contractile vacuole near ventral wall of infundibulum; a C‐shaped macronucleus; P3 three‐rowed; 30–35 and 9–11 transverse silverlines above and below the trochal band, respectively. The SSU rDNA sequences of five Pseudovorticella species, namely P. annulata, P. monilata, P. parakenti, P. spathulata sp. n., and P. cf. vestita, plus that of Zoothamnium hartwigi, are reported for the first time and their evolutionary relationships are investigated. Five undefined Pseudovorticella forms are considered might be conspecific with P. monilata. Two congeners are conspecific with P. spathulata sp. n. Phylogenetic analyses based on SSU rDNA sequences reveal that Pseudovorticella is not monophyletic and Z. hartwigi clusters with its congeners as expected.     

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.     

Morphology,morphogenesis and molecular phylogeny of a new marine ciliate,Trichototaxis marina n. sp. (Ciliophora,Urostylida)

The live morphology, infraciliature and morphogenesis of a new urostylid ciliate, Trichototaxis marina n. sp., collected from coastal water in Qingdao, China, were studied based on the observations of live and silver stained specimens. The new species is characterised as follows: body very flexible and contractile, slight to brick-reddish in colour due to irregularly-shaped, brick-red pigments; ca. 70 adoral membranelles; about 17 frontal cirri arranged in a bicorona; average 67 midventral pairs, the right base of each pair being conspicuously larger than the left base; five to seven transverse cirri; constantly two frontoterminal, one buccal and two pretransverse ventral cirri; two or three left marginal rows; right and innermost left marginal rows with 56–92 and 66–106 cirri, respectively; six bipolar dorsal kineties; more than 100 macronuclear nodules. The characteristic morphogenetic feature in T. marina is the development of the left marginal rows, that is, only one left marginal row is newly built the other one or two being retained from the parental cell. Phylogenetic analyses based on small subunit ribosomal gene sequence data reveal a close relationship of T. marina with members of family Pseudokeronopsidae.     

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.     

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.     

Morphology,morphogenesis and molecular phylogeny of a freshwater ciliate,Monomicrocaryon euglenivorum euglenivorum (Ciliophora,Oxytrichidae)

The morphology, morphogenesis and molecular phylogeny of the oxytrichid ciliate, Monomicrocaryon euglenivorum euglenivorum (Kahl, 1932) Foissner, 2016, isolated from freshwater in a seaside park, Guangzhou, China, were investigated. Monomicrocaryon euglenivorum euglenivorum can be recognized as follows: caudal cirri in midline of body; dorsal kinety 1 without a one-kinetid-wide gap; transverse cirri acicular or rod-shaped with a fringed distal end; right marginal row commences at level of buccal vertex or anterior to buccal vertex. The main events during binary fission are as follows: (1) the proter retains the parental adoral zone of membranelles entirely; (2) frontoventral-transverse cirral anlagen I–VI 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 postoral ventral, two pretransverse ventral and five transverse cirri, respectively; (3) dorsal morphogenesis is in the typical Oxytricha-pattern, but fragmentation of dorsal kinety 3 is indistinct; and (4) three caudal cirri are formed, one at the posterior end of each of dorsal kineties 1, 2 and 4. Phylogenetic analyses based on SSU rDNA sequences showed that M. euglenivorum euglenivorum clustered with Kleinstyla dorsicirrata and Heterourosomoida lanceolata rather than with its congener M. elegans. The genus Monomicrocaryon is not monophyletic in this study; however, its monophyly is not rejected by the AU test.     

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 and phylogeny of a new urostylid ciliate, Monocoronella carnea n. g., n. sp. (Ciliophora, Hypotricha) from Daya Bay, Southern China

The morphology, infraciliature and small subunit ribosomal RNA gene-based phylogeny of an urostylid ciliate, Monocoronella carnea n. g., n. sp., found in coastal areas off Daya Bay, Southern China, were investigated. The new genus Monocoronella n. g. is recognized by the following features: having conspicuous frontal cirri forming a long and single corona; buccal and frontoterminal cirri present; single marginal row on each side; adoral zone, midventral complex and transverse cirri in Pseudokeronopsis mode. The type species M. carnea n. sp. is diagnosed by the combination of marine habitat and brown-reddish color of the cortical granules. Phylogenetic analyses for the new taxon indicate that Monocoronella n. g. is most closely related to Bergeriella, and is located within the core Urostylida clade. A misidentification in previous literature was recognized and a new species, Monocoronella dragescoi n. sp. [Basionym: Holosticha (Keronopsis) monilata (Kahl 1928) sensu Dragesco (1970) et sensu Dragesco and Dragesco-Kernéis (1986), non sensu Kahl (1928)], was suggested.     

Phylogenetic Analyses Support Validity of Genus Eodinium (Ciliophora,Entodiniomorphida, Ophryoscolecidae)

The validity of genus Eodinium has been historically disputed due to morphological similarities with Diplodinium (absence of skeletal plates as well as adoral and dorsal ciliary zones at the same body level). To address this issue, the 18S rDNA of four Eodinium posterovesiculatum morphotypes and four Diplodinium anisacanthum morphotypes were sequenced and phylogenetically analyzed. The different inference methods suggest the existence of a last common ancestor of Eodinium and Ostracodinium that is not shared with Diplodinium, strongly supporting the validity of genus Eodinium. Since skeletal plates are present in all members of genus Ostracodinium, the most parsimonious is a secondary loss of skeletal plates in E. posterovesiculatum. This work represents a breakthrough in the taxonomy and phylogeny of the family Ophryoscolecidae indicating that the skeletal plates may not reflect evolutionary divergence within this group of ciliates as traditionally proposed.     

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”.     

Phylogeny of the order Tintinnida (Ciliophora, Spirotrichea) inferred from small- and large-subunit rRNA genes

Concatenated sequences of small- and large-subunit rRNA genes were used to infer the phylogeny of 29 species in six genera of Tintinnida. We confirmed previous results on the positions of major clusters and the grouping of various genera, including Stenosemella, the paraphyletic Tintinnopsis, the newly investigated Helicostomella, and some species of the polyphyletic Favella. Tintinnidium and Eutintinnus were found to be monophyletic. This study contributes to tintinnid phylogenetic reconstruction by increasing both the number of species and the range of genetic markers analyzed.