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,Morphogenesis, and Molecular Phylogeny of Neobakuella aenigmatica n. sp. (Ciliophora,Spirotrichea, Bakuellidae)

The morphology and morphogenesis of a new ciliate species, Neobakuella aenigmatica n. sp., which was discovered in an estuary in Korea, were investigated, using live observation, protargol impregnation, and scanning electron microscopy. This new species is characterized by a large (185–300 × 55–105 μm in vivo), elongate‐ellipsoidal, flexible but not contractile body. It has ellipsoidal, yellowish cortical granules, 1.3 × 1.0 μm in size. The species has invariably 3 frontal and 2 frontoterminal cirri, about 5–10 buccal and 1–6 parabuccal cirri, 7 midventral rows, and 1 right and 2–4 left marginal rows. The outer left marginal row(s) consists of 1–7 short rows of cirri. The nuclear apparatus comprises 130 macronuclear nodules and 2 spherical micronuclei on average. The dorsal ciliature consists of 3 dorsal kineties. The leftmost left marginal row(s) likely develops from anlagen originating from both the rightmost and leftmost left marginal row(s). The molecular phylogenetic tree based on SSU rDNA suggests the nonmonophyly of the genus Neobakuella.     

Morphology and Morphogenesis of a Novel Saline Soil Hypotrichous Ciliate,Gonostomum sinicum nov. spec. (Ciliophora,Hypotrichia, Gonostomatidae), Including a Report on the Small Subunit rDNA Sequence

Morphology, cirral pattern, and morphogenesis of the new saline soil hypotrich, Gonostomum sinicum nov. spec. collected from Longfeng Wetland in Daqing, north China, were studied, using detailed live observations and protargol‐stained specimens. The new species is characterized as follows: (i) a size in vivo of 100–125 × 30–40 μm, (ii) colorless cortical granules, 0.5 μm across, arranged in short rows, (iii) an adoral zone composed of 28–33 membranelles, (iv) three or four frontoventral rows, one of which extends onto the postoral area, (v) left and right marginal rows composed of 18–27 and 21–35, cirri, respectively, and (vi) usually two transverse cirri. Morphogenesis is as usual for the genus Gonostomum, i.e. the cirral primordia II–VI are primary primordia which split into two sets for proter and opisthe in division middle stages, except for anlage I which develops independently. However, the number of frontoventral transverse anlagen is either five or six not only in different individuals but even in proter and opisthe of the same divider. The phylogenetic analyses based on SSU rDNA sequences showed that the genus Gonostomum is nonmonophyletic, indicating that the patterns of cirri and dorsal kineties are homoplasious characters. The new species G. sinicum nov. spec. is perhaps closely related to Cotterillia bromelicola and two congeners.     

Morphology and Molecular Phylogeny of Two New Ciliates,Holostichides heterotypicus n. sp. and Holosticha muuiensis n. sp. (Ciliophora: Urostylida)

Two new urostylid species, Holostichides heterotypicus n. sp. and Holosticha muuiensis n. sp., were discovered in South Korea. Morphological and phylogenetic analyses were carried out to confirm that these species are new to science. Holostichides heterotypicus is mainly characterized by the following combination of features: 110–205 μm long in vivo; 5–10 frontoterminal cirri; 6–8 midventral pairs with 2–3 midventral cirral rows; cortical granules present; four bipolar dorsal kineties; and 6–9 caudal cirri. Ontogenetic features of H. heterotypicus are similar to those of H. typicus. Phylogenetic analyses revealed that H. heterotypicus was distantly separated from bakuellid genera Apobakuella, Bakuella, Metaurostylopsis, and Neobakuella. This result is supported by the following features: transverse cirri (present in the other four bakuellids vs. absent in Holostichides) and caudal cirri (absent in the other four bakuellids vs. present in Holostichides). Holosticha muuiensis n. sp. is mainly distinguished from its congeners by the following combination of features: 100–185 long in vivo; shortened undulating membrane; cortical granules lacking; contractile vacuole absent; 51–66 adoral zone of membranelles; 42–60 macronuclear nodules; and five bipolar dorsal kineties. In the phylogenetic tree, Holosticha muuiensis n. sp. clustered with a Holosticha group (containing Holosticha diademata, Holosticha foissneri, and Holosticha heterofoissneri).     

A New Subspecies of Oxytricha granulifera (Hypotrichia: Oxytrichidae) from Mexico,with Notes on its Morphogenesis and Phylogenetic Position

The genus Oxytricha Bory de Saint‐Vincent in Lamouroux, Bory de Saint‐Vincent and Deslongchamps, 1824 comprises about 38 species distributed worldwide and has been considered to be a nonmonophyletic group. Based on living observations, protargol preparations, and a small subunit ribosomal RNA (SSU rRNA) gene sequence, we describe a new subspecies Oxytricha granulifera chiapasensis n. subsp. This new taxon is morphologically characterized by undulating membranes basically in a Stylonychia‐pattern, six dorsal kineties, size in vivo ca. 60–120 × 20–40 μm, 21–30 right and 21–31 left marginal cirri, 22–29 adoral membranelles, and spherical cortical granules arranged in longitudinal rows on the dorsal side. In terms of the SSU rRNA gene sequence, the new subspecies differs from populations of O. granulifera from GENBANK by 7–35 nucleotides. Phylogenetic analyses showed that Oxytricha granulifera gene sequences were nested into three groups, with the new subspecies included in one of them. Oxytricha granulifera chiapasensis n. subsp. is different from Oxytricha granulifera granulifera Foissner and Adam, 1983 and Oxytricha granulifera quadricirrata Blatterer and Foissner, 1988 based on: (i) undulating membranes in Stylonychia‐pattern, (ii) formation of a sixth dorsal kinety during morphogenesis, (iii) the adoral membranelles number, and (iv) inhabiting freshwater habitats.     

Rubrioxytricha guamensis nov. spec. (Ciliophora,Spirotricha), a Novel Hypotrich Ciliate from Guam (United States), Micronesia

The oxytrichid ciliate Rubrioxytricha guamensis nov. spec. isolated from water samples collected from a small freshwater pond near the Hagåtña River in Hagåtña, Guam (United States territory), Micronesia, was investigated, using live observation and protargol impregnation. The morphology, ontogenesis, and molecular phylogeny inferred from the small‐subunit rRNA gene sequences were studied. The new species is mainly characterized by a cell size of about 100 × 35 μm in vivo, two elongate ellipsoidal macronuclear nodules and two micronuclei, a single contractile vacuole, a colorless cytoplasm, yellowish cortical granules, arranged in short rows and in small groups, an adoral zone occupying about 34% of body length and comprising 27 membranelles on average, about 27 cirri each in the right and left marginal rows, 18 frontoventral transverse cirri, four dorsal kineties including one dorsomarginal row, and one or two caudal cirri at the posterior end of dorsal kinety 3. The ontogenesis of the new species is similar to that of Rubrioxytricha indica Naqvi et al., 2006. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the family Oxytrichidae Ehrenberg, 1838, where it clustered with other Rubrioxytricha species, viz., R. tsinlingensis, R. ferruginea, and R. haematoplasma.     

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.     

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).     

Morphology of Clapsiella magnifica gen. n., sp. n., a new hypotrichous ciliate with a curious dorsal ciliary pattern

The present work describes the morphology and infraciliature of a new hypotrichous ciliate, Clapsiella magnifica gen. n., sp. n., found in rewetted soil from a temporal pond in Argentina. It was studied by means of live observation and protargol impregnation. Its main diagnostic features are: Flexible hypotrich measuring 250–320 μm × 70–140 μm in vivo; two macronuclear nodules and 4–6 micronuclei. Single contractile vacuole. Cytoplasm transparent, cortical granules absent. Somatic ciliature composed of a tricorona of cirri, three buccal(?) cirri, 6–9 ventral rows, 3–5 right marginal(?) rows, one left marginal row, and 12–17 transverse cirri. Dorsal pattern rather complicated, with about 14 kineties and kinety fragments, with scattered kinetids among them; 17–28 caudal cirri arranged in three rows on dorsal kineties 1, 3, and 7. Remarkably, dorsal kinetids have two or four basal bodies, bearing a stiff bristle arising from left anterior basal body. Adoral zone composed of 70–92 membranelles, occupying about 40% of body length in protargol preparations; paroral and endoral curved, resembling a cyrtohymenid pattern. The peculiar dorsal ciliary arrangement and the unique combination of other characters require the establishment of a new genus for this new species, which is considered incertae sedis in the Hypotricha but possibly related to the oxytrichids.     

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 SSU rRNA gene sequences of three Frontonia species,including a description of F. subtropica spec. nov. (Ciliophora,Peniculida)

The morphology and infraciliature of three Frontonia species, F. subtropica spec. nov., F. canadensis Roque and Puytorac, 1972, and F. magna Fan et al., 2011, isolated from coastal waters in southern China sea, were investigated using living observation and silver impregnation methods. Frontonia subtropica spec. nov. is recognized by the combination of the following characters: body elliptical in outline with right margin depressed in anterior third, about 180–230 μm × 60–80 μm in vivo; 104–114 somatic kineties; peniculi 1–3 each with four kineties; five vestibular and five postoral kineties; one centrally located elongate-elliptical macronucleus; single contractile vacuole located left-dorsally in posterior third of body. We also provide improved diagnoses for F. canadensis and F. magna based on current and previous reports. The small subunit (SSU) rRNA gene was sequenced for all three species. Comparisons with sequences of morphologically similar congeners clearly support the validity each species.     

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.     

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.     

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 Phylogenetic Position of an Unusual Stentor polymorphus (Ciliophora: Heterotrichea) Without Symbiotic Algae

We investigated the live morphology, infraciliature, and small subunit rRNA gene sequences of an unusual population of Stentor polymorphus without symbiotic algae that was isolated from the southeastern region of Brazil. The morphological and molecular data confirmed the identity of this strain as S. polymorphus. The Brazilian S. polymorphus organism is 850–2,000 μm in length in vivo and has colorless cortical granules, a moniliform macronucleus with 6–12 nodules, somatic ciliature composed of 50–60 kineties, a single contractile vacuole located to the left of the cytostome, and a conspicuous oral pouch, and it does not build a lorica. Based on the phylogenetic analyses, the Brazilian S. polymorphus was located within a cluster consisting of four other S. polymorphus sequences, with high support values using both the Bayesian inference and maximum likelihood algorithms. Our study presents the first report of a S. polymorphus population without its symbionts under natural conditions. On the basis of our findings, we propose that the presence or absence of symbiotic algae should not be used as a taxonomic character for the identification of Stentor species.     

Morphology and Molecular Phylogeny of a New Hypotrich Ciliate,Pseudourostyla guizhouensis sp. nov. from Southern China,with Notes on a Chinese Population of Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012 (Ciliophora,Hypotricha)

The morphology and molecular phylogeny of a soil hypotrich ciliate, Pseudourostyla guizhouensis sp. nov., collected from southern China, were investigated. Pseudourostyla guizhouensis sp. nov. has an elongate elliptical body measuring 180–310 × 65–85 μm in vivo; invariably two right and three or four left marginal rows; six or seven dorsal kineties; adoral zone consisting of 57–70 membranelles; 12–16 frontal cirri, one buccal cirrus, 13–20 midventral pairs, two frontoterminal cirri, two pretransverse cirri, and five to seven transverse cirri. Morphogenesis during physiological regeneration indicates that the marginal rows of each side originate from a common anlage that differentiates into several rows. Molecular phylogenetic analysis based on SSU rDNA sequence data reveals that P. guizhouensis sp. nov. clusters with the type species P. cristata (Jerka‐Dziadosz, 1964) Borror, 1972 and that the genus Pseudourostyla is monophyletic. The morphological characters of another soil hypotrich ciliate, Hemicycliostyla franzi (Foissner, 1987) Paiva et al., 2012, are also described based on a Chinese (Guizhou) population.     

Pelagostrobilidium liui n. sp. (Ciliophora,Choreotrichida) from the Coastal Waters of Northeastern Taiwan and an Improved Description of Pelagostrobilidium minutum Liu et al., 2012

The number of somatic kineties in Pelagostrobilidium ranges from 4 to 6 according to the present state of knowledge. This study investigates Pelagostrobilidium liui n. sp. using live observation, protargol stain, and small subunit rDNA data sequencing. Pelagostrobilidium liui n. sp. is characterized by having a spherical‐shaped body, four somatic kineties, with kinety 2 spiraled around the left side of body, about six elongated external membranelles, and invariably no buccal membranelle. It differs from its most similar congener, Pelagostrobilidium minutum Liu et al., 2012 , in (i) cell shape; (ii) macronucleus width; (iii) oral apparatus; (iv) anterior orientation of kinety 2; (v) location where kinety 2 commences; (vi) arrangement of kinety 1; (vii) distance between the anterior cell end and the locations where kineties commence; and (viii) the presence of 12 different bases (including two deletions) in the small subunit rDNA sequences. The diagnosis of P. minutum Liu et al., 2012 is also improved to include the following new characteristics: invariably four somatic kineties; kineties 2 and 4 alone commence at the same level; kinety 2 originates from right anterior cell half on ventral side, extends sinistrally posteriorly, over kinety 1, around left posterior region, terminates near posterior cell end on dorsal side; kinety 1 commences below anterior third of kinety 2.