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.     

Redescription of two little-known urostyloid ciliates,Anteholosticha randani () and A. antecirrata (Ciliophora,Urostylida)

The morphology of two little-known urostyloid ciliates, Anteholosticha randani (Grolière, 1975) Berger, 2003 and A. antecirrata Berger, 2006, collected from freshwater biotopes in southern China, was studied based on live observations and protargol staining. Anteholosticha randani is characterized by its bipartite adoral zone and short, longitudinally aligned undulating membranes. One early stage of reorganization/morphogenesis, one early-middle stage of reorganization and one middle stage of morphogenesis are also reported. Anteholosticha antecirrata is characterized by its large body size in vivo (200–400 × 40–80 μm), a row of buccal cirri and conspicuous, yellow-green cortical granules. Phylogenetic analyses based on SSU rDNA sequence data reveal that A. antecirrata may share a most recent common ancestor with Urostyla grandis and Bakuella granulifera, whereas A. randani branches independently and is sister to a large clade that includes Pseudourostyla, Pseudokeronopsis, Caudiholosticha and several species of Anteholosticha.     

Morphology and molecular phylogeny of a new hypotrich ciliate,Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora,Hypotrichia)

A new urostylid ciliate, Anteholosticha songi nov. spec., isolated from forest soil in Tibet, and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991, isolated from a freshwater pond in the USA, are investigated in terms of their morphology, ontogenesis, and molecular biology. Anteholosticha songi nov. spec. is characterized by a slender to ellipsoidal body measuring 160–205 × 40–55 μm in vivo; rod-shaped yellowish cortical granules arranged in irregular short rows; four dorsal kineties; adoral zone consisting of 35–40 membranelles; three frontal, one buccal, one parabuccal, two frontoterminal, two pretransverse, and four to six transverse cirri and 14–25 midventral pairs; 12–22 ellipsoidal macronuclear nodules longitudinally arranged in pairs left of cell mid-line. Supplemental information on morphogenesis in Holosticha pullaster is also presented. The phylogenetic relationship of Anteholosticha and Holosticha inferred from SSU rDNA sequence data are concordant with previous studies and showing that Holosticha is monophyletic whereas Anteholosticha is polyphyletic and should be split into two or more genera.     

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

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

Morphology,morphogenesis and molecular phylogeny of Lamtostyla granulifera sinensis subsp. nov. (Ciliophora,Hypotrichia) from a wetland in China

The morphology, morphogenesis and molecular phylogeny of a hypotrichous ciliate, Lamtostyla granulifera sinensis subsp. nov., isolated from northern China, were investigated. This population appeared highly similar in morphology to L. granulifera Foissner, 1997. However, on detailed investigation some non-overlapping features were identified, i.e., the body shape and the arrangement of the cortical granules. These differences suggested the separation at subspecies level. Furthermore, the morphogenesis of the new subspecies is described, which is characterized by: (1) the posterior part of the parental adoral zone of membranelles is renewed; (2) the amphisiellid median cirral row is formed from two anlagen; and (3) the frontoventral-transverse cirral anlagen II to VI generate one transverse cirrus each. Phylogenetic analyses based on SSU rDNA sequence data show that Lamtostyla species are scattered in different clades. The monophyly of the genus Lamtostyla is also rejected by the AU test in this study.     

Ontogenesis and molecular phylogeny of a new marine urostylid ciliate, Anteholosticha petzi n. sp. (Ciliophora, Urostylida)

A new urostylid ciliate, Anteholosticha petzi n. sp., isolated from the northern China seas, was studied using live observation and protargol impregnation. It differs from all the congeners mainly in the highly flexible and variable body shape, its red-brown to brick-red colour, and the possession of three types of cortical granules. Analysis of morphometric and molecular data confirm the validity of A. petzi as a distinct species. Anteholosticha petzi n. sp. is characterized as follows: by having 3 frontal, 1 buccal, 2 frontoterminal, 2 pretransverse, and 8-11 transverse cirri; a midventral row that comprises 10-16 cirral pairs and extends ca. 60% of cell length; and 3 bipolar dorsal kineties. Ontogenesis is similar to that in Anteholosticha warreni except for the formation of the oral primordium and is characterized by: (1) the parental adoral membranelles are completely renewed and the oral primordium is formed de novo; and (2) the anlagen for the frontal-ventral-transverse cirri are formed as primary primordia. The small subunit rRNA gene sequence of A. petzi n. sp. was also compared with that of its congeners with the sequence differences ranging from 0.39% to 8.62%. Phylogenetic trees based on the SSU rRNA gene sequence were constructed, indicating the nonmonophyly of the genus Anteholosticha, as supported by the approximately unbiased test.     

Two Urosoma species (Ciliophora,Hypotrichia): A multidisciplinary approach provides new insights into their ultrastructure and systematics

The general morphology and ultrastructure of two soil hypotrichous ciliates, Urosoma emarginata and U. salmastra, were investigated using light microscopy, scanning electron microscopy and transmission electron microscopy. Phylogenetic analyses, based on the newly sequenced small subunit ribosomal (SSU) rRNA genes, were conducted on three U. emarginata populations and one U. salmastra population. Our findings support for the validity of Perilemmaphora Berger, 2008, a rankless taxon comprising spirotrich ciliates having a perilemma. The cortical granules of both species are extrusomes representing a new type of mucocyst in U. emarginata and possibly a new type of pigmentocyst in U. salmastra. Additionally, the lithosomes were revealed as subglobose structures composed of a low electron-dense, homogeneous inner part and an electron-dense outer part. The ultrastructural features of the cortical granules, together with ontogenetic and molecular phylogenetic data, suggest that the genus Urosoma might need to be divided. It is posited that ultrastructural features of hypotrichous ciliates in general may have important taxonomic value warranting further investigation.     

Morphology and molecular phylogeny of two new brackish-water species of Amphisiella (Ciliophora,Hypotrichia), with notes on morphogenesis

Two new species of the taxonomically confused genus Amphisiella were isolated from brackish-water habitats in southern China, and their morphology and morphogenesis were investigated. The genes coding for small subunit (SSU) rRNA were sequenced in each species and included in a phylogenetic analysis with all data available from nominal species of Amphisiella and related hypotrichs. Both new species are diagnosed by an elongate body shape, yellow-brown/grey-brown cell color, one kind of cortical granule, and two macronuclear nodules. The cortical granules in A. pulchra sp. n. are grouped in small clusters which are irregularly distributed both ventrally and dorsally; in A. candida sp. n., they are irregularly and sparsely distributed ventrally and form longitudinal stripes dorsally. The ontogenetic processes of both species show similarities to those of other congeners. Phylogenetic trees based on SSU rRNA gene sequences suggest that the family Amphisiellidae is a paraphyletic assemblage. The results further demonstrate that two isolates identified as Amphisiella annulata (DQ832260 and GU170843) are very likely cryptic species, and a sequence identified as A. milnei (DQ845293) may belong to a genus other than Amphisiella.     

Morphology,morphogenesis, and molecular phylogeny of a new freshwater ciliate,Gonostomum jangbogoensis n. sp. (Ciliophora,Hypotricha), from Victoria Land,Antarctica

In a study on ciliate diversity, we discovered the new hypotrich species, Gonostomum jangbogoensis n. sp., in freshwater from Terra Nova Bay, Victoria Land, southeast Antarctica. We describe its morphology and morphogenesis using standard methods, and the SSU rRNA gene phylogeny is provided as well. Morphology of Gonostomum jangbogoensis n. sp. is characterized as follows: slender to elongated body shape; grayish under low magnification; cortical granules present; 32–41 adoral membranelles; 3 enlarged frontal cirri; 1 buccal cirrus; 2 frontoterminal cirri; 3 or 4 frontoventral cirral pairs, 2 pretransverse cirri, 6–7 transverse cirri; 13–19 left and 18–26 right marginal cirri; 17–23 paroral kinetids; 3 dorsal kineties; 3 caudal cirri; 2 macronuclear nodules with 1–3 micronuclei. The morphogenesis of the new species confirms that it has at least seven frontal-ventral-transverse cirral anlagen, which is also reported in Gonostomum sp. 1 sensu Shin from Korea. Even though these two populations occur very far from each other, the morphometric data prove that this character state, the seven cirral anlagen, is a stable feature across these populations and might be an apomorphy. The phylogenetic analyses show that the genus Gonostomum is non-monophyletic and that the new species is a sister to G. bromelicola.     

Reevaluation of the ‘well-known’ Paraurostyla weissei complex,with notes on the ontogenesis of a new Paraurostyla species (Ciliophora,Hypotrichia)

Two hypotrichous ciliates, Paraurostyla wuhanensis nov. spec. from Wuhan (China) and a new North American population of the Paraurostyla weissei complex, were studied based on live observations and protargol impregnation. Paraurostyla wuhanensis nov. spec. differs from the congeners by the combined features of having six or seven frontal cirri, 2–4 frontoventral cirri, 5–7 ventral rows, and yellow-greenish cortical granules. Ontogenesis proceeds as in the type species, except that fewer frontoventral cirri are formed in the new species. The morphology of the new population of the Paraurostyla weissei complex corresponds well with other American populations. In the phylogenetic trees based on the 18S rRNA gene, Paraurostyla sequences nest in a large clade together with Apoamphisiella and Notohymena. Monophyly of Paraurostyla is rejected by the results of the approximately unbiased test analyses. Morphological, morphogenetic, and phylogenetic analyses show a close relationship between the North American populations of the Paraurostyla weissei complex and Apoamphisiella, indicating that the taxonomic position of the former needs to be reassigned. A new combination, viz. Apoamphisiella polymicronucleata (Merriman, 1937) comb. nov., a reevaluation of the Paraurostyla weissei complex, and emended diagnoses of Paraurostyla and Apoamphisiella, are provided.     

Morphology,ontogenesis and molecular phylogeny of a new saline soil ciliate,Uroleptoides salina nov. spec. (Ciliophora,Hypotrichia)

The morphology, morphogenesis and molecular phylogeny of a new saline soil hypotrich ciliate, Uroleptoides salina nov. spec., discovered from China, was investigated. The new species is characterized as follows: body 150–215 × 40–50 μm in vivo, slender and highly flexible; usually four ellipsoidal macronuclear nodules; contractile vacuole absent; cortical granules absent; endosymbiotic algae present; amphisiellid median cirral row consists of 14–25 cirri and terminates about 47% down length of body; usually three buccal cirri and 3–13 cirri left of anterior portion of amphisiellid median cirral row; 3–5 transverse cirri. Morphogenesis during binary fission is characterized by: (1) the parental adoral zone of membranelles is retained completely, parental paroral contributes to the formation of the undulating membranes anlage for the proter; (2) the oral primordium of the opisthe is formed apokinetally; and (3) the amphisiellid median cirral row is formed from two anlagen. Phylogenetic analyses based on SSU rDNA sequence data show that Uroleptoides salina nov. spec. has a close relationship with its morphologically similar species, U. longiseries, U. magnigranulosus, Orthamphisiella breviseries, and Parabistichella variabilis.     

Morphology,morphogenesis and molecular phylogeny of a novel saline soil ciliate,Urosoma quadrinucleatum n. sp. (Ciliophora,Hypotrichia)

The morphology and morphogenesis of a new saline soil hypotrichous ciliate, Urosoma quadrinucleatum n. sp., collected from northwestern China, were studied based on live observations and protargol stained specimens. The new species is characterized as follows: size in vivo 90–130 × 20–30 μm; body outline elongate-elliptical with both ends broadly rounded; four macronuclear nodules; cortical granules present; paroral in front of endoral; usually 16 frontal-ventral-transverse cirri. Urosoma quadrinucleatum n. sp. has almost the same morphogenetic characteristics as its congeners U. gigantea and U. salmastra, and differs from other three congeners whose morphogenesis is known in the formation of the frontal-ventral-transverse cirral anlagen as well as the development of marginal and dorsal kineties anlagen. The sequence differences among U. quadrinucleatum n. sp. and other Urosoma species further support the validity of the present organism as novel species. Further, U. quadrinucleatum n. sp. clusters with U. salmastra in the phylogenetic analyses based on small subunit ribosomal RNA gene (SSU rDNA) sequence data.     

OOCYTE DIFFERENTIATION IN THE SEA URCHIN, ARBACIA PUNCTULATA, WITH PARTICULAR REFERENCE TO THE ORIGIN OF CORTICAL GRANULES AND THEIR PARTICIPATION IN THE CORTICAL REACTION

This paper presents morphological evidence on the origin of cortical granules in the oocytes of Arbacia punctulata and other echinoderms. During oocyte differentiation, those Golgi complexes associated with the production of cortical granules are composed of numerous saccules with companion vesicles. Each element of the Golgi complex contains a rather dense homogeneous substance. The vesicular component of the Golgi complex is thought to be derived from the saccular member by a pinching-off process. The pinched-off vesicles are viewed as containers of the precursor(s) of the cortical granules. In time, they coalesce and form a mature cortical granule whose content is bounded by a unit membrane. Thus, it is asserted that the Golgi complex is involved in both the synthesis and concentration of precursors utilized in the construction of the cortical granule. Immediately after the egg is activated by the sperm the primary envelope becomes detached from the oolemma, thereby forming what we have called the activation calyx (see Discussion). Subsequent to the elaboration of the activation calyx, the contents of cortical granules are released (cortical reaction) into the perivitelline space. The discharge of the constituents of a cortical granule is accomplished by the union of its encompassing unit membrane, in several places, with the oolemma.     

Morphology,ontogenesis, and molecular phylogeny of an Indian population of Cyrtohymena (Cyrtohymenides) shii,including remarks on the subgenus

The freshwater ciliate Cyrtohymena (Cyrtohymenides) shii (Shi et al., 1997) Shao et al., 2012 (Hypotricha, Oxytrichidae), isolated from Barsey Rhododendron Sanctuary of The Eastern Himalayas, is slightly flexible, measures about 150 μm × 50 μm in life and possesses citrine cortical granules randomly distributed singly and in small clusters. Cells of our Indian population have five or six dorsal kineties arising from multiple fragmentation of the third dorsal anlage. The subgenus Cyrtohymenides includes species with multiple dorsal kinety fragmentation namely C. (C.) aspoecki (type species), C. (C.) australis, and the present species. Ventral morphogenesis of the genus Cyrtohymena has been reported only for the type species C. muscorum. Notable features of the Indian population include formation of frontal anlagen from four parental cirri, two more parental cirri possibly contribute to these anlagen later, and the formation of primary primordia which later split transversely to form two sets, one for each daughter cell. 18S rDNA sequence of the Indian population matches with those of two populations of C. citrina; it also clusters with Afrokeronopsis aurea, a neokeronopsid, with which it interestingly shares some morphological features, supporting the CEUU hypothesis.     

New contributions to the taxonomy of urostylid ciliates (Ciliophora,Hypotrichia), with establishment of a new genus and new species

Hypotrichia, one of the most complex and highly differentiated groups in Ciliophora, has been the object of extensive studies, especially in recent years. Nevertheless, methodological difficulties and insufficient faunistic studies have limited our understanding of their biodiversity and phylogeny. In this study, one novel urostylid ciliate, Pseudoholosticha zhaoi nov. gen., nov. spec. and two populations of Anteholosticha monilata (Kahl, 1928) Berger, 2003, type species of the latter genus, are studied using an integrative approach (live observation, protargol impregnation, scanning electron microscopy, and phylogenetic analysis) to provide further insights into the diversity, classification, and phylogeny of this group of ciliates. Pseudoholosticha nov. gen. can be separated from other morphologically similar genera mainly by the absence of buccal and caudal cirri. A key to 12 morphologically similar genera and illustrations of their cirral patterns are provided. The validation of the new genus and new species is supported by both morphological and phylogenetic analyses. The first 18S rRNA gene sequence of A. monilata, with detailed morphological data, provided a reliable clarification of A. monilata populations and corroborated the phylogenetic position of the type species of the polyphyletic genus Anteholosticha.     

Morphology and molecular phylogeny of Oxytricha seokmoensis sp. nov. (Hypotrichia: Oxytrichidae), with notes on its morphogenesis

A new hypotrichous ciliate, Oxytricha seokmoensis sp. nov., was discovered in a soil from a forest in South Korea and described based on the observations of living and stained specimens. In addition, phylogenetic analyses were performed using the small subunit ribosomal RNA (18S rRNA) gene sequence. Morphologically, the new species is similar to the O. granulifera-complex in terms of ciliary structure and arrangement of cortical granules, but dorsal kineties 3 and 4 (not completely separated vs. separated) and macronuclear nodules in the cyst (separated vs. fused) differ. Oxytricha seokmoensis is most similar to O. pulvillus, but can be distinguished by the number of adoral membranelles (30–40 vs. 23–27), contractile vacuole (present vs. absent), number of left (27–37 vs. 17–25) and right (27–35 vs. 18–23) marginal cirri, and lepidosomes on the cyst surface (present vs. absent). In a phylogenetic tree, O. seokmoensis is distinctly separated from the O. granulifera clade, but is sister to the Paroxytricha clade. In addition, O. seokmoensis and P. longigranulosa have the smallest genetic difference (d = 0.015, 23 of 1579 nt difference). This close relationship is supported by incomplete dorsal kinety 3 fragmentation and separated macronuclear nodules in resting cysts.