Redescription of Rigidohymena inquieta (Stokes, 1887) Berger, 2011 as Metahymena inquieta gen. nov., comb. nov. (Ciliophora, Hypotricha) Based on Morphology, Morphogenesis, and Molecular Phylogeny.

The morphology and morphogenesis of Rigidohymena inquieta (Stokes, 1887) Berger, 2011, isolated from a lawn soil in the campus of the University of Ulsan, Korea, was studied, using live observation and protargol impregnation. The molecular phylogeny was studied based on the SSU rRNA gene sequences. The morphology of the Korean population of R. inquieta matches the previously known populations; however, the morphogenetic pattern shows differences to the species R. candens in the involvement of cirrus V/3 in the anlagen formation. A novel genus namely Metahymena gen. nov. has been erected for the present species based on the ontogenetic difference, and the new combination Metahymena inquieta gen. nov., comb. nov. is proposed. The morphology, morphogenesis, distribution, and phylogeny of M. inquieta are presented. The morphologic and morphogenetic data corroborate the phylogenetic analyses as M. inquieta clusters among the stylonychid ciliates in a clade distant from Rigidohymena candens.

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.

Description and Molecular Phylogeny of a Novel Hypotrich Ciliate from the Soil of Marche Region, Italy; Including Notes on the MOSYSS Project.

The morphology and morphogenesis during cell division of a new stylonychine hypotrich, Rigidocortex quadrinucleatus n. sp., were investigated using live observation and protargol staining. The new species was isolated from soil samples collected from an organic farm in the Marche Region, Italy, in framework of the MOSYSS project. Rigidocortex quadrinucleatus is characterized as follows: cell size about 180 × 80 µm in vivo; four ellipsoidal macronuclear nodules; 44 adoral membranelles: 18 fronto-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse, and five transverse cirri; dorsal kinety 3 with multiple fragmentation; resting cyst with hyaline ridges. Rigidocortex quadrinucleatus mainly differs from the type species R. octonucleatus in having four (vs. eight) macronuclear nodules. Rigidocortex quadrinucleatus can be easily confused with Sterkiella cavicola since both have a rather similar ventral ciliature; however, they can be separated by the slightly higher number of cirri in the left marginal row that runs along the posterior cell's margin in R. quadrinucleatus. Morphogenesis on the ventral surface is highly similar to that of Sterkiella species, but differs significantly on the dorsal surface (multiple vs. simple fragmentation of dorsal kinety 3). Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to S. cavicola.

Two Gonostomatid Ciliates from the Soil of Lombardia, Italy; including Note on the Soil Mapping Project.

Two gonostomatid ciliates, Gonostomum paronense n. sp. and G. strenuum, isolated from the soil sample of paddy field, Lombardia, Italy, were investigated using live observation and protargol impregnation. Gonostomum paronense n. sp. is mainly characterized by a tailed body, frontoventral cirri arranged in pairs, and presence of pretransverse and transverse cirri. Morphologically and morphometrically, the new species is similar to Gonostomum namibiense in having a tailed body and frontoventral cirral pairs; however, it differs mainly in the number of frontoventral cirral pairs (seven vs. three). Phylogenetic analyses based on the SSU rDNA sequences show that the new species is more closely related to G. namibiense than to G. strenuum, supporting the morphological classification based on the cirral pattern and the tailed body. However, due to the poor nodal support and absence of gene sequence of the type species Gonostomum, a more robust phylogeny of this group still remains unresolved. The biometric data of the Italian population of Gonostomum strenuum overlap with those from other known populations. Both species were collected from the industrial area of Parona, in the framework of the "Soil Mapping, Lombardia" project in which, for the first time in Italy, soil ciliates were used as bioindicators of soil quality.

Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy.

The terrestrial oxytrichid ciliate Pseudouroleptus plestiensis n. sp., isolated from soil samples collected from the uplands of Colfiorito (Umbria region, Italy), was investigated using live observation and protargol impregnation. The morphology, morphogenesis and molecular phylogeny inferred from small-subunit (SSU) rRNA gene sequences were studied. The novel species is mainly characterized by the following: a cell size of about 145×35 µm in vivo; two ellipsoidal macronuclear nodules and two to four micronuclei; adoral zone about 26% of body length with a mean of 30 membranelles; about 40 cirri in the right marginal row and 38 in the left marginal row; left fronto-ventral row consisting of about 27-40 cirri, right fronto-ventral row of about three to seven cirri forming a short row to the right of the rear portion of the left fronto-ventral row; one parabuccal cirrus ( = III/2), one buccal and one post-peristomial cirrus; and four dorsal kineties with caudal cirri at the end of kineties 1 and 2. The morphogenesis of the novel species is similar to that of Pseudouroleptus caudatus. Phylogenetic analyses based on SSU rRNA gene sequences consistently placed the novel species within the family Oxytrichidae Ehrenberg, 1838, clustering with P. caudatus and the genus Strongylidium. The results from the present study contribute to the expanding knowledge of the diversity of ciliates in Italian soil.

Morphology, morphogenesis, and molecular phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a "Stylonychine Oxytrichidae" (Ciliophora, Hypotrichida) without transverse cirri.

A terrestrial oxytrichid ciliate Paraparentocirrus sibillinensis n. gen., n. sp., which was found in soil samples of a beech forest stand within the National Park of Sibillini Mountains, Italy, was investigated using live observation and protargol impregnation. The morphology of interphase, morphogenesis, and molecular phylogeny inferred from SSU rDNA sequences of this ciliate were studied. Paraparentocirrus n. gen., is mainly characterized by a semirigid body, an undulating membrane in the Oxytricha pattern, six fronto-ventral (FV) rows, the absence of transverse cirri, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kinety 4. During morphogenesis, oral primordia develop through the proliferation of basal bodies from some cirri of FV rows 4 and 5, and FV row 6 takes part in the anlagen formation of the proter. The dorsal morphogenesis was typical of oxytrichids, with simple fragmentation of dorsal kinety 3, and the dorsomarginal rows developed from the right marginal row. Phylogenetic analyses based on the SSU rDNA sequences support the classification of this new genus in the stylonychines.

Chemical offense by means of toxicysts in the freshwater ciliate, Coleps hirtus.

Coleps hirtus is a small common freshwater ciliate belonging to the protostomatid group, its body covered by calcified plates assembled to form an armor. Coleps feeds on bacteria, algae, flagellates, living and dead ciliates, animal and plant tissues. To assist its carnivorous feeding the ciliate is equipped with offensive extrusomes (toxicysts), clustering mainly in and around its oral aperture. In this study, we isolated the discharge of the toxicysts from living cells, evaluating its cytotoxic effects against various ciliate species, and demonstrating that it is essential for the effectiveness of Coleps' predatory behavior. The analysis of the toxicyst discharge performed by liquid chromatography-electrospray-mass spectrometry and gas chromatography-mass spectrometry, revealed the presence of a mixture of 19 saturated, monounsaturated and polyunsaturated free fatty acids with the addition of a minor amount of a diterpenoid (phytanic acid).

Description of a new oxytrichid ciliate, Oxytricha buxai n. sp. and redescription of O. quadricirrata Blatterer and Foissner, 1988 based on morphology and 18S rDNA analyses.

The morphology of a new oxytrichid ciliate, Oxytricha buxai n. sp., isolated from a soil sample collected from the Buxa Tiger Reserve, West Bengal, India, was studied based on live observation and protargol impregnation. The new species is characterised by a body size of 85×35 µm in vivo, two macronuclear nodules with one or two micronuclei attached at variable positions, a few colourless cortical granules scattered throughout cortex, adoral zone of membranelles about 35% of body length with 26 membranelles on average, about 18 cirri in left and 16 cirri in right marginal row, right marginal row starts at the level of buccal vertex, usually 18 frontoventral transverse cirri, five dorsal kineties including one dorsomarginal row, three caudal cirri. Further, a redescription based on live and protargol-impregnated specimens of Oxytricha quadricirrata Blatterer and Foissner, 1988, isolated from a moss sample collected from the Kangra district, Himachal Pradesh, India, is provided. The Indian population of O. quadricirrata is similar in morphology to the type population. However, the dorsal side shows some variation, i.e., the presence of a second dorsomarginal row with one or two bristles and incomplete fragmentation of dorsal kinety 3 (vs single dorsomarginal row and complete fragmentation). The resting cyst is spherical and about 20 µm across, with a wrinkled surface. Morphogenesis is in typical Oxytricha pattern. Based on 18S rDNA, phylogenetic analyses show Oxytricha to be a polyphyletic genus. Further, O. quadricirrata clusters away from O. granulifera, thereby supporting the validity of the former.

Characterization of a Novel Hypotrich Ciliate From Heavy Metal-Contaminated Industrial Outlet in Onsan, Ulsan, South Korea.

Very few studies exist on the description of protozoan ciliates from industrially contaminated sites. In this study, we report a description of a novel hypotrich ciliate isolated from water samples collected from an industrially contaminated outlet in Onsan, Ulsan, South Korea. The oxytrichid ciliate, Histriculus tolerans n. sp., was investigated using live observation and protargol impregnation. The morphology, morphogenesis, and molecular phylogeny inferred from small-subunit (SSU) rRNA gene sequences were studied. The new species is mainly characterized by a cell size of about 70 × 40 µm in vivo, two elongate ellipsoidal macronuclear nodules and one or two micronuclei, adoral zone of about 51% of body length with 32 membranelles on average, about 34 cirri in the right and 24 cirri in the left marginal row, 18 frontoventral transverse cirri, six dorsal kineties including two dorsomarginal rows, and dorsal kinety 1 with 26 bristles. Morphogenesis is similar to that of the type species, i.e., Histriculus histrio, except that oral primordium does not contribute to anlage II of the proter. Phylogenetic analyses, based on small-subunit rRNA gene sequences, consistently place the new species within the family Oxytrichidae, clustering with H. histrio.

First record of Stenostomum sphagnetorum (Platyhelminthes, Catenulida) from India.

Catenulida are mostly inhabitants of freshwater ecosystems, like ponds, streams, though the marine species are few (Larsson and Willems, 2010). About 110 species of catenulids are known worldwide, with most of the studies conducted in South America (Marcus, 1945a, 1945b; Noreña et al., 2005), North America (Kepner and Carter 1931; Nuttycombe and Waters, 1938) and Scandinavian Peninsula (Luther, 1960, Larsson and Willems, 2010; Larsson et al., 2008). The diversity of catenulids from India has not been studied intensively; however some reports on other turbellaria exists for the country (Annandale, 1912; Whitehouse, 1913; Kapadia, 1947; Basil and Fernando, 1975; Apte and Pitale, 2011; Kalita and Goswami, 2012; Venkataraman et al., 2015). The genus Stenostomum, however, has been studied extensively around the world with identification of over 60 species (Tyler et al., 2006-2016). This is first report of the genus from India. The present study was part of the project to catalogue the diversity of free living protozoan ciliates from the Hooghly stretch of the Ganga River during which the flatworms were found. The worms were studied based on the live observations, with recognition of characters which led to its identification. This study serves to fill knowledge gap in the freshwater flatworms from India.

Morphology and ontogeny of Tetmemena pustulata indica nov. subspec. (Ciliophora, Hypotricha), from the Thane Creek, Mumbai, India.

The morphology and ontogenesis of a novel stylonychid ciliate, Tetmemena pustulata indica nov. subspec., were investigated using live observation and protargol preparation. The new subspecies was isolated from a water sample collected from the Thane Creek, Mumbai, India. At first glance, T. pustulata indica looked very similar in morphology and ontogenesis to the well-known Tetmemena pustulata, however, on detailed investigation some non-overlapping features in the ciliature were identified, i.e., the numbers of cirri in marginal rows, adoral membranelles, and bristles in dorsal kineties. These morphometric differences justified the separation at subspecies level. Furthermore, the resting cysts are different, i.e., with smooth external layer in T. pustulata indica whereas spinous in the nominotypical subspecies T. pustulata pustulata. The Venezuelan population of T. pustulata described by Foissner corresponds very well with the Indian population in the ciliature; however, the information on the resting cyst is lacking for the former. Our study presents an example of a cyst subspecies among hypotrich ciliates, and thus extends the concept which has been mainly reported for spathidiids.

Shedding light on the polyphyletic behavior of the genus Sterkiella: The importance of ontogenetic and molecular phylogenetic approaches.

Present study, investigates a poorly known species of the genus Sterkiella, i.e., S. tricirrata, based on two populations isolated from soil samples collected from the Colfiorito Regional Park, Umbria Region, Italy and from the Silent Valley National Park, India. Both populations showed a highly similar morphology, however different ontogenetic pattern in between. The study confirms the validity of the species S. tricirrata which was considered to be a species within the Sterkiella histriomuscorum complex. The main ontogenetic difference between S. tricirrata and other species of the genus Sterkiella is the different mode of formation of anlagen V and VI of the proter in the former. In the phylogenetic analyses, Sterkiella tricirrata clusters with Sterkiella sinica within the stylonychine oxytrichids, in a clade away from the type species (Sterkiella cavicola) of the genus Sterkiella. The study highlights the importance of ontogenetic as well as molecular data in shedding light on the polyphyletic behavior of the genus Sterkiella. A detailed description of S. tricirrata based on morphology, ontogenesis and molecular phylogenetic methods is presented. Further, the improved diagnosis has been provided for the genus Sterkiella and the poorly known species S. tricirrata.

Discovery of a new hypotrich ciliate from petroleum contaminated soil.

Pollution after oil spill represents extreme habitat for survival and is a major concern for loss of species diversity in the affected area. In this study, we investigated soil samples collected from a petrochemical industry, Ulsan, South Korea. The soil was in the phase of recovery from the contamination of crude oil spill. Detailed investigation, based on morphology, ontogenesis, and molecular phylogenetic methods, resulted in discovery of a novel hypotrich ciliate, i.e., Metasterkiella koreana n. gen., n. sp., which is morphologically characterized by a semirigid body, undulating membranes in Oxytricha pattern, 18 frontal-ventral-transverse cirri with cirrus V/3 placed posteriorly, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kineties 1, 2, and 4. Interestingly, during ontogenesis, formation of three common anlagen for the proter and the opisthe and involvement of cirrus V/3 in anlagen formation was observed. The dorsal ontogenesis was typical of oxytrichids, i.e., simple fragmentation of dorsal kinety 3 and formation of dorsomarginal rows close to the right marginal row. The new species was found to be similar with Sterkiella subtropica, except for some minor differences in morphometry, and at gene level with only one base pair difference. In phylogenetic analyses, based on SSU rRNA gene sequence, M. koreana cluster in a clade away from Sterkiella species, which could be explained by the differences in the morphogenetic pattern between these two genera. It is proposed that S. subtropica probably belongs to Metasterkiella; however, we do not perform changes and wait for the reinvestigation of its morphogenetic pattern.

Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy.

The morphology of Rigidosticha italiensis n. gen., n. sp., which was found in a soil sample collected from an uncultivated field in Lombardia, Italy, was investigated using live observation and protargol staining. Rigidosticha n. gen. is characterised by a rigid body, undulating membranes resembling a Steinia pattern, oxytrichid frontal ciliature, distinct mid-ventral cirral pairs, transverse cirri, one right and one left row of marginal cirri, absence of dorsal kinety 3 fragmentation, more than two dorsomarginal rows, and caudal cirri. The new species shows the following features: size in vivo 230-330×100-170µm, on average 230×115µm in protargol preparations; two ellipsoidal macronuclear nodules; 51 adoral membranelles; one buccal cirrus; one parabuccal cirrus; two frontoterminal cirri; 16 mid-ventral cirral pairs; and three transverse cirri. Rigidosticha mainly differs from Rigidothrix, Afrophrya, Uroleptus, and Territricha, in having the undulating membranes in Rigidosticha (vs. oxytrichid and cyrtohymenid) pattern. The oxytrichid frontal ciliature and midventral pattern in the present species further support the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis.

Fine-tune investigations on three stylonychid (Ciliophora, Hypotricha) ciliates.

The morphology and morphogenesis of two stylonychid ciliates, Stylonychia ammermanniGupta et al., 2001 and Tetmemena bifaria (Stokes, 1887) Berger, 2001, isolated from soil samples of Lombardia region, Northern Italy, and water samples from Cheonggancheon stream, South Korea, were investigated. The Italian population of S. ammermanni was found to be very similar in morphology to the Indian type population and the junior synonym S. harbinensisShi and Ammermann, 2004 since most of the morphometric data overlapped. On the contrary, the Korean population of S. ammermanni showed some non-overlapping differences in ciliature suggesting a separation at subspecies level, i.e., S. ammermanni ammermanni and S. ammermanni koreana nov. subspec. Furthermore, the resting cyst of the Italian population of S. ammermanni has many ring-shaped structures on the surface which, however, were not observed in the Korean population. Phylogenetic analyses based on the SSU rRNA gene sequences show that the Italian and Korean populations of S. ammermanni fit well into the S. mytilus complex and moderately support the subspecies separation. Additionally, we split Tetmemena bifaria into two subspecies, viz., T. bifaria bifaria and T. bifaria minima nov. subspec. based on differences in the number of adoral membranelles and number of cirri in the marginal rows of the Italian and the Argentinian populations in comparison with the populations described by Wirnsberger et al. (1985).

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India.

The morphology and morphogenesis during cell division of Sterkiella tetracirrata n. sp., isolated from a soil sample collected from the Silent Valley National Park, Kerala, India, were investigated using live observation, protargol staining and scanning electron microscopy. The new species differs from its congeners by the following combination of features: cell size in vivo 85-110×35-50µm, on average 84×37µm in protargol preparations; four ellipsoidal macronuclear nodules; 31 adoral membranelles; 17 frontal-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse and invariably four transverse cirri; resting cyst with separate macronuclear nodules. Sterkiella tetracirrata differs from the similar species S. terricola in the number of transverse cirri (invariably 4 vs. 3) and in the number of adoral membranelles (24-35 vs. 22 or 23). Morphogenesis resembles that of its congeners S. nova and S. histriomuscorum. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to either S. cavicola or S. histriomuscorum. The analyses support the morphological evidence (e.g., similarity in the oral apparatus and the dorsal kinety pattern) that Gastrostyla and Pattersoniella evolved from a Sterkiella-like ancestor.