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.

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.

Morphological Redescriptions and Molecular Phylogeny of Three Stentor Species (Ciliophora: Heterotrichea: Stentoridae) from Korea.

The morphologies of the three freshwater stentorid ciliates in Korea, Stentor coeruleus (Pallas, 1766); Stentor muelleri Ehrenberg, 1831, and Stentor tartari Murthy Bai, 1974, were investigated based on live observations and protargol impregnation. The Korean population of S. tartari exhibits the following characteristics: body size 200-355 × 85-135 µm in vivo, 62-106 somatic kineties, 8-13 peristomial kineties, 110-180 adoral membranelles, mostly two macronuclear nodules and 5-18 micronuclei, reddish and colorless cortical granules and the presence of symbiotic algae. We identified S. tartari based on unique characteristics compared to close congeners. Korean populations of S. coeruleus and S. muelleri are congruent with previously described populations in most aspects of their morphologies. Here, for the first time, we report molecular gene sequence information for S. tartari. Small subunit (SSU) rRNA gene sequence-based phylogeny indicates that S. tartari, which has multiple macronuclei, forms a monophyletic group with other Stentor species having a single macronucleus. Our findings based on morphology and SSU rRNA gene sequence information corroborate the hypothesis that the elongated macronucleus evolved from the compact single or multi macronucleus state.

Segnochrobactrum spirostomi gen. nov., sp. nov., isolated from the ciliate Spirostomum yagiui and description of a novel family, Segnochrobactraceae fam. nov. within the order Rhizobiales of the class Alphaproteobacteria.

A bacterial strain, designated Sp-1T, was isolated from the heterotrich ciliate Spirostomum yagiui collected from a reservoir located in Ulsan, Republic of Korea. Cells of Sp-1T were Gram stain-negative, rod-shaped, non-spore-forming, non-motile and contained poly-ß-hydroxybutyrate granules. Phylogenetic analyses based on 16S rRNA gene sequences indicated that Sp-1T constituted a distinct phylogenetic lineage within different families in the order Rhizobiales with a pairwise sequence similarity of 95 % to the species of the genus Ochrobactrum: Ochrobactrum anthropi ATCC 49188T and Ochrobactrum cytisi ESC1T (family Brucellaceae). The major cellular fatty acids were C19 : 0 cyclo ω8c (44.4 %) and C16 : 0 (32.1 %). The identified sole isoprenoid quinone was ubiquinone-10 (Q-10). The major polar lipids produced were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, two unidentified phospholipids and three unidentified lipids. The genome size was about 5.4 Mbp and the DNA G+C content was 68.2 mol%. Sp-1T exhibited the highest average nucleotide identity value of 76.6 % and in silico DNA-DNA hybridization value of 22.1 % with Pseudoxanthobacter soli DSM 19599T (family Xanthobacteraeae). This strain is distinguishable from closely related members of the order Rhizobiales by its differential phenotypic, chemotaxonomic, genomic and phylogenetic characteristics. On the basis of evidence from polyphasic taxonomic analysis, we concluded that Sp-1T represents a novel species in a novel genus within the order Rhizobiales, for which the name Segnochrobactrum spirostomi gen. nov., sp. nov. is proposed. The type strain is Sp-1T (=KCTC 62036T=JCM 32162T). We also describe a novel family, Segnochrobactraceae fam. nov., to encompass the proposed novel genus and species.

Deciphering phylogenetic relationships and delimiting species boundaries using a Bayesian coalescent approach in protists: A case study of the ciliate genus Spirostomum (Ciliophora, Heterotrichea).

The ciliate genus Spirostomum comprises eight morphospecies, inhabiting diverse aquatic environments worldwide, where they can be used as water quality indicators. Although Spirostomum species are relatively easily identified using morphological methods, the previous nuclear rDNA-based phylogenies indicated several conflicts in morphospecies delineation. Moreover, the single locus phylogenies and previous analytical approaches could not unambiguously resolve phylogenetic relationships among Spirostomum morphospecies. Here, we attempt to investigate species boundaries and evolutionary history of Spirostomum taxa, using 166 new sequences from multiple populations employing one mitochondrial locus (CO1 gene) and two nuclear loci (rRNA operon and alpha-tubulin gene). In accordance with previous studies, relationships among the eight Spirostomum morphospecies were poorly supported statistically in individual gene trees. To overcome this problem, we utilised for the first time in ciliates the Bayesian coalescent approach, which accounts for ancestral polymorphisms, incomplete lineage sorting, and recombination. This strategy enabled us to robustly resolve deep relationships between Spirostomum species and to support the hypothesis that taxa with compact macronucleus and taxa with moniliform macronucleus each form a distinct lineage. Bayesian coalescent-based delimitation analyses strongly statistically supported the traditional morphospecies concept but also indicated that there are two S. minus-like cryptic species and S. teres is non-monophyletic. Spirostomum teres was very likely defined by a set of ancestral features of lineages that also gave rise to S. yagiui and S. dharwarensis. However, molecular data from type populations of the morphospecies S. minus and S. teres are required to unambiguously resolve the taxonomic problems.

New contributions to Gruberia lanceolata (Gruber, 1884) Kahl, 1932 based on analyses of multiple populations and genes (Ciliophora, Heterotrichea, Gruberiidae).

Gruberia Kahl, 1932 is a species-poor genus comprising only seven named species. Most of these species have not been reinvestigated since the original reports. In the present work, we investigated the taxonomy and phylogeny of Gruberia lanceolata (Gruber, 1884) Kahl, 1932 based on analyses of morphology and multiple gene sequences from four South Korean populations. This species is mainly characterized by a well-developed peristome region, segmented paroral membrane, and moniliform macronucleus. Some morphological features were not stable among the four populations investigated, such as body shape and size, cell color, and the ratio of oral length to body length. However, our molecular analyses of four different genetic markers - three nuclear DNA markers (18S rDNA, ITS1-5.8S-ITS2 region, D1D2 of 28S rDNA) and one mitochondrial (mt) marker (CO1 gene) - indicated that all Korean populations examined were the same species. Based on our present findings and historic works, we propose that G. calkinsi, G. aculeata, and G. beninensis are junior synonyms of G. lanceolata.

Diversification dynamics of rhynchostomatian ciliates: the impact of seven intrinsic traits on speciation and extinction in a microbial group.

Ciliates are a suitable microbial model to investigate trait-dependent diversification because of their comparatively complex morphology and high diversity. We examined the impact of seven intrinsic traits on speciation, extinction, and net-diversification of rhynchostomatians, a group of comparatively large, predatory ciliates with proboscis carrying a dorsal brush (sensoric structure) and toxicysts (organelles used to kill the prey). Bayesian estimates under the binary-state speciation and extinction model indicate that two types of extrusomes and two-rowed dorsal brush raise diversification through decreasing extinction. On the other hand, the higher number of contractile vacuoles and their dorsal location likely increase diversification via elevating speciation rate. Particular nuclear characteristics, however, do not significantly differ in their diversification rates and hence lineages with various macronuclear patterns and number of micronuclei have similar probabilities to generate new species. Likelihood-based quantitative state diversification analyses suggest that rhynchostomatians conform to Cope's rule in that their diversity linearly grows with increasing body length and relative length of the proboscis. Comparison with other litostomatean ciliates indicates that rhynchostomatians are not among the cladogenically most successful lineages and their survival over several hundred million years could be associated with their comparatively large and complex bodies that reduce the risk of extinction.

Morphology and molecular phylogeny of three heterotrichid species (Ciliophora, Heterotrichea), including a new species of Anigsteinia.

Heterotrichs are generally larger than ciliates of other groups with a seemingly cosmopolitan distribution, and recent studies have demonstrated that they exhibit great biodiversity. In the present work, we investigated the morphology and small subunit rRNA (SSU rRNA) gene sequences of three heterotrichous species, including that of a new one, Anigsteinia paraclarissima spec. nov. The new organism is morphologically very similar to A. clarissima, however, it can be distinguished from the latter by the larger dimensions and more somatic kineties (25-32 vs. 18-26), and its sequence similarity of SSU rRNA gene is 97.14% which indicate that it is a distinct species. Detailed morphological and molecular data for Blepharisma bimicronucleatum are supplied together for the first time in this study. In addition, the morphology of a poorly known species, Spirostomum yagiui is redescribed and an improved species diagnosis is provided. Finally, based on phylogenetic analyses of SSU rRNA gene sequences data, the Spirostomum group contains two main clades based on the type of the macronucleus. Spirostomum yagiui was newly sequenced here and clustered with the other S. yagiui populations and positioned within the Spirostomum assemblage. The Anigsteinia clade, including A. paraclarissima and A. clarissima, clustered within the family Spirostomidae.

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.

Molecular phylogeny and species delimitation within the ciliate genus Spirostomum (Ciliophora, Postciliodesmatophora, Heterotrichea), using the internal transcribed spacer region.

Morphological and molecular delimitation of Spirostomum species is currently under debate. We addressed species boundaries within the genus Spirostomum, using the ITS1-5.8S-ITS2 region and the secondary structure of the ITS2 molecule, and 18S and 28S (D1D2) sequences additionally. The Spirostomum ITS region is among the shortest within the ciliates hitherto studied. The Spirostomum ITS2 molecule matches the "ring model", but exhibits only two helices radiating from a common loop. According to comparative analyses, they very likely correspond to helices II and III of other eukaryotes. Our phylogenetic analyses of the ITS region revealed a complex genealogical structure within the genus Spirostomum. However, boundaries among Spirostomum species could not be unambiguously determined either by phylogenetic trees, networks or sequence divergence cutoffs, because ITS2 sequences transcended species boundaries of the following morphospecies: S. ambiguum, S. minus, S. subtilis and S. teres. According to molecular diversity analysis, this is very likely caused by polymorphism in S. minus and S. teres, and by the lack of variability in S. ambiguum and S. subtilis. No compensatory base changes (CBCs) were detected in helices of the ITS2 molecule between different Spirostomum species, documenting that CBC analysis per se is not able to effectively discriminate Spirostomum species.

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%.

Two New Species of Zoothamnium (Ciliophora, Peritrichia) from Korea, with New Observations of Z. parahentscheli Sun et al., 2009.

Three peritrichous ciliates, Zoothamnium arcuatum n. sp., Z. grossi n. sp., and Z. parahentscheli Sun et al., 2009, were collected from an estuary of the Taehwagang River, Korea. All these species were investigated based on live observations and silver staining, and their small subunit (SSU) rRNA gene was also sequenced. Zoothamnium arcuatum can be identified by a goblet-shaped colony, double-layered peristomial lip, and abstomally shortened row 3 of infundibular polykinety 3 (P3). Zoothamnium grossi is morphologically characterized by an alternately branched stalk with the lowest secondary stalk diverging from the main part of colony, asymmetrically bell-shaped zooids, and three short, parallel ciliary rows in P3. Phylogenetic analysis revealed that the three Zoothamnium species described in this paper clustered with other members of the family Zoothamniidae, as expected.

Morphological and molecular characterization of the name-bearing type species Rimaleptus binucleatus (Kahl, 1931), with a phylogenetic re-analysis of dileptid evolutionary history (Ciliophora: Litostomatea: Rhynchostomatia).

We collected Rimaleptus binucleatus from soil in the surroundings of the town of Ulsan, South Korea. Its morphology and 18S rRNA gene were studied using standard methods. This possibly widely distributed species is characterized by: (i) a size of about 170-400×20-65µm; (ii) a narrowly to cylindrically dileptid body with proboscis occupying about 30-65% of body length; (iii) two dorsal contractile vacuoles; (iv) two size groups of rod-shaped extrusomes; and (v) about 18-29 ciliary rows, 4-6 of them anteriorly differentiated into a staggered dorsal brush. Phylogenetic analyses of five new rhynchostomatian 18S rRNA gene sequences supported monophylies of the orders Tracheliida and Dileptida, but revealed that the genera Rimaleptus and Pseudomonilicaryon are polyphyletic. Monophyly of genera with two macronuclear nodules was consistently rejected, but monophylies of dileptids with many scattered macronuclear nodules and of dileptids with moniliform macronucleus and multi-rowed dorsal brush could not be excluded by statistical topology tests. Nevertheless, phylogenetic network analyses indicated considerable conflict in the phylogenetic signal provided by the 18S rRNA gene to resolve unambiguously relationships among dileptid genera.

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.

Phylogenetic relationships of the ciliate class Heterotrichea (Protista, Ciliophora, Postciliodesmatophora) inferred from multiple molecular markers and multifaceted analysis strategy.

The ciliate class Heterotrichea is defined by somatic dikinetids bearing postciliodesmata, by an oral apparatus consisting of a paroral membrane and an adoral zone of membranelles, as well as by features of nuclear division involving extramacronuclear microtubules. Although phylogenetic interrelationships among heterotrichs have been analyzed several times, deeper nodes of the heterotrichean tree of life remain poorly resolved. To cast more light on the evolutionary history of heterotricheans, we performed phylogenetic analyses of multiple loci (18S rRNA gene, ITS1-5.8S rRNA-ITS2 region, and 28S rRNA gene) using traditional tree-building phylogenetic methods and statistical tree topology tests as well as phylogenetic networks, split spectrum analysis and quartet likelihood mapping. This multifaceted approach has shown that (1) Peritromus is very likely an adelphotaxon of all other heterotrichs; (2) Spirostomum and Anigsteinia are sister taxa and their common monophyletic origin is strongly supported by a uniquely posteriorly-thickened paroral membrane; (3) the monotypic family Chattonidiidae should be suppressed because its type genus clusters within the family Condylostomatidae; and (4) new families are needed for Gruberia and Fabrea because their affiliation with Spirostomidae and Climacostomidae, respectively, is not supported by molecular phylogenies nor the fine structure of the paroral membrane.

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.