Phylogeny and species delimitation of ciliates in the genus Spirostomum (Class, Heterotrichea) using single-cell transcriptomes.

Ciliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages over a billion years ago. The extensive evolutionary timespan of ciliate has led to enormous genetic and phenotypic changes, contributing significantly to their high level of diversity. Recent analyses based on molecular data have revealed numerous cases of cryptic species complexes in different ciliate lineages, demonstrating the need for a robust approach to delimit species boundaries and elucidate phylogenetic relationships. Heterotrich ciliate species of the genus Spirostomum are abundant in freshwater and brackish environments and are commonly used as biological indicators for assessing water quality. However, some Spirostomum species are difficult to identify due to a lack of distinguishable morphological characteristics, and the existence of cryptic species in this genus remains largely unexplored. Previous phylogenetic studies have focused on only a few loci, namely the ribosomal RNA genes, alpha-tubulin, and mitochondrial CO1. In this study, we obtained single-cell transcriptome of 25 Spirostomum species populations (representing six morphospecies) sampled from South Korea and the USA, and used concatenation- and coalescent-based methods for species tree inference and delimitation. Phylogenomic analysis of 37 Spirostomum populations and 265 protein-coding genes provided a robustious insight into the evolutionary relationships among Spirostomum species and confirmed that species with moniliform and compact macronucleus each form a distinct monophyletic lineage. Furthermore, the multispecies coalescent (MSC) model suggests that there are at least nine cryptic species in the Spirostomum genus, three in S. minus, two in S. ambiguum, S. subtilis, and S. teres each. Overall, our fine sampling of closely related Spirostomum populations and wide scRNA-seq allowed us to demonstrate the hidden crypticity of species within the genus Spirostomum, and to resolve and provide much stronger support than hitherto to the phylogeny of this important ciliate genus.

Codon Usage Bias Analysis in Macronuclear Genomes of Ciliated Protozoa.

Ciliated protozoa (ciliates) are unicellular eukaryotes, several of which are important model organisms for molecular biology research. Analyses of codon usage bias (CUB) of the macronuclear (MAC) genome of ciliates can promote a better understanding of the genetic mode and evolutionary history of these organisms and help optimize codons to improve gene editing efficiency in model ciliates. In this study, the following indices were calculated: the guanine-cytosine (GC) content, the frequency of the nucleotides at the third position of codons (T3, C3, A3, G3), the effective number of codons (ENc), GC content at the 3rd position of synonymous codons (GC3s), and the relative synonymous codon usage (RSCU). Parity rule 2 plot analysis, Neutrality plot analysis, ENc plot analysis, and correlation analysis were employed to explore the main influencing factors of CUB. The results showed that the GC content in the MAC genomes of each of 21 ciliate species, the genomes of which were relatively complete, was lower than 50%, and the base compositions of GC and GC3s were markedly distinct. Synonymous codon analysis revealed that the codons in most of the 21 ciliates ended with A or T and four codons were the general putative optimal codons. Collectively, our results indicated that most of the ciliates investigated preferred using the codons with anof AT-ending and that codon usage bias was affected by gene mutation and natural selection.

The widely reported but poorly studied ciliate family Folliculinidae (Protozoa, Ciliophora, Heterotrichea): a revision with notes on its taxonomy, morphology and phylogenetic relationships.

Ciliates of the heterotrich family Folliculinidae are widespread in various habitats and are distinguished by their transparent loricae of various shapes, conspicuous peristomial lobes, and dimorphic life cycles. They usually attach firmly to the surface of substrates, feed on bacteria and microalgae, and play a significant role in energy flow and material cycling in the microbial food web. However, little is known regarding their biodiversity and systematics. In this work, we establish the terminology of the family Folliculinidae and select six crucial features for genus recognition. Based on previous studies, we revise the classification of Folliculinidae, supply improved diagnoses for each of the 33 folliculinid genera, and provide a key to their identification. Moreover, phylogenetic analyses based on small subunit ribosomal DNA (SSU rDNA) sequences revealed that the family is monophyletic and comprises two subclades (subclades I II) which can be identified by the flexibility of their peristomial lobes and the sculpturing of their necks. Furthermore, we investigate the evolutionary relationships of folliculinids using the six chosen generic features. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00152-z.

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.

Overview of the Diversity, Phylogeny and Biogeography of Strombidiid Oligotrich Ciliates (Protista, Ciliophora), With a Brief Revision and a Key to the Known Genera.

Strombidiids are common free-living ciliates that have colonized coastal and open oceanic waters across the world. In recent years, numerous new taxa and gene sequences of strombidiids have been reported, revealing a large diversity of both their morphologic and genetic features. Here, we compare the taxonomic characters of all genera in the family Strombidiidae, provide a key to their identification, and investigate their molecular phylogeny. In addition, we analyze their regional distribution based on faunal data accumulated in China and attempt to infer their global distribution based on SSU rRNA gene sequence data. The current work revises the systematics of strombidiids based on morphologic, phylogenetic, and biogeographic evidence and provides a genus-level review of marine strombidiids.

Progress in studies on the diversity and distribution of planktonic ciliates (Protista, Ciliophora) in the South China Sea.

As an important component of microzooplankton, ciliates play a key role in matter cycling and energy flow in marine planktonic ecosystems. Studies of planktonic ciliate have been extensive in the South China Sea (SCS) over the last 20 years. Here, we summarize the recent progress on the diversity and distribution of this group in the SCS. This includes that in: (1) the waters covering the intertidal zone of the northern SCS, most studies have focused on taxonomy, with 71 species collected, identified, and described (with ~ 40% new species); (2) neritic waters distribution patterns have been examined at a regional scale, with ciliates displaying significant spatial variations and seasonal dynamics; (3) in oceanic waters, there has been a focus on ciliate distribution in north, centre, and south regions, where mesoscale physical processes play roles in controlling distributions, and noticeable vertical variations occur. More generally, some studies examine the influences of environment variables on ciliates, and indicate that chlorophyll a concentration is commonly positively correlated with ciliates abundance. In addition, some significant findings are summarized, the limitations of past studies are considered, and recommendations are made for future work on planktonic ciliates in SCS.

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.

Pseudaeromonas paramecii sp. nov., isolated from the ciliate Paramecium caudatum and emendation of the genus Pseudaeromonas.

A Gram-stain-negative, oxidase-negative and catalase-negative, motile, rod-shaped bacterial strain, designated PCS8T, hosted by the ciliate Paramecium caudatum was investigated by using a polyphasic approach. Strain PCS8T was observed to be able to grow at 12-44 °C (optimum, 36-37 °C), at pH 6.0-10.0 (optimum, 7.0) and in the presence of 0-3 % NaCl (optimum, 1-2 %). It could hydrolyse starch and aesculin and produce acid from d-sorbitol, myo-inositol, glycerol and l-rhamnose. The sequence similarity of the new isolate was 96.9 % with respect to Pseudaeromonas pectinilytica and 96.3 % with respect to Pseudaeromonas sharmana. Phylogenetically, strain PCS8T falls within the cluster comprising the Pseudaeromonas species. The predominant cellular fatty acids of strain PCS8T were C16 : 0 (35.8 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 35.1 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 10.8 %). This novel strain also contained various fatty acids that are not detected in other members of the genus Pseudaeromonas, such as C16 : 0 3-OH, C18 : 1ω9c and summed feature 5 (C18 : 2ω6,9c and/or C18 : 0 ante). Strain PCS8T contained ubiquinone-8 as the sole respiratory quinone and phosphatidylethanolamine and phosphatidylglycerol as major polar lipids. The G+C content of the genomic DNA of the type strain was 66.5 mol%. Based on the distinct phenotypic, phylogenetic, chemotaxonomic and G+C content results, strain PCS8T represents a currently undescribed species within the genus Pseudaeromonas in the family Aeromonadaceae, for which we suggest the name Pseudaeromonas paramecii sp. nov. with the type strain PCS8T (=KCTC 62038T=JCM 32226T). An emended description of the genus Pseudaeromonas is also provided.

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.

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.

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).

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.

An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates.

Peritrichs are a major group of ciliates with worldwide distribution. Yet, its internal phylogeny remains unresolved owing to limited sampling. Additionally, ecological distributions of peritrichs are poorly known. We performed substantially expanded phylogenetic analyses of peritrichs that incorporated SSU rDNA sequences of samples collected from three continents, revealing a number of new relationships between and within major lineages that greatly challenged the classic view of the group. Interrogation of a dataset comprising new environmental sequences from an estuary and the open ocean generated with high throughput sequencing and clone libraries plus putative environmental peritrich sequences at Genbank, produced a comprehensive tree of peritrichs from a variety of habitats and revealed unique ecological distribution patterns of several lineages for the first time. Also, evidence of adaptation to extreme environments in the Astylozoidae clade greatly broadened the phylogenetic range of peritrichs capable of living in extreme environments. Reconstruction of ancestral states revealed that peritrichs may have transitioned repeatedly from freshwater to brackish/marine/hypersaline environments. This work establishes a phylogenetic framework for more mature investigations of peritrichs in the future, and the approach used here provides a model of how to elucidate evolution in the context of ecological niches in any lineage of microbial eukaryotes.

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%.