Highly divergent morphology but a close molecular phylogenetic relationship between two little-known ciliate genera Actinobolina and Papillorhabdos (Protozoa: Ciliophora: Litostomatea) with description of two new species.

Free-living litostomatean ciliates, prominent microeukaryote predators commonly encountered in freshwater and marine habitats, play vital roles in maintaining energy flow and nutrient cycles. Nevertheless, understanding their biodiversity and phylogenetic relationships remains challenging due to insufficient morphological information and molecular data. As a new contribution to this group, three haptorian ciliates, including two new species (Actinobolina bivacuolata sp. nov. and Papillorhabdos foissneri sp. nov.) and the insufficiently described type species, Actinobolina radians, were isolated from wetlands around Lake Weishan, China and investigated by a combination of living morphology, stained preparations, and 18S rRNA gene sequence data. An illustrated key of the valid species within the two genera is provided. In addition, we reveal the phylogenetic positions of these two genera for the first time. Although they differ in all key morphologic characters such as general appearance (ellipsoidal with numerous tentacles vs. cylindrical), extrusomes (stored in tentacles vs. anchored to pellicle), circumoral kinety (present vs. absent), composition of somatic kineties (kinetosome clusters vs. monokinetids), and number of dorsal brush rows (1 vs. 4), they both cluster in a fully supported clade in the phylogenetic tree, which indicates that the biodiversity and additional molecular markers of this group need further exploration.

New evidence of consistency between phylogeny and morphology for two taxa in ciliated protists, the subclasses Oligotrichia and Choreotrichia (Protista, Ciliophora).

Marine planktonic ciliates are largely oligotrichs and choreotrichs, which are two subclasses of the class Spirotrichea. The current phylogenetic assignments of oligotrichs and choreotrichs are inconsistent with previous results based on morphological features, probably hindered by the limited information from a single gene locus. Here we provide 53 new sequences from small subunit ribosomal RNA (SSU rDNA), ITS1-5.8S rDNA-ITS2, and large subunit ribosomal RNA (LSU rDNA) gene loci in 25 oligotrich and choreotrich species. We also predict RNA secondary structures for the ITS2 regions in 55 species, 48 species of which are reported for the first time. Based on these novel data, we make a more comprehensive phylogenetic reconstruction, revealing consistency between morphological taxonomy and an updated phylogenetic system for oligotrichs and choreotrichs. With the addition of data from ciliature patterns and genes, the phylogenetic analysis of the subclass Oligotrichia suggests three evolutionary trajectories, among which: 1) Novistrombidium asserts an ancestral ciliary pattern in Oligotrichia; 2) the subgenera division of Novistrombidium and Parallelostrombidium are fully supported; 3) the three families (Tontoniidae, Pelagostrombidiidae and Cyrtostrombidiidae) all evolved from the most diverse family Strombidiidae, which explains why strombidiids consistently form polyphyletic clades. In the subclass Choreotrichia, Strombidinopsis likely possesses an ancestral position to other choreotrichs, and both phylogenetic analysis and RNA secondary structure prediction support the hypothesis that tintinnids may have evolved from Strombidinopsis. The results presented here offer an updated hypothesis for the evolutionary history of oligotrichs and choreotrichs based on new evidence obtained by expanding sampling of molecular information across multiple gene loci.

High salinity gradients and intermediate spatial scales shaped similar biogeographical and co-occurrence patterns of microeukaryotes in a tropical freshwater-saltwater ecosystem.

Microeukaryotes play key ecological roles in the microbial web of aquatic ecosystems. However, large knowledge gaps urgently need to be filled regarding the biogeography with associated shaping mechanisms and co-occurrence patterns of microeukaryotes under freshwater-saltwater gradients, especially true in tropical regions. Here, we investigated microeukaryotes of six mixed freshwater-saltwater regions in the Pearl River Estuary and surrounding coasts in southern China, with salinity ranging 0.1-32.0% and distances spanned up to 500 km, using molecular ecological methods. Results indicate that the biogeography of abundant and rare microeukaryotic communities was similar, both their co-occurrence patterns and biogeographical patterns were driven by deterministic and stochastic processes. The environmental factors with higher selective pressure than dispersal limitation meant that the role of deterministic process in structuring communities was more significant than that of stochastic process, and salinity played important role in structuring both microeukaryotic communities and networks. The abundant communities had stronger influence on entire microeukaryotic communities and seemed to be more sensitive to environmental changes than their rare counterparts, while rare ones had stronger interspecific relationships. Finally, the geographic scale and environmental gradients of study regions should firstly be clarified in future research on the ecological processes of microeukaryotes before conclusions are drawn.

Large-scale phylogenomic analysis provides new insights into the phylogeny of the class Oligohymenophorea (Protista, Ciliophora) with establishment of a new subclass Urocentria nov. subcl.

The class Oligohymenophorea is one of the most diverse assemblage of ciliated protists, which are particularly important in fundamental biological studies including understanding the evolutionary relationships among the lineages. Phylogenetic relationships within the class remain largely elusive, especially within the subclass Peniculia, which contains the long-standing problematic taxa Urocentrum and Paranassula. In the present study, we sequenced the genomes and/or transcriptomes of six non-culturable oligohymenophoreans using single-cell sequencing techniques. Phylogenomic analysis was performed based on expanded taxon sampling of 85 taxa, including 157 nuclear genes encoding 36,953 amino acids. The results indicate that: (1) urocentrids form an independent branch that is sister to the clade formed by Scuticociliatia and Hymenostomatia, which, together with the morphological data, supports the establishment of a new subclass, Urocentria n. subcl., within Oligohymenophorea; (2) phylogenomic analysis and ortholog comparison reveal a close relationship between Paranassula and peniculines, providing corroborative evidence for removing Paranassula from Nassulida and elevating it as an order, Paranassulida, within the subclass Peniculia; (3) based on the phylogenomic analyses and morphological data, we hypothesize that Peritrichia is the earliest diverging clade within Oligohymenophorea while Scuticociliatia and Hymenostomatia share the most common ancestor, followed successively by Urocentria and Peniculia. In addition, stop codon analyses indicate that oligohymenophoreans widely use UGA as the stop codon, while UAR are reassigned to glutamate (peritrichs) or glutamine (others), supporting the evolutionary hypothesis.

Redescription and SSU rRNA gene-based phylogeny of an anaerobic ciliate, Plagiopyla ovata Kahl, 1931 (Ciliophora, Plagiopylea).

The morphology and molecular phylogeny of Plagiopyla ovata Kahl, 1931, a poorly known anaerobic ciliate, were investigated based on a population isolated from sand samples collected from the Yellow Sea coast at Qingdao, PR China. Details of the oral ciliature are documented for the first time to our knowledge and an improved species diagnosis is given. The small subunit ribosomal RNA (SSU rRNA) gene was newly sequenced and phylogenetic analyses revealed that P. ovata clusters within the monophyletic family Plagiopylidae. However, evolutionary relationships within both the family Plagiopylidae and the genus Plagiopyla remain obscure owing to undersampling, the lack of sequence data from known species and low nodal support or unstable topologies in gene trees. A key to the identification of the species of the genus Plagiopyla with validly published names is also supplied.

The insights into the systematic relationship of Gastrostyla-affinitive genera, with report on a new saline soil ciliate genus and new species (Protozoa, Ciliophora).

BACKGROUND: Hypotrichia are a group with the most complex morphology and morphogenesis within the ciliated protists. The classification of Gastrostyla-like species, a taxonomically difficult group of hypotrichs with a common ventral cirral pattern but various dorsal and ontogenetic patterns, is poorly understood. Hence, systematic relationships within this group and with other taxa in the subclass Hypotrichia remain unresolved. RESULTS: 18S rRNA gene sequence of a new Gastrostyla-like taxon was obtained. Phylogenetic analyses based on the 18S rRNA gene sequences indicate that this ciliate represents a new genus that is closely related to Heterourosomoida and Kleinstyla within the oxytrichid clade of the Hypotrichia. However, the position of this cluster remains unresolved. All three genera deviate from the typical oxytrichids by their incomplete (or lack of) dorsal kinety fragmentation during morphogenesis. Morphology and morphogenesis of this newly discovered form, Heterogastrostyla salina nov. gen., nov. spec., are described. Heterogastrostyla nov. gen., is characterised as follows: more than 18 fronto-ventral-transverse cirri, cirral anlagen V and VI develop pretransverse cirri, and dorsal ciliature in Urosomoida-like pattern. CONCLUSIONS: Similar to the CEUU-hypothesis about convergent evolution of urostylids and uroleptids, we speculate that the shared ventral cirral patterns of Gastrostyla-like taxa might have resulted from convergent evolution.

Morphology and Molecular Phylogeny of Two Freshwater Oligotrich Ciliates (Protozoa, Ciliophora, Oligotrichia), Pelagostrombidium fallax (Zacharias, 1895) Krainer, 1991 and Limnostrombidium viride (Stein, 1867) Krainer, 1995, with Brief Notes on Stomatogenesis.

The morphology of two oligotrich ciliates, Pelagostrombidium fallax (Zacharias, 1895) Krainer, 1991 and Limnostrombidium viride (Stein, 1867) Krainer, 1995, collected from a freshwater pond in northern China, was studied based on live observation and protargol staining. Currently, undescribed features in the girdle kinety in P. fallax are (a) sparsely spaced single argyrophilic basal bodies in the shoulder region and (b) a U-shape formed below the buccal peristome. An improved diagnosis for P. fallax is supplied, based on previous and present morphological descriptions. In addition, certain ontogenetic stages of P. fallax are reported for the first time. During division, two new embryonic bodies are successively generated de novo, the first one goes to the proter, the second one to the opisthe; the new canal derives from the old canal. The morphology of L. viride is redescribed in detail; the Chinese population is highly consistent with populations in Europe. The SSU rRNA gene of P. fallax was sequenced for the first time. Phylogenetic analyses, based on SSU rRNA gene sequence data, reveal that P. fallax and L. viride cluster with Strombidium species while the result of an AU test did not reject the possibility of monophyly of the family Pelagostrombidiidae.

Further analyses on the phylogeny of the subclass Scuticociliatia (Protozoa, Ciliophora) based on both nuclear and mitochondrial data.

So far, the phylogenetic studies on ciliated protists have mainly based on single locus, the nuclear ribosomal DNA (rDNA). In order to avoid the limitations of single gene/genome trees and to add more data to systematic analyses, information from mitochondrial DNA sequence has been increasingly used in different lineages of ciliates. The systematic relationships in the subclass Scuticociliatia are extremely confused and largely unresolved based on nuclear genes. In the present study, we have characterized 72 new sequences, including 40 mitochondrial cytochrome oxidase c subunit I (COI) sequences, 29 mitochondrial small subunit ribosomal DNA (mtSSU-rDNA) sequences and three nuclear small subunit ribosomal DNA (nSSU-rDNA) sequences from 47 isolates of 44 morphospecies. Phylogenetic analyses based on single gene as well as concatenated data were performed and revealed: (1) compared to mtSSU-rDNA, COI gene reveals more consistent relationships with those of nSSU-rDNA; (2) the secondary structures of mtSSU-rRNA V4 region are predicted and compared in scuticociliates, which can contribute to discrimination of closely related species; (3) neither nuclear nor mitochondrial data support the monophyly of the order Loxocephalida, which may represent some divergent and intermediate lineages between the subclass Scuticociliatia and Hymenostomatia; (4) the assignments of thigmotrichids to the order Pleuronematida and the confused taxon Sulcigera comosa to the genus Histiobalantium are confirmed by mitochondrial genes; (5) both nuclear and mitochondrial data reveal that the species in the family Peniculistomatidae always group in the genus Pleuronema, suggesting that peniculistomatids are more likely evolved from Pleuronema-like ancestors; (6) mitochondrial genes support the monophyly of the order Philasterida, but the relationships among families of the order Philasterida remain controversial due to the discrepancies between their morphological and molecular data.

Morphological Characterizations of Four Species of Parallelostrombidium (Ciliophora, Oligotrichia), with a Note on the Phylogeny of the Genus.

The morphology and phylogeny of four oligotrichid ciliates, Parallelostrombidium paraellipticum sp. n., P. dragescoi sp. n., P. jankowskii (Xu et al. 2009) comb. n., and P. kahli (Xu et al. 2009) comb. n., are described or redescribed based on live observation, protargol stained material, and SSU rRNA gene sequences. The new species P. paraellipticum sp. n. is characterized by its obovoidal cell shape, adoral zone composed of 17-21 collar, 9-11 buccal, and two thigmotactic membranelles, and extrusomes attached in one row along the girdle kinety. The new species P. dragescoi sp. n. is distinguished from its congeners by its obovoidal cell shape and a lack of thigmotactic membranelles. Based on ciliary patterns recognizable in the original slides, Omegastrombidium jankowskii Xu et al. 2009 and O. kahli Xu et al. 2009 should be transferred to the genus Parallelostrombidium Agatha 2004. Phylogenetic analyses based on SSU rRNA gene sequence data demonstrate that all four new sequences cluster with previously described congeners. The genus Parallelostrombidium is separated into two clusters, suggesting its non-monophyly and probably corresponding to the two subgenera proposed by Agatha and Strüder-Kypke (2014), as well as their morphological difference (cell dorsoventrally flattened vs. unflattened).

A Contribution to the Morphology and Phylogeny of Chlamydodon, with Three New Species from China (Ciliophora, Cyrtophoria).

Three new cyrtophorian ciliates isolated from coastal areas of China were described based on morphological and genetic data. The Chlamydodon mnemosyne-like species Chlamydodon similis sp. n. differs from its congeners mainly by its number of somatic kineties. Chlamydodon oligochaetus sp. n. is distinguished from its congeners mainly by having fewer somatic kineties, and/or an elongated body shape. Chlamydodon crassidens sp. n. is characterized mainly by an inverted triangular body shape, a posteriorly interrupted cross-striated band (5-6 µm wide), and a large cytostome. Moreover, we provided small-subunit (SSU) rDNA sequences of C. similis sp. n. and C. oligochaetus sp. n. Maximum likelihood (ML) and Bayesian inference (BI) consistently placed C. similis sp. n. as a sister to C. paramnemosyne, but showed different branching position of C. oligochaetus sp. n., which may be due to a low taxon sampling in the Chlamydodontidae and/or an insufficient resolution of the marker gene at species level.

Further consideration on the phylogeny of the Ciliophora: Analyses using both mitochondrial and nuclear data with focus on the extremely confused class Phyllopharyngea.

Most ciliate phylogenetic analyses have largely relied on the nuclear small subunit ribosome DNA (nSSU-rDNA) locus. However, single locus or multi-loci from the same genome or chromosome may not be sufficient enough to elucidate phylogenetic relationships among ciliate taxa. Therefore, in addition to nSSU-rDNA, the mitochondrial small subunit ribosome DNA (mtSSU-rDNA) was applied in this study. We expanded the taxon sampling especially within the class Phyllopharyngea. Phylogenetic analyses based on nSSU-rDNA and mtSSU-rDNA, independently, as well as concatenated were performed and revealed the following: (1) mtSSU-rDNA is more variable than nSSU-rDNA, and is better at elucidating relationships at lower levels, e.g. intra-/inter-specific or generic relationships; (2) the validity of the two genera Mirodysteria and Spirodysteria is challenged based on their similar morphology with Dysteria and the analyses from both mtSSU-rDNA and nSSU-rDNA; (3) Brooklynella is confirmed to be an intermediate taxon between Dysteriidae and Hartmannulidae, and may represent a distinct family; (4) Trithigmostoma should remain in Chilodonellidae; (5) the separation of Paraspathidium from Litostomatea is supported and it groups with prostomateans and plagiopyleans. In summary, results from mtSSU-rDNA corroborated those of nSSU-rDNA for highly supported clades, and the mtSSU-rDNA tree with its secondary structure gave topologies that could be explained by the morphology; therefore it can be useful in some cases towards better resolution of robust phylogenies.

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

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

Morphology and Molecular Phylogeny of Two Poorly Known Species of Protocruzia (Ciliophora: Protocruziida).

The ciliate genus Protocruzia is a highly confused group, which was formerly placed in the class Heterotrichea or Karyorelictea, and is according to the most recent system tentatively assigned to the class Spirotrichea. In the present study, the morphology, ciliary pattern, and molecular phylogeny of two poorly known species, Protocruzia tuzeti Villeneuve-Brachon, 1940, and Protocruzia granulosa Kahl, 1933, isolated from coastal waters of China, were investigated. Protocruzia tuzeti differs from its congeners mainly in possessing 6 adoral membranelles, 8-11 somatic kineties, and postoral dikinetids. Protocruzia granulosa is characterized by its extremely slender body, three postoral kineties, and 13 or 14 somatic kineties. The morphogenesis of P. granulosa is similar to that of P. tuzeti, especially in the parakinetal mode of stomatogenesis and the reorganization of the parental paroral membrane; however, more than one somatic kinety joins in the formation of the oral primordium in P. granulosa. Phylogenetic analyses based on small subunit ribosomal RNA gene revealed that six Protocruzia species form a fully supported clade that does not belong to any ciliate class; therefore, our data support the establishment of the class Protocruziea Gao et al. (Sci. Rep., 6, 2016, 24874).

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Utility of combining morphological characters, nuclear and mitochondrial genes: An attempt to resolve the conflicts of species identification for ciliated protists.

Ciliates comprise a highly diverse protozoan lineage inhabiting all biotopes and playing crucial roles in regulating microbial food webs. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. Here, we use the species-rich taxon Frontonia and employ both nuclear and mitochondrial loci. We attempt to assess the level of genetic diversity and evaluate the potential of each marker in delineating species of Frontonia. Morphological features and ecological characteristics are also integrated into genetic results, in an attempt to resolve conflicts of species identification based on morphological and molecular methods. Our studies reveal: (1) the mitochondrial cox1 gene, nuclear ITS1 and ITS2 as well as the hypervariable D2 region of LSU rDNA are promising candidates for species delineation; (2) the cox1 gene provides the best resolution for analyses below the species level; (3) the V2 and V4 hypervariable regions of SSU rDNA, and D1 of LSU rDNA as well as the 5.8S rDNA gene do not show distinct barcoding gap due to overlap between intra- and inter-specific genetic divergences; (4) morphological character-based analysis shows promise for delimitation of Frontonia species; and (5) all gene markers and character-based analyses demonstrate that the genus Frontonia consists of three groups and monophyly of the genus Frontonia is questionable.

Morphology, Cell Division, and Phylogeny of Uroleptus longicaudatus (Ciliophora, Hypotricha), a Species of the Uroleptus limnetis Complex.

A Chinese population of the little-known freshwater hypotrich Uroleptus longicaudatus was investigated with emphasis on its living morphology and infraciliature. The characteristic, tripartite body consists of a narrowed (cephalized) anterior portion, a slender trunk, and a long, slender, and strongly contractile tail occupying up to 30% of body length. Contracted specimens with a tail length of about 12% closely resemble Uroleptus limnetis which has, like U. longicaudatus, its type locality on the East Coast of the United States so that it cannot be excluded that these two species are synonymous. Thus, we propose to subsume these and few other little-known species, which are not clearly distinguishable at the present state of knowledge, as U. limnetis complex. The morphogenesis of U. longicaudatus proceeds as in most congeners. The phylogenetic analyses reveal that Uroleptus is a monophyletic group, but due to the lack of detailed morphological data of the populations sequenced so far, the relationships within this taxon remain obscure. For the objective determination of the tail length of hypotrichs, we propose the "1/3-method", which says that the tail commences at that body width which corresponds one-third of the maximum width. Paruroleptus ophryoglena Gelei, 1954 is transferred to Uroleptus: Uroleptus ophryoglena (Gelei, 1954) comb. nov.

Morphology, Ontogeny, and Molecular Phylogeny of Two Freshwater Species of Deviata (Ciliophora, Hypotrichia) from Southern China.

The morphology and partial morphogenesis of two freshwater hypotrichous ciliates, Deviata brasiliensis Siqueira-Castro et al., 2009 and Deviata rositae Küppers et al., 2007, isolated from southern China, were investigated using live observation and protargol staining. Our populations resemble the original ones in terms of their live characters and ciliary patterns. The main determinable morphogenetic features of D. brasiliensis basically correspond with those of the type population. However, the origin of anlage V for either proter or opisthe is ambiguous: whether anlage V for the proter originates from parental frontoventral row 2 (the same as in the original population) or parental frontoventral row 3 (the same as in Deviata abbrevescens) or even de novo is not clear; the anlage V for the opisthe is possibly derived from frontoventral row 3 and further migrates to frontoventral row 2, like that in D. abbrevescens. In addition, the SSU rRNA gene was first sequenced for both species. Molecular phylogenetic analyses suggest that the genus Deviata is non-monophyletic and has a close relationship with Perisincirra paucicirrata.

Morphology and morphogenesis of a novel mangrove ciliate, Sterkiella subtropica sp. nov. (Protozoa, Ciliophora, Hypotrichia), with phylogenetic analyses based on small-subunit rDNA sequence data.

A novel marine hypotrichous ciliate, Sterkiella subtropica sp. nov., was recently isolated from a mangrove wetland in Hong Kong. Its morphology, morphogenesis and systematic position have been investigated. The novel species is diagnosed by combined features of morphology, ciliature and nuclear apparatus, while its ontogenetic events present a stable pattern: (i) the six streaks of the undulating membrane (UM) and cirral anlagen are segmented in a 1 : 3 : 3 : 3 : 4 : 4 pattern from left to right, and form three frontal, four frontoventral, one buccal, five ventral and five transverse cirri; (ii) the dorsal structure is similar to most other oxytrichids; that is, in a '4+2' pattern with three caudal cirri being formed. Based on the small-subunit rDNA sequence, the novel species is different from its congeners by between 21 and 35 bp, with sequence identities from 0.978 to 0.987. All molecular trees exhibited a similar topology: the monophyly of species of the genus Sterkiella is not completely supported in our analyses, and approximately unbiased tests (both including and excluding the novel species) also reject the possibility that Sterkiella is a monophyletic lineage, as indicated by the morphology-based classification.

Morphology and phylogeny of two species of Loxodes (Ciliophora, Karyorelictea), with description of a new subspecies, Loxodes striatus orientalis subsp. n.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35-55 vs. 50-70) and a variable number of macronuclei (2-4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.