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.

Morphology and phylogenetic position of three anaerobic ciliates from the classes Odontostomatea and Muranotrichea (Ciliophora).

The diversity of the classes Odontostomatea and Muranotrichea, which contain solely obligate anaerobes, is poorly understood. We studied two populations of Mylestoma sp., one of Saprodinium dentatum (Odontostomatea), two of Muranothrix felix sp. nov., and one of Muranothrix sp. (Muranotrichea) employing live observation, protargol impregnation, scanning electron microscopy, and 18S rRNA gene sequencing. Conspecificity of Mylestoma sp., described here, with a previously described species of this genus cannot be excluded since no species have been studied with modern methods. Phylogenetically, the genus Mylestoma is closely related to the odontostomatid Discomorphella pedroeneasi, although the phylogenetic position of class Odontostomatea itself remains unresolved. The newly described muranotrichean species, Muranothrix felix sp. nov., is morphologically similar to M. gubernata but can be distinguished by its fewer macronuclear nodules and fewer adoral membranelles; moreover, it is clearly distinguished from M. gubernata by its 18S rRNA gene sequence. Another population, designated here as Muranothrix sp., most likely represents a separate species.

Morphology, Morphogenesis, and Phylogeny of Urosoma caudata (Ehrenberg, 1833) Berger, 1999 (Ciliophora, Hypotrichia) based on a Chinese Population.

We report the morphology and morphogenesis of Urosoma caudata (Ehrenberg, 1833) Berger, 1999 based on in vivo observation and protargol impregnation and provide an improved diagnosis of U. caudata based on previous and current work. Urosoma caudata differs from its congeners mainly by the combination of the following features: tail-like posterior end, colorless cortical granules, and two macronuclear nodules. Urosoma caudata shares most of the ontogenetic features with its congeners: the oral primordium of the opisthe develops apokinetally, and the frontal-ventral-transverse cirral anlagen develop in five streaks. However, a unique morphogenetic characteristic is recognizable: the anlagen of three dorsal kineties occur de novo to the left of the parental structures differing from their intrakinetal origin in other Urosoma species. The first record of the 18S rRNA gene sequence for the species is also provided. Phylogenetic analyses based on 18S rRNA gene sequence data suggest that the genus Urosoma is a nonmonophyletic group.

Phylogeny of the ciliate family Psilotrichidae (Protista, Ciliophora), a curious and poorly-known taxon, with notes on two algae-bearing psilotrichids from Guam, USA.

BACKGROUND: The classification of the family Psilotrichidae, a curious group of ciliated protists with unique morphological and ontogenetic features, is ambiguous and poorly understood particularly due to the lack of molecular data. Hence, the systematic relationship between this group and other taxa in the subclass Hypotrichia remains unresolved. In this paper the morphology and phylogenetics of species from two genera of Psilotrichida are studied to shed new light on the phylogeny and species diversity of this group of ciliates. RESULTS: The 18S rRNA gene sequences of species from two psilotrichid genera were obtained. In the phylogenetic trees, the available psilotrichid sequences are placed in a highly supported clade, justifying the establishment of the family Psilotrichidae. The morphology of two little-known species, packed with green algae, including a new species, Hemiholosticha kahli nov. spec., and Psilotrichides hawaiiensis Heber et al., 2018, is studied based on live observation, protargol impregnation, and scanning electron microscopy. Both species are easily recognized by their green coloration due to the intracellular algae, and a comprehensive discussion as to the possible roles of the intracellular algae is provided. CONCLUSIONS: The 18S rRNA gene phylogeny supports the morphological argument that Hemiholosticha, Psilotrichides and Urospinula belong to the same family, Psilotrichidae. However, the single-gene analysis, not surprisingly, does not resolve the deeper relationships of Psilotrichidae within the subclass Hypotrichia. Two little-known psilotrichid genera with green algae were collected from the same puddle on the island of Guam, indicating a high species diversity and broader geographic distribution of this group of ciliates than previously supposed. Phylogenetic inferences from transcriptomic and/or genomic data will likely be necessary to better define the systematic position and evolution of the family Psilotrichidae. Further studies are also needed to clarify the role of the intracellular eyespot-bearing algae in these ciliates.

A Simple and Rapid Cryopreservation Technique for Ciliates: A Long-Term Storage Procedure Used for Marine Scuticociliates.

Pseudocohnilembus persalinus is a free-living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, long-term laboratory maintenance for this species is mainly achieved by subculture that requires rigorous culture environments and, too often, cultures of the organism die out for a variety of reasons. Successful transport of viable cultures also poses problems for researchers. This study describes a simple and rapid protocol for long-term cryopreservation of P. persalinus. The effects of physiological states of individuals before freezing, the type and concentration of cryoprotectant, and optimal temperatures for freezing and thawing were assessed. A cryopreservation protocol, using a mixture of 30% glycerol and 70% concentrated P. persalinus cell culture, incorporating rate-controlled freezing at -80 °C before liquid nitrogen storage, maintained a high recovery efficiency after 8 wk of storage. These results suggest that broader application of this protocol to build a cryopreserved marine protozoa culture bank for biological studies may be possible.

Resolving relationships at the animal-fungal divergence: A molecular phylogenetic study of the protist trichomycetes (Ichthyosporea, Eccrinida).

Trichomycetes is a group of microorganisms that was considered a class of fungi comprising four orders of commensal, gut-dwelling endosymbionts obligately associated with arthropods. Since molecular phylogenies revealed two of those orders (Amoebidiales and Eccrinales="protist trichos") to be closely related to members of the protist class Ichthyosporea (=Mesomycetozoea), trichomycetes have been considered an ecological association of both early-diverging fungi and protists. Understanding of the taxonomy, evolution, and diversity of the protist trichos is lacking largely due to the difficulties inherent in species collection that have contributed to undersampling and understudy. The most recent classification divides the protist trichos between two families, Amoebidiidae and Eccrinidae (suborder Trichomycina, order Eccrinida). However, there is no comprehensive molecular phylogeny available for this group and major questions about the systematics of protist trichos remain unanswered. Therefore, we generated 18S and 28S rDNA sequences for 106 protist tricho samples and combined them with publicly available Eccrinida sequences for phylogenetic analyses. We also sequenced a conserved protein-coding gene (heat-shock 70 protein) to obtain a multigene data set. We conducted ancestral state reconstruction (ASR) and Bayesian tip-association significance test (BaTS) analyses by mapping six morphological and ecological characters onto the resulting phylogenetic trees. Our results demonstrate: (1) several ecological and morphological character states (habitat, host type, host stage at time of infestation, location within host, spore production, and growth form) are significantly correlated with the phylogeny, and (2) two additional protist tricho families should be incorporated into the taxonomy to reflect phylogenetic relationships. Our data suggest that an integrated strategy that combines morphological, ecological, and molecular characters is needed to further resolve and clarify the systematics of the Eccrinida.

Molecular Phylogeny and Taxonomy of a New Freshwater Hymenostomatid from Northeastern China, with the Establishment of a New Genus Anteglaucoma gen. n. (Protista, Ciliophora, Oligohymenophorea).

The morphology, infraciliature and SSU rDNA sequence of a new freshwater hymenostomatid ciliate, Anteglaucoma harbinensis gen. nov., spec. nov., collected from a farmland pond in Harbin, China, were investigated. The new genus Anteglaucoma is characterized as follows: small to medium-sized Glaucomidae with oral apparatus in anterior one-third of cell; paroral membrane composed of almost longitudinally arranged dikinetids; three adoral membranelles nearly equal in length and arranged almost longitudinally in parallel; silverline pattern tetrahymenid. The improved diagnosis of family Glaucomidae Corliss 1971 is provided based on the previous and present work. The type species Anteglaucoma harbinensis spec. nov. is defined by having 32-35 somatic kineties; four or five postoral kineties; membranelle 1 and membranelle 2 having five or six kinetosomal rows, membranelle 3 having three kinetosomal rows; single macronuclear nodule; contractile vacuole on average 15% from posterior body end; locomotion characterized by crawling with a rather hectic jerking motion; freshwater habitat. Phylogenetic analyses show that Anteglaucoma clusters in the family Glaucomidae and groups with the genera Glaucoma. The molecular and morphological data indicate that Glaucomidae is related to the family Bromeliophryidae in the phylogenetic trees.

Multigene-based phylogeny of the ciliate families Amphisiellidae and Trachelostylidae (Protozoa: Ciliophora: Hypotrichia).

Phylogenetic analyses of ciliated protozoa have been increasingly relied on multigene information, which was revealed to provide more robust interpretations than single-gene information. Previous studies showed that Amphisiellidae was an extremely divergent group within the order Stichotrichida, with species widely dispersed throughout the stichotrichid assemblage, while Trachelostylidae, excluding gonostomatid species, is a monophyletic group within the order Sporadotrichida. In the present study, we provide 38 new sequences of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes to infer phylogenetic relationships among all taxa available in Amphisiellidae and Trachelostylidae. The results indicate that: (1) Amphisiellidae is polyphyletic, with Amphisiella, Hemiamphisiella, Orthoamphisiella, Uroleptoides, and Urospinula distributing in different clades; (2) Amphisiella is separated into two clades in phylogenetic trees, corroborated by difference in cortical granule distribution. Thus, cortical granule pattern and distribution may be strong diagnostic features to divide Amphisiella species into two subgenera; (3) the monophyly of Trachelostylidae sensu Berger (2008) is strongly supported, suggesting it is a well-defined family; (4) Gonostomatidae is confirmed to be a valid family.

Reductitherus cryptostomus n. gen., n. sp. (Ciliophora: Armophorea: Clevelandellida), a remarkable new nyctotherid from an Australian cockroach, Parapanesthia gigantea (Blaberidae: Panesthiinae).

Ciliates of the family Nyctotheridae (Armophorea: Clevelandellida) are frequent intestinal symbionts of various invertebrates and some poikilotherm vertebrates. Depending on the classification scheme, there are between 15 and 18 recognized genera of Nyctotheridae, the majority of which exhibit a rather uniform morphology. They have round to ellipsoidal cells with an adoral zone of membranelles that begins anteriorly in an adoral groove and continues posteriorly into the buccal cavity where it extends deep into the cell in the peristomial funnel. The taxonomy of the Nyctotheridae is primarily based on the number and location of kinetal sutures. The only known divergence from the relatively conservative nyctortherid body plan are the bizarre symbionts of Panesthiinae cockroaches, ciliates of the family Clevelandellidae, which forms a clade nested within the Nyctotheridae genus Nyctotherus. In this study we report another ciliate that diverges morphologically from the canonical Nyctotheridae body plan, and which is also found in Panesthiinae hosts. The novel ciliate Reductitherus cryptostomus n. gen., n. sp. differs from the rest of Nyctotheridae by absence of the anterior adoral groove, a shortened adoral zone completely enclosed in a notably small buccal cavity, and two strongly reduced kinetal sutures, one left anterodorsal and the other right posterodorsal.

Protargol synthesis: an in-house protocol.

The protargol staining method has proved to be indispensable for revealing the cellular structures of a variety of protozoa, especially the flagellates and ciliates. Protargol provides permanent stains of a variety of cellular structures: nuclei, extrusomes, basal bodies, and microfilamentous constituents of cells. Together with the older silver nitrate methods, protargol impregnations have provided the basis for the detailed descriptions of nearly all ciliates to date. The performance of commercially available preparations has varied widely. Recently, suppliers have stopped stocking the effective forms of protargol resulting in a worldwide shortage. Thus, it has become necessary for protistologists to explore on-site synthesis of this critically important agent. An optimum protocol for synthesis of protargol should be rapid, relatively inexpensive, simple enough to be done by non-chemists, and achievable without specialized equipment. In this article, the authors briefly review the interesting history of protargol and describe a protocol, based on the early studies of neuroanatomists, that yields a protargol producing impregnations of ciliates comparable to those obtained with previously available commercial preparations.

Anaerocyclidiidae fam. nov. (Oligohymenophorea, Scuticociliatia): A newly recognized major lineage of anaerobic ciliates hosting prokaryotic symbionts.

The research on anaerobic ciliates, to date, has mainly been focused on representatives of obligately anaerobic classes such as Armophorea or Plagiopylea. In this study, we focus on the anaerobic representatives of the subclass Scuticociliatia, members of the class Oligohymenophorea, which is mainly composed of aerobic ciliates. Until now, only a single anaerobic species, Cyclidium porcatum (here transferred to the genus Anaerocyclidium gen. nov.), has been described both molecularly and morphologically. Our broad sampling of anoxic sediments together with cultivation and single cell sequencing approaches have shown that scuticociliates are common and diversified in anoxic environments. Our results show that anaerobic scuticociliates represent a distinctive evolutionary lineage not closely related to the family Cyclidiidae (order Pleuronematida), as previously suggested. However, the phylogenetic position of the newly recognized lineage within the subclass Scuticociliatia remains unresolved. Based on molecular and morphological data, we establish the family Anaerocyclidiidae fam. nov. to accommodate members of this clade. We further provide detailed morphological descriptions and 18S rRNA gene sequences for six new Anaerocyclidium species and significantly broaden the described diversity of anaerobic scuticociliates.

Rediscovery of Remarkably Rare Anaerobic Tentaculiferous Ciliate Genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea).

Free-living anaerobic ciliates are of considerable interest from an ecological and an evolutionary standpoint. Extraordinary tentacle-bearing predatory lineages have evolved independently several times within the phylum Ciliophora, including two rarely encountered anaerobic litostomatean genera, Legendrea and Dactylochlamys. In this study, we significantly extend the morphological and phylogenetic characterization of these two poorly known groups of predatory ciliates. We provide the first phylogenetic analysis of the monotypic genus Dactylochlamys and the three valid species of Legendrea based on the 18S rRNA gene and ITS-28S rRNA gene sequences. Prior to this study, neither group had been studied using silver impregnation methods. We provide the first protargol-stained material and also a unique video material including documentation, for the first time, of the hunting and feeding behavior of a Legendrea species. We briefly discuss the identity of methanogenic archaeal and bacterial endosymbionts of both genera based on 16S rRNA gene sequences, and the importance of citizen science for ciliatology from a historical and contemporary perspective.

Genealogical analyses of multiple loci of litostomatean ciliates (Protista, Ciliophora, Litostomatea).

The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of free-living and endocommensal species. However, their traditional morphology-based classification conflicts with 18S rRNA gene phylogenies indicating (1) a deep bifurcation of the Litostomatea into Rhynchostomatia and Haptoria+Trichostomatia, and (2) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea. To test whether 18S rRNA molecules provide a suitable proxy for litostomatean evolutionary history, we used eighteen new ITS1-5.8S rRNA-ITS2 region sequences from various free-living litostomatean orders. These single- and multiple-locus analyses are in agreement with previous 18S rRNA gene phylogenies, supporting that both 18S rRNA gene and ITS region sequences are effective tools for resolving phylogenetic relationships among the litostomateans. Despite insertions, deletions and mutational saturations in the ITS region, the present study shows that ITS1 and ITS2 molecules can be used to infer phylogenetic relationships not only at species level but also at higher taxonomic ranks when their secondary structure information is utilized to aid alignment.

Hypotrichidium tisiae (Gelei, 1929) Gelei, 1954: a unique hypotrichid ciliate having a highly specialized developmental pattern during binary division.

In sharp contrast to their pelagic relatives, the oligotrichs, the overwhelming majority of hypotrich ciliates inhabit the benthos. Only a few species, including those of the genus Hypotrichidium Ilowaisky, 1921, have adapted to a planktonic lifestyle. The ontogenetic mode of the highly differentiated ciliate, Hypotrichidium tisiae (Gelei, 1929) Gelei, 1954, is unknown. In this study, the interphase morphology and the ontogenetic process of this species are investigated. Accordingly, the previously unidentified ciliary pattern of Hypotrichidium is redefined. The main morphogenetic features are as follows: (1) The parental adoral zone of membranelles is inherited completely by the proter and the oral primordium of the opisthe arises in a deep pouch. (2) Five frontoventral cirral anlagen (FVA) are formed: FVA I contributes to the single frontal cirrus, FVA II-IV generate three frontoventral cirral rows, FVA V migrates and forms postoral ventral cirri. (3) All marginal cirral row anlagen develop de novo: each of the two left anlagen forms a single cirral row, while the single right anlage fragments into anterior and posterior parts. (4) Two dorsal kinety anlagen occur de novo, with the right one fragmenting to form kineties 2 and 3. (5) Two long caudal cirral rows are formed at the ends of dorsal kineties 1 and 3. On the basis of the morphogenetic features and phylogenetic analyses, the assignment of Hypotrichidium to the family Spirofilidae Gelei, 1929 within Postoralida is supported. The establishment of separate families for the slender "tubicolous" spirofilids and the highly helical spirofilids is also validated. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00148-9.

Taxonomy and molecular phylogeny of two poorly known ciliate genera, Balantidion and Acropisthium (Protista: Ciliophora: Litostomatea), including a new species of Balantidion.

The class Litostomatea Small & Lynn, 1981 is a morphologically diverse ciliate group including hundreds of free-living and endocommensal species. The genera Acropisthium Perty, 1852 and Balantidion Eberhard, 1862 previously consisted of one free-living freshwater species each. Here, we not only highlight additional morphological features of the two type species, but also investigate a new species, Balantidion foissneri sp. nov., isolated from a river flowing through Lake Weishan, China, based on complementary methods, i.e., living morphology, stained preparations, and 18S rRNA gene sequence data. Balantidion foissneri sp. nov. can be distinguished from the type species, B. pellucidum Eberhard, 1862, by the body size (115-170 × 50-80 µm vs. 70-100 × 25-45 µm in B. pellucidum), oral bulge (distinct vs. indistinct), extrusome shape (filiform vs. rod-shaped), and the number of somatic kineties (46-60 vs. 25-40). In Balantidion species, pre-encystment trophonts show similarly-shaped polymorphic cytoplasmic lepidosomes destined to adorn the outer surface of the resting cyst. Based on the current knowledge, assignment of Balantidion to the family Acropisthiidae Foissner & Foissner, 1988 is proposed. In addition, phylogenetic analyses based on molecular data show that the two Balantidion species form a fully-supported clade to which Acropisthium mutabile has a sister relationship.

Phylogeny and classification of the Litostomatea (Protista, Ciliophora), with emphasis on free-living taxa and the 18S rRNA gene.

The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of species ranging from aerobic, free-living predators to anaerobic endocommensals. This is traditionally reflected by classifying the Litostomatea into the subclasses Haptoria and Trichostomatia. The morphological classifications of the Haptoria conflict with the molecular phylogenies, which indicate polyphyly and numerous homoplasies. Thus, we analyzed the genealogy of 53 in-group species with morphological and molecular methods, including 12 new sequences from free-living taxa. The phylogenetic analyses and some strong morphological traits show: (i) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea and (ii) three distinct lineages (subclasses): the Rhynchostomatia comprising Tracheliida and Dileptida; the Haptoria comprising Lacrymariida, Haptorida, Didiniida, Pleurostomatida and Spathidiida; and the Trichostomatia. The curious Homalozoon cannot be assigned to any of the haptorian orders, but is basal to a clade containing the Didiniida and Pleurostomatida. The internal relationships of the Spathidiida remain obscure because many of them and some "traditional" haptorids form separate branches within the basal polytomy of the order, indicating one or several radiations and convergent evolution. Due to the high divergence in the 18S rRNA gene, the chaeneids and cyclotrichiids are classified incertae sedis.

Morphology, morphometrics, and molecular characterization of Bryophrya gemmea n. sp. (Ciliophora, Colpodea): implications for the phylogeny and evolutionary scenario for the formation of oral ciliature in the order Colpodida.

We studied the morphology, morphometry, resting, and reproductive cysts, as well as the molecular phylogeny of Bryophrya gemmea n. sp., a colpodid ciliate that was discovered in ephemeral puddles in Idaho, northwest United States. This new species is distinguished from congeners by the irregularly pentagonal adoral organelles, four to five vestibular kineties, the single micronucleus, and one to three rows of brightly refractive protuberant interkinetal cortical granules to the right of the preoral suture. Resting cysts have two distinct membranes and an outer mucous coat. As typical for most colpodids, reproduction occurs in division cysts but details of ontogenesis are unknown. The 18S rRNA gene sequence shows only weak support for the phylogenetic relationship between Bryophrya and the bryophryid genus Notoxoma previously inferred from morphologic characters. Further, our molecular phylogenies classify bryophryids rather basal within the order Colpodida, not supporting ordinal status suggested by morphologists. Based on molecular data and morphologic characters, the colpodid genus Ilsiella is removed from the family Marynidae and placed in a new family, Ilsiellidae. Considering the molecular data, an evolutionary scenario for the formation of colpodid oral structures from a cyrtolophosidid ancestor through a bryophryid intermediate is proposed.

Phylogenetic Position of Three Well-known Ciliates from the Controversial Order Loxocephalida Jankowski, 1980 (Scuticociliatia, Oligohymenophorea) and Urozona buetschlii (Schewiakoff, 1889) with Improved Morphological Descriptions.

Loxocephalids represent a nonmonophyletic assemblage of oligohymenophorean ciliates with morphological features common to both scuticociliates (mode of ontogenesis) and hymenostomes (morphology of the oral structures). In phylogenetic analyses that include both nuclear and mitochondrial molecular markers, relationships among loxocephalid ciliates are still largely unresolved. With the aim to clarify the phylogeny of the controversial order Loxocephalida Jankowski, 1980, we provide the first 18S rRNA gene sequences of three morphologically well-described ciliates currently included in the order, namely Cinetochilum margaritaceum, Zitheron hovassei, and Zitheron muscorum comb. nov. We also provide the first 18S rRNA gene sequence for the monotypic genus Urozona. Here we present improved diagnoses for these species based on updated morphologic data. Our molecular data circumscribe the family Cinetochilidae Perty, 1852 and support the exclusion of genus Cinetochilides from family Cinetochilidae; therefore, we establish a family Cinetochilididae fam. nov. We consider Urozona as incertae sedis in Oligohymenophorea at the current state of knowledge.

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

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

Taxonomy and Molecular Phylogeny of Two New Urostylid Ciliates (Protozoa: Ciliophora) From Chinese Wetlands and Establishment of a New Genus.

Hypotrich ciliates with evolutionary novelties are continually being discovered, challenging the current taxonomic system and attracting increased attention. In the present work, two new urostylid ciliates, Heterobakuella bergeri gen. nov., sp. nov. and Anteholosticha perezuzae sp. nov., isolated from Chinese wetland samples, were identified based on morphology and 18S rRNA gene sequences. Heterobakuella gen. nov. is defined by three frontal cirri, single buccal cirrus, one parabuccal cirrus, midventral complex composed of cirral pairs and one cirral row, one left and two right marginal cirral rows, transverse and pretransverse cirri present, caudal and frontoterminal cirri absent. Heterobakuella can be easily distinguished from the morphologically most similar genus, Apobakuella, mainly by the single buccal cirrus (vs. one buccal cirral row) and one parabuccal cirrus (vs. several parabuccal cirral rows originated from different anlagen). Phylogenetic analyses show that H. bergeri branches within the clade formed by Bergeriella ovata, Monocoronella carnea, Anteholosticha gracilis, and Neourostylopsis spp., rather than the clade represented by Apobakuella. The other species, A. perezuzae, is mainly characterized by a distinctly slender body shape with an average length:width ratio about 7, distinctively shaped biconcave and greenish cortical granules, as well as one or two pretransverse cirri. Phylogenetic analyses indicate the genus Anteholosticha is non-monophyletic.