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.

Redescription and molecular phylogeny of the freshwater metopid, Castula strelkowi (Jankowski, 1964) from the Czech Republic and synonymization of Pileometopus with Castula.

The relationships of the mainly free living, obligately anaerobic ciliated protists belonging to order Metopida continue to be clarified and now comprise three families: Metopidae, Tropidoatractidae, and Apometopidae. The most species-rich genus of the Metopidae, Metopus has undergone considerable subdivision into new genera in recent years as more taxa are characterized by modern morphologic and molecular methods. The genus, Castula, was established to accommodate setae-bearing species previously assigned to Metopus: C. setosa and C. fusca, and one new species, C. flexibilis. Another new species, C. specialis, has been added since. Here we redescribe another species previously included in Metopus, using morphologic and molecular methods, and transfer it to Castula as C. strelkowi n. comb. (original combination Metopus strelkowi). We also reassess the monotypic genus, Pileometopus, which nests within the strongly supported Castula clade in 18S rRNA gene trees and conclude that it represents a morphologically divergent species of Castula.

Redescription and molecular characterization of Loxocephalus luridus Eberhard, 1862 based on Czech populations: Implications for order Loxocephalida Jankowski, 1980.

The phylogenetic and taxonomic affinities of lineages currently assigned to the non-monophyletic ciliate order Loxocephalida Jankowski (1980) within subclass Scuticociliatia Small (1967) remain unresolved. In the current study, we redescribe the morphology of the type species, Loxocephalus luridus Eberhard (1862) based on two Czech populations and include the first scanning and transmission electron microscopy images of the species. We provide the first 18S rRNA gene sequences for L. luridus and consider its phylogenetic position. Our results support the separation of Dexiotricha from Loxocephalus; however, the former genus is recovered as non-monophyletic. The monophyly of genus Dexiotricha and that of Loxocephalus + Dexiotricha is rejected. Loxocephalus luridus, together with Dexiotricha species, nests within a fully supported clade with Conchophthirus species, long presumed to belong to the Pleuronematida. Haptophrya is recovered as sister to this clade. The monophyly of the Astomatia Schewiakoff (1896) including Haptophrya is rejected. No clear morphologic synapomorphy is identified for the fully supported clade consisting of Haptophrya, Dexiotricha, Loxocephalus, and Conchophthirus.

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.

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.

Enchelyothrix muria n. gen, n. sp., (Ciliophora, Litostomatea, Spathidiida), an extreme halotroph spathidiid from Maharloo Lake, Iran.

Although free-living ciliated protists (Ciliophora) commonly inhabit aquatic and terrestrial biotopes of mild to moderate salinity (PSS-practical salinity scale S = 10-150), very few have adapted to life at more extreme salinities (>150 to saturation). Such extreme halophiles or halotrophs are of interest from the standpoints of evolution, cell physiology, ecology, and even astrobiology. In this work, we present the morphology, 18S rRNA gene sequence, and phylogenetic analysis of a novel spathidiid ciliate (Ciliophora, Litosatomatea) that thrives in saturated brines of Maharloo Lake, Iran. Based on its unique combination of morphologic features, its molecular characterization, and its unusual ecology, it is assigned to a newly erected monotypic genus incertae sedis in order Spathidiida. The new species differs from other spathidiids by: a cell with a very short acutely pointed tail, the absence of a circumoral kinety with, instead, inclined, interrupted circumoral kinetofragments at the anterior end of somatic kineties and brush rows abutting the oral bulge, the absence of a contractile vacuole, a heteromorphic dorsal brush comprising five or six rows, and an extreme (at or near saturation) hypersaline habitat. We briefly discuss the persistent uncertainties regarding the phylogenetic relationships within the order Spathidiida.

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.

Morphologic and molecular characterization of Apertospathula pilata n. sp., a novel freshwater spathidiid (Ciliophora, Litostomatea) from Idaho, USA.

Order Spathidiida Foissner and Foissner, 1988 comprises a large group of morphologically diverse, primarily predatory, free living ciliates, the phylogeny of which has remained stubbornly unresolved. Families Arcuospathidiidae and Apertospathulidae are two morphologically similar groups established on the basis of differences in the morphology of the oral bulge and circumoral kinety. While Arcuospathidiidae is non-monophyletic in 18S rRNA gene analyses, the Apertospathulidae has been represented by only a single Apertospathula sequence in public databases. In this report, a novel freshwater species, Apertospathula pilata n. sp. is described on the basis of living observation, silver impregnation, and scanning electron microscopy. The phylogeny of the new species is assessed based on the rRNA cistron. The main features distinguishing A. pilata n. sp. from all congeners are: the oral bulge extrusomes (filiform, up to 25 µm long), the combination of body size (130-193 µm) and shape (spatulate), the extensive oral bulge length (41% of the cell length after protargol impregnation), and multiple micronuclei (one to five, two on average). The monophyly of Apertospathulidae Foissner, Xu and Kreutz, 2005 is rejected.

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.

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.

Diversity and Phylogenetic Position of Bothrostoma Stokes, 1887 (Ciliophora: Metopida), with Description of Four New Species.

Bothrostoma is a genus of anaerobic ciliates in family Metopidae comprising four species, all described based solely on the morphology of living and fixed cells. Unlike other metopids, cells of Bothrostoma are not twisted anteriorly, have a flattened preoral dome, a very prominent sail-like paroral membrane, and an adoral zone of distinctive, very narrow, curved membranelles confined to a wide, non-spiraling peristome on the ventral side. We examined 20 populations of Bothrostoma from hypoxic freshwater sediments. We provide morphological characterization and 18S rRNA gene sequences of four new species, namely B. bimicronucleatum sp. nov., B. boreale sp. nov., B. kovalyovi sp. nov., and B. robustum sp. nov., as well as B. undulans (type species), B. nasutum, and B. ovale comb. nov. (original combination Metopus undulans var. ovalis Kahl, 1932). Except for B. nasutum, Bothrostoma species show low genetic variability among geographically distant populations. Intraspecific phenotypic variability might be driven by environmental conditions. In phylogenetic analyses, Bothrostoma is not closely related to Metopus sensu stricto and forms a moderately supported clade with Planometopus, here referred to as BoPl clade. The anterior axial torsion of the body, typical of other Metopidae, appears to have been lost in the last common ancestor of the BoPl clade.

Morphology and phylogeny of two anaerobic freshwater ciliates: Brachonella comma sp. nov. and the widely distributed but little-known caenomorphid, Ludio parvulus Penard, 1922.

Hypoxic, sulfidic freshwater sediments typically support a diffuse consortium of distinctive ciliated protists, including caenomorphids, metopids, and odontostomatids among others. A recent resurgence of interest in these important members of sapropelic food webs has resulted in the description of many new species and an effort, still in its infancy, to characterize them from a morphologic, molecular, and metabolic standpoint and to determine their phylogenetic relationships. Their seemingly invariable association with prokaryotic endosymbionts and, less commonly, ectosymbionts has become a focus for many researchers. In this report, based on morphologic and molecular data, we describe a Brachonella species (Ciliophora, Metopida) new to science and analyze its phylogeny. We also provide a morphologic and molecular characterization of the smallest representative of the Caenomorphidae Poche, 1913, Ludio parvulus Penard, 1922. The phylogenetic analysis confirms the inclusion of this species in the Caenomorphidae.

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.

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.

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.

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.

The Function and Evolution of Motile DNA Replication Systems in Ciliates.

DNA replication is a ubiquitous and conserved cellular process. However, regulation of DNA replication is only understood in a small fraction of organisms that poorly represent the diversity of genetic systems in nature. Here we used computational and experimental approaches to examine the function and evolution of one such system, the replication band (RB) in spirotrich ciliates, which is a localized, motile hub that traverses the macronucleus while replicating DNA. We show that the RB can take unique forms in different species, from polar bands to a "replication envelope," where replication initiates at the nuclear periphery before advancing inward. Furthermore, we identify genes involved in cellular transport, including calcium transporters and cytoskeletal regulators, that are associated with the RB and may be involved in its function and translocation. These findings highlight the evolution and diversity of DNA replication systems and provide insights into the regulation of nuclear organization and processes.

Description of Three New Genera of Metopidae (Metopida, Ciliophora): Pileometopus gen. nov., Castula gen. nov., and Longitaenia gen. nov., with Notes on the Phylogeny and Cryptic Diversity of Metopid Ciliates.

We report the discovery of three new species of freshwater metopid ciliates, Pileometopus lynni gen. et sp. nov., Castula flexibilis gen. et sp. nov., and Longitaenia australis gen. et sp. nov. Based on morphologic features and the 18S rRNA gene phylogeny, we transfer two known species of Metopus to the new genus Castula, as C. fusca (Kahl, 1927) comb. nov. and C. setosa (Kahl, 1927) comb. nov. and another known species is herein transferred to the new genus Longitaenia, as L. gibba (Kahl, 1927) comb. nov. Pileometopus is characterized by a turbinate body shape, a dorsal field of densely spaced dikinetids, a bipartite paroral membrane, and long caudal cilia. A distinctive morphologic feature of Castula species is long setae arising over the posterior third of the body (as opposed to a terminal tuft). Longitaenia spp. are characterized by an equatorial cytostome and long perizonal ciliary stripe relative to the cell length. Based on phylogenetic analyses of 18S rRNA gene sequences, we identify and briefly discuss strongly supported clades and intraspecific genetic polymorphism within the order Metopida.

Genomics of New Ciliate Lineages Provides Insight into the Evolution of Obligate Anaerobiosis.

Oxygen plays a crucial role in energetic metabolism of most eukaryotes. Yet adaptations to low-oxygen concentrations leading to anaerobiosis have independently arisen in many eukaryotic lineages, resulting in a broad spectrum of reduced and modified mitochondrion-related organelles (MROs). In this study, we present the discovery of two new class-level lineages of free-living marine anaerobic ciliates, Muranotrichea, cl. nov. and Parablepharismea, cl. nov., that, together with the class Armophorea, form a major clade of obligate anaerobes (APM ciliates) within the Spirotrichea, Armophorea, and Litostomatea (SAL) group. To deepen our understanding of the evolution of anaerobiosis in ciliates, we predicted the mitochondrial metabolism of cultured representatives from all three classes in the APM clade by using transcriptomic and metagenomic data and performed phylogenomic analyses to assess their evolutionary relationships. The predicted mitochondrial metabolism of representatives from the APM ciliates reveals functional adaptations of metabolic pathways that were present in their last common ancestor and likely led to the successful colonization and diversification of the group in various anoxic environments. Furthermore, we discuss the possible relationship of Parablepharismea to the uncultured deep-sea class Cariacotrichea on the basis of single-gene analyses. Like most anaerobic ciliates, all studied species of the APM clade host symbionts, which we propose to be a significant accelerating factor in the transitions to an obligately anaerobic lifestyle. Our results provide an insight into the evolutionary mechanisms of early transitions to anaerobiosis and shed light on fine-scale adaptations in MROs over a relatively short evolutionary time frame.