Divergent marine anaerobic ciliates harbor closely related Methanocorpusculum endosymbionts.

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont internal transcribed spacer region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life.

Single cell transcriptomics reveals UAR codon reassignment in Palmarella salina (Metopida, Armophorea) and confirms Armophorida belongs to APM clade.

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates - Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general.

Morphology, morphogenesis, and molecular characterization of Castula specialis sp. nov. (Ciliophora, Armophorea, Metopida).

The morphology, morphogenesis, and molecular phylogeny of a new metopid ciliate, Castula specialis sp. nov., comprising three strains from geographically distant (China, Mexico, Czech Republic) anoxic freshwater habitats, were studied based on microscopic observation of live and protargol-stained specimens as well as SSU rRNA gene sequence data. The new species is characterized as follows: size in vivo 105-220 × 25-70 µm, body oblong to elongated ellipsoidal and asymmetrical; preoral dome distinctly projecting beyond the body; 32-46 adoral membranelles; 31-52 somatic kineties; and 4-7 setae. This study brings the first morphogenetic investigation of a member of the genus Castula. The morphogenesis of the type population (China) of the new species proceeds as in Metopus spp. comprising drastic changes in body shape and a pleurotelokinetal stomatogenesis; however, the main difference is the origin of the opisthe's paroral membrane that derives from all perizonal rows and some adjacent dome kineties. Phylogenetically, the genus Castula is paraphyletic.

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.

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.

Anaerobic ciliates as a model group for studying symbioses in oxygen-depleted environments.

Anaerobiosis has independently evolved in multiple lineages of ciliates, allowing them to colonize a variety of anoxic and oxygen-depleted habitats. Anaerobic ciliates commonly form symbiotic relationships with various prokaryotes, including methanogenic archaea and members of several bacterial groups. The hypothesized functions of these ecto- and endosymbionts include the symbiont utilizing the ciliate's fermentative end products to increase the host's anaerobic metabolic efficiency, or the symbiont directly providing the host with energy by denitrification or photosynthesis. The host, in turn, may protect the symbiont from competition, the environment, and predation. Despite rapid advances in sampling, molecular, and microscopy methods, as well as the associated broadening of the known diversity of anaerobic ciliates, many aspects of these ciliate symbioses, including host specificity and coevolution, remain largely unexplored. Nevertheless, with the number of comparative genomic and transcriptomic analyses targeting anaerobic ciliates and their symbionts on the rise, insights into the nature of these symbioses and the evolution of the ciliate transition to obligate anaerobiosis continue to deepen. This review summarizes the current body of knowledge regarding the complex nature of symbioses in anaerobic ciliates, the diversity of these symbionts, their role in the evolution of ciliate anaerobiosis and their significance in ecosystem-level 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.

The Little-known Freshwater Metopid Ciliate, Idiometopus turbo (Dragesco and Dragesco-Kernéis, 1986) nov. gen., nov. comb., Originally Discovered in Africa, Found on the Micronesian Island of Guam.

Class Armophorea Lynn, 2004 includes two orders of mainly free-living anaerobic ciliates, Armophorida Jankowski, 1980 and Metopida Jankowski, 1980 and, a third, the exclusively endosymbiotic Clevelandellida. Kahl described the majority of free-living metopid species early in the 20th century. Excepting Jankowski in the 1960's, little further interest was shown in this group. Metopus turbo Dragesco and Dragesco-Kernéis, 1986, from a West African pond, was more recently described. Although not explicitly described as endemic, Dragesco believed in some degree of endemism of sub-Saharan ciliates. Our discovery of M. turbo on Guam, Micronesia was unexpected. Metopus turbo was identified by live observation, protargol impregnation, and scanning electron microscopy, providing reasonable evidence of conspecificity when compared with the original description. Outstanding morphologic features include the stout shape and transversely situated bandform macronucleus. The morphology of Metopus turbo differs markedly from that of the Metopus/Brachonella clade and is distant from Metopus es in phylogenetic analyses. The 18S rDNA sequence of the Guam M. turbo differs from that of M. es, type species of Metopus, by >8% and shows distinct morphologic differences from the genus Atopospira to which it is sister. These facts suggest that transfer to a new genus, Idiometopus gen. nov. is indicated.

Morphologic and molecular characterization of Brachonella pulchra (Kahl, 1927) comb. nov. (Armophorea, Ciliophora) with comments on cyst structure and formation.

In this article we provide morphologic and morphometric data based on in vivo observation, protargol impregnation, scanning electron microscopy and an 18S rRNA gene sequence for another member of the genus Brachonella, Brachonella pulchra comb. nov. (basionym: Metopus pulcher Kahl, 1927). We also provide preliminary data on resting cyst structure and formation in Brachonella pulchra and discuss the possible taxonomic usefulness of these structures.

Tropidoatractidae fam. nov., a Deep Branching Lineage of Metopida (Armophorea, Ciliophora) Found in Diverse Habitats and Possessing Prokaryotic Symbionts.

We report a discovery of a novel family of anaerobic ciliates, Tropidoatractidae fam. nov. Phylogenetic analyses based on the 18S rRNA gene show that the family Tropidoatractidae corresponds to the previously reported clade of environmental sequences closely related to the lineage consisting of orders Metopida and Clevelandellida. The family comprises two genera, Tropidoatractus and Palmarella, and five species, two of which are newly described herein. Tropidoatractidae are cosmopolitan Metopida with sparse somatic and oral ciliature, deep, cup-like buccal cavity, and hyaline cortex with interkinetal ridges. Moreover, all species occur in two morphotypes, slender and stout. They inhabit microoxic or anoxic freshwater, brackish, and marine sediments and possess anaerobic mitochondrion-related organelles and various prokaryotic symbionts. The discovery of Tropidoatractidae provides valuable information about the evolution of Armophorea and gives us insights to the diversity and ecological preferences of anaerobic ciliates in general.

Morphologic and molecular characterization of seven species of the remarkably diverse and widely distributed metopid genus Urostomides Jankowski, 1964 (Armophorea, Ciliophora).

The free-living ciliates of the order Metopida Jankowski, 1980 are pivotal players in the microbial food web of the sulfuretum, acting as hosts to prokaryotic endo- and ectosymbionts. They are also of interest in the study of the function and evolution of their mitochondrion-related organelle, the hydrogenosome. The taxonomy and phylogeny of this group remains confused, due, in large part, to the fact that most of its taxa have not been characterized by modern methods including molecular sequencing. In this report we provide morphologic and molecular characterization of seven taxa from the poorly-known resurrected genus Urostomides obtained in the course of broad geographic sampling. Foissner (2016) established the family Apometopidae to include Apometopus (a junior synonym of Urostomides) and Cirranter Jankowski, 1964. These two genera differ from all other metopid genera in having a four-rowed perizonal ciliary stripe, the only currently recognizable morphologic synapomorphy for the family. The members of Urostomides show remarkable morphologic diversity. The genus has a broad geographic distribution, occurring on six continents. Urostomides species form a strongly supported clade in phylogenetic analyses. Relationships within the genus itself are less clearly resolved. The diagnoses of Apometopidae and Urostomides are emended.

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.

Redescription and molecular phylogeny of the type species for two main metopid genera, Metopus es (Müller, 1776) Lauterborn, 1916 and Brachonella contorta (Levander, 1894) Jankowski, 1964 (Metopida, Ciliophora), based on broad geographic sampling.

Metopid ciliates occupy terrestrial, freshwater, and marine habitats worldwide, playing important roles as predominant consumers of bacteria, flagellates, algae, and diatoms in hypoxic environments. Metopus and Brachonella are the most species-rich metopid genera, however most of their species have not been studied by modern methods Here, we report the morphologic, morphometric and molecular characterization, and phylogeny of Metopus es and Brachonella contorta, both types of their respective genera, collected in a broad global sampling effort. Five strains of M. es and three strains of B. contorta were studied in detail, providing the first correlation of morphology, morphometrics, and 18S rRNA gene sequencing for both. We submitted 29 new 18S rRNA gene sequences to GenBank. Phylogenetic analyses yielded trees of similar topology. A strongly supported Metopus es clade is sister to the Brachonella contorta clade. Our analysis shows genus Metopus is not monophyletic. The monophyly of Brachonella cannot yet be determined due to lack of sequences for other species of this genus in molecular databases. Both species appear to have a global distribution. Metopus es was not found in Africa, probably reflecting low sampling effort. Strains of both species showed low 18S rRNA gene sequence divergence despite wide geographic separation.