Global perspective of environmental distribution and diversity of Perkinsea (Alveolata) explored by a meta-analysis of eDNA surveys.

Perkinsea constitutes a lineage within the Alveolata eukaryotic superphylum, mainly composed of parasitic organisms. Some described species represent significant ecological and economic threats due to their invasive ability and pathogenicity, which can lead to mortality events. However, the genetic diversity of these described species is just the tip of the iceberg. Environmental surveys targeting this lineage are still scarce and mainly limited to the Northern Hemisphere. Here, we aim to conduct an in depth exploration of the Perkinsea group, uncovering the diversity across a variety of environments, including those beyond freshwater and marine ecosystems. We seek to identify and describe putative novel organisms based on their genetic signatures. In this study, we conducted an extensive analysis of a metabarcoding dataset, focusing on the V4 region of the 18S rRNA gene (the EukBank dataset), to investigate the diversity, distribution and environmental preferences of the Perkinsea. Our results reveal a remarkable diversity within the Perkinsea, with 1568 Amplicon Sequence Variants (ASVs) identified across thousands of environmental samples. Surprisingly, we showed a substantial diversity of Perkinsea within soil samples (269 ASVs), challenging the previous assumption that this group is confined to marine and freshwater environments. In addition, we revealed that a notable proportion of Perkinsea ASVs (428 ASVs) could correspond to putative new organisms, encompassing the well-established taxonomic group Perkinsidae. Finally, our study shed light on previously unveiled taxonomic groups, including the Xcellidae, and revealed their environmental distribution. These findings demonstrate that Perkinsea exhibits far greater diversity than previously detected and surprisingly extends beyond marine and freshwater environments. The meta-analysis conducted in this study has unveiled the existence of previously unknown clusters within the Perkinsea lineage, solely identified based on their genetic signatures. Considering the ecological and economic importance of described Perkinsea species, these results suggest that Perkinsea may play a significant, yet previously unrecognized, role across a wide range of environments, spanning from soil environments to the abyssal zone of the open ocean with important implications for ecosystem functioning.

Identification of glycine betaine as a host-derived molecule required for the vegetative proliferation of the protozoan parasite Perkinsus olseni.

Perkinsus olseni is an industrially significant protozoan parasite of Manila clam, Ruditapes philippinarum. So far, various media, based on Dulbecco's Modified Eagle Medium and Ham's F-12 nutrient mixture with supplementation of fetal bovine serum (FBS), have been developed to proliferate the parasitizing trophozoite stage of P. olseni. The present study showed that P. olseni did not proliferate in FBS-deficient Perkinsus broth medium (PBMΔF), but proliferated well in PBMΔF supplemented with tissue extract of host Manila clams, indicating that FBS and Manila clam tissue contained molecule(s) required for P. olseni proliferation. Preliminary characterization suggested that the host-derived molecule(s) was a heat-stable molecule(s) with a molecular weight of less than 3 kDa, and finally a single molecule required for the proliferation was purified by high-performance liquid chromatography processes. High-resolution electrospray ionization mass spectrometry and nuclear magnetic resonance analyses identified this molecule as glycine betaine (=trimethylglycine), and the requirement of this molecule for P. olsseni proliferation was confirmed by an assay using chemically synthesized, standard glycine betaine. Although glycine betaine was required for the proliferation of all examined Perkinsus species, supplementation of glycine betaine precursors, such as choline and betaine aldehyde, enhanced the proliferation of 4 Perkinsus species (P. marinus, P. chesapeaki, P. mediterraneus and P. honshuensis), but not of 2 others (P. olseni and P. beihaiensis). Thus, it was concluded that the ability to biosynthesise glycine betaine from its precursors varied among Perkinsus species, and that P. olseni and P. beihaiensis lack the ability required to biosynthesize glycine betaine for proliferation.

Characterisation and Cultivation of New Lineages of Colponemids, a Critical Assemblage for Inferring Alveolate Evolution.

There are several alveolate groups outside the well-studied trio - ciliates, dinoflagellates, and apicomplexans - that are crucial for understanding the evolution of this major taxon. One such assemblage is the "colponemids", which are eukaryotrophic biflagellates, usually with a ventral groove associated with the posterior flagellum. Previous phylogenetic studies show colponemids forming up to three distinct deep branches within alveolates (e.g. sister groups to Myzozoa or all other alveolates). We have developed dieukaryotic (predator-prey) cultures of four colponemid isolates. One represents the first stable culture of the halophile Palustrimonas (feeding on Pharyngomonas), while SSU rDNA phylogenies show the other isolates as two distinct new lineages. Neocolponema saponarium gen. et sp. nov. is a swimming alkaliphile with a large groove, which feeds on a kinetoplastid. Loeffela hirca gen. et sp. nov. is halophilic, has a subtle groove, usually moves along surfaces, and feeds on Pharyngomonas and Percolomonas. Prey capture in both new genera is raptorial, involves a specialized structure/region to the right of the proximal posterior flagellum, and presumed extrusomes. The relationships amongst Myzozoa, ciliates, and the (now) five described colponemid clades are unresolved, signaling that colponemid diversity represents both a challenge and important resource for tracing deep alveolate evolution.

The photic-aphotic divide is a strong ecological and evolutionary force determining the distribution of ciliates (Alveolata, Ciliophora) in the ocean.

The bulk of knowledge on marine ciliates is from shallow and/or sunlit waters. We studied ciliate diversity and distribution across epi- and mesopelagic oceanic waters, using DNA metabarcoding and phylogeny-based metrics. We analyzed sequences of the 18S rRNA gene (V4 region) from 369 samples collected at 12 depths (0-1000 m) at the Bermuda Atlantic Time-series Study site of the Sargasso Sea (North Atlantic) monthly for 3 years. The comprehensive depth and temporal resolutions analyzed led to three main findings. First, there was a gradual but significant decrease in alpha-diversity (based on Faith's phylogenetic diversity index) from surface to 1000-m waters. Second, multivariate analyses of beta-diversity (based on UniFrac distances) indicate that ciliate assemblages change significantly from photic to aphotic waters, with a switch from Oligotrichea to Oligohymenophorea prevalence. Third, phylogenetic placement of sequence variants and clade-level correlations (EPA-ng and GAPPA algorithms) show Oligotrichea, Litostomatea, Prostomatea, and Phyllopharyngea as anti-correlated with depth, while Oligohymenophorea (especially Apostomatia) have a direct relationship with depth. Two enigmatic environmental clades include either prevalent variants widely distributed in aphotic layers (the Oligohymenophorea OLIGO5) or subclades differentially distributed in photic versus aphotic waters (the Discotrichidae NASSO1). These results settle contradictory relationships between ciliate alpha-diversity and depth reported before, suggest functional changes in ciliate assemblages from photic to aphotic waters (with the prevalence of algivory and mixotrophy vs. omnivory and parasitism, respectively), and indicate that contemporary taxon distributions in the vertical profile have been strongly influenced by evolutionary processes. Integration of DNA sequences with organismal data (microscopy, functional experiments) and development of databases that link these sources of information remain as major tasks to better understand ciliate diversity, ecological roles, and evolution in the ocean.

Comparative biological traits of perkinsozoan parasitoids infecting marine dinoflagellates.

The number of perkinsozoan parasitoid species known to infect dinoflagellates has increased to 11 over the last two decades. However, most of the current knowledge about the autecology of perkinsozoan parasitoids of dinoflagellates has derived from studies of one or two species, thereby making it difficult to directly compare their biological traits at the same time and even their potentials as biological control agents if they are to be exploited to mitigate harmful dinoflagellate blooms in the field. This study investigated total generation time, the number of zoospores produced per sporangium, zoospore size, swimming speed, parasite prevalence, zoospore survival and success rate, and host range and susceptibility for five perkinsozoan parasitoids. Four of the species (Dinovorax pyriformis, Tuberlatum coatsi, Parvilucifera infectans, and P. multicavata) were from the family Parviluciferaceae and one (Pararosarium dinoexitiosum) was from the family Pararosariidae, with dinoflagellate Alexandrium pacificum employed as a common host. Distinct differences in the biological traits of the five perkinsozoan parasitoid species were found, suggesting that the fitness of these parasitoids for the common host species differs. These results thus offer useful background information for the understanding of the impacts of parasitoids on the natural host population and for the design of numerical modeling including the host-parasitoid systems and biocontrol experiments in the field.

Mitochondrial metabolism of the facultative parasite Chilodonella uncinata (Alveolata, Ciliophora).

BACKGROUND: Chilodonella uncinata is an aerobic ciliate capable of switching between being free-living and parasitic on fish fins and gills, causing tissue damage and host mortality. It is widely used as a model organism for genetic studies, but its mitochondrial metabolism has never been studied. Therefore, we aimed to describe the morphological features and metabolic characteristics of its mitochondria. METHODS: Fluorescence staining and transmission electron microscopy (TEM) were used to observe the morphology of mitochondria. Single-cell transcriptome data of C. uncinata were annotated by the Clusters of Orthologous Genes (COG) database. Meanwhile, the metabolic pathways were constructed based on the transcriptomes. The phylogenetic analysis was also made based on the sequenced cytochrome c oxidase subunit 1 (COX1) gene. RESULTS: Mitochondria were stained red using Mito-tracker Red staining and were stained slightly blue by DAPI dye. The cristae and double membrane structures of the mitochondria were observed by TEM. Besides, many lipid droplets were evenly distributed around the macronucleus. A total of 2594 unigenes were assigned to 23 functional classifications of COG. Mitochondrial metabolic pathways were depicted. The mitochondria contained enzymes for the complete tricarboxylic acid (TCA) cycle, fatty acid metabolism, amino acid metabolism, and cytochrome-based electron transport chain (ETC), but only partial enzymes involved in the iron-sulfur clusters (ISCs). CONCLUSIONS: Our results showed that C. uncinata possess typical mitochondria. Stored lipid droplets inside mitochondria may be the energy storage of C. uncinata that helps its transmission from a free-living to a parasitic lifestyle. These findings also have improved our knowledge of the mitochondrial metabolism of C. uncinata and increased the volume of molecular data for future studies of this facultative parasite.

A comparative ultrastructural study on the nanoscale extrusomes of tintinnids (Alveolata, Ciliophora, Spirotricha) and their phylogenetic significance.

The capsules, putative extrusomes in tintinnid ciliates, are known since 1971. Based on their ultrastructure, shape, and size, five capsule types were distinguished and suggested to be of phylogenetic significance. However, detailed morphometric data and transmission electron micrographs are lacking to verify former conclusions. In the current study, comprehensive analyses of transmission electron microscopic data were performed, investigating 14 species from 13 genera and more than seven families collected in European coastal waters and in the Northeast Pacific. Our data suggest two main capsule types (large and ampulliform vs small and ellipsoidal/ovoidal) each including two subtypes characterised by their internal structures. Species groupings inferred from the capsule (sub-)types emerge also as closely related in gene trees. Additionally, the ampulliform type unites the Undellidae, Xystonellidae, and Tintinnid clade 2, while the shared possession of the small ellipsoidal type proposes a close relationship of Tintinnid clade 11 with the Rhabdonellidae and Cyttarocylididae. Thus, the capsules provide promising features to shed light on several unresolved evolutionary relationships among tintinnid genera and families; yet, information on capsules is still missing for many monophyletic groupings. Finally, we provide the first ultrastructural clues for the extrusive character of these organelles.

Amphibian Perkinsea.

Vanessa Smilansky and Thomas A. Richards introduce Perkinsea - a lineage of freshwater parasitic protists that infect certain amphibians and cause of severe Perkinsea infection.

The new chytridiomycete Paradinomyces triforaminorum gen. et sp. nov. co-occurs with other parasitoids during a Kryptoperidinium foliaceum (Dinophyceae) bloom in the Baltic Sea.

A new chytrid genus and species was isolated and cultured from samples obtained in the Baltic Sea during a dinoflagellate bloom event. This species is characterized by having a spherical sporangium without papillae and zoospores of 2-3 µm in diameter that are released through 3 discharge pores. Molecular phylogeny based on ribosomal operon showed its sister position to the Dinomyces cluster in Rhizophydiales. Zoospores lack fenestrated cisternae but contain a paracrystalline inclusion, found in a Rhizophydiales representative for the first time. Additionally, the kinetid features are uncommon for Rhizophydiales and only observed in Dinomyces representatives so far. These morphological features and its phylogenetic relationships justify the description of the new genus and speciesParadinomyces triforaminorum gen. nov. sp. nov. belonging to the family Dinomycetaceae. The chytrid was detected during a high-biomass bloom of the dinoflagellate Kryptoperidinium foliaceum. Laboratory experiments suggest this species is highly specific and demonstrate the impact it can have on HAB development. The chytrid co-occurred with three other parasites belonging to Chytridiomycota (Fungi) and Perkinsea (Alveolata), highlighting that parasitic interactions are common during HABs in brackish and marine systems, and these multiple parasites compete for similar hosts.

Morphological diversity and molecular phylogeny of five Paramecium bursaria (Alveolata, Ciliophora, Oligohymenophorea) syngens and the identification of their green algal endosymbionts.

Paramecium bursaria is a mixotrophic ciliate species, which is common in stagnant and slow-flowing, nutrient-rich waters. It is usually found living in symbiosis with zoochlorellae (green algae) of the genera Chlorella or Micractinium. We investigated P. bursaria isolates from around the world, some of which have already been extensively studied in various laboratories, but whose morphological and genetic identity has not yet been completely clarified. Phylogenetic analyses of the SSU and ITS rDNA sequences revealed five highly supported lineages, which corresponded to the syngen and most likely to the biological species assignment. These syngens R1-R5 could also be distinguished by unique synapomorphies in the secondary structures of the SSU and the ITS. Considering these synapomorphies, we could clearly assign the existing GenBank entries of P. bursaria to specific syngens. In addition, we discovered synapomorphies at amino acids of the COI gene for the identification of the syngens. Using the metadata of these entries, most syngens showed a worldwide distribution, however, the syngens R1 and R5 were only found in Europe. From morphology, the syngens did not show any significant deviations. The investigated strains had either Chlorella variabilis, Chlorella vulgaris or Micractinium conductrix as endosymbionts.

Architecture and tube structure of Sabellaria spinulosa (Leuckart, 1849): Comparison with the Mediterranean S. alveolata congener.

The Atlantic-Mediterranean polychaete Sabellaria spinulosa (Leuckart, 1849) lives in agglutinated tubes forming discrete reef-like bioconstructions on shallow-water bottoms beaten by waves where sediment particles are constantly resuspended. Tubes are built with sand grains glued by a proteinaceous cement. Analyses of a S. spinulosa reef sample of this worm collected off the Casarza coast (central Adriatic Sea) allowed the description of its tube architecture and gluing modality. The tube consists of three layers of agglutinated sand: (a) a thin inner layer with sandy particles arranged side by side with a flat side facing the tube lumen (b) a thick middle layer with larger isodiametric and squat grains with empty pores in between; and (c) a thin discontinuous outer layer of heterometric clasts, prevalently large and flat, diverging towards the opening. This fits the general tube construction known for S. alveolata and in general for tubes of the sabellariidae family, but compared to Sabellaria alveolata, S. spinulosa possesses a smaller tube with a wall about 1/3 thick; the agglutinated sandy elements are finer, and some number of muddy particles participates in the tube construction. Morphological and epifluorescence observations revealed that biocement portions are irregularly and haphazardly distributed compared with those of S. alveolata that consist of drops and strips of glue carefully placed. Adjacent tubes leave empty interspaces in between them only locally filled by loose sand, extremely reduced to absent in S. alveolata.

Rumen ciliates (Alveolata, Ciliophora) associated with goats: checklist, geographic distribution, host specificity, phylogeny and molecular dating.

Although the diversity (~35 species) and worldwide distribution of goats (Ruminantia, Bovidae, Caprinae) are significant, studies on the diversity of symbiont ciliates in these mammals are scarce in comparison to other ruminants. The present work is a review and checklist of species based on taxonomic, morphologic, and ecologic studies of rumen ciliate protozoa in goats, presenting geographic distribution and hosts, as well as estimating the macroevolutionary relationships of the species observed in the studies. To that end, all of the available literature on databases was reviewed, the schematic drawings were made based on information present in the original description of the taxa, and the phylogenetic relationships were inferred based on Maximum Likelihood and Bayesian Inference analyses. According to our review, 72 species and 14 genera of ciliates have been associated with goats. Through the analysis of the association between ciliate genera and caprine hosts, it was shown that ciliates are more associated with domestic animals (Capra hircus14 genera) than wild ones (Rupicapra rupicaprasix genera, Capra ibexone genus, Capra pyrenaicaone genus). Thirteen countries were identified in the distribution map as having had reports of ciliate species associated with goats. The interaction networks of ciliates and their hosts showed that the species of ciliates associated with goats also occur in other herbivore mammal species. The recovered phylogenetic hypotheses show that the ciliate species in goats form a non-monophyletic group with maximum and minimum ages of ~8.2My and ~2.4My. We have also found that a large portion of the studies on the diversity of ciliates in goats does not employ all necessary techniques in an integrative way, despite it being essential for detailed descriptions and better knowledge of this fraction of biodiversity.

Fractal analysis highlights analogies in arenaceous tubes of Sabellaria alveolata (Metazoa, Polychaeta) and agglutinated tests of foraminifera (Protista).

Bioconstructions of Sabellaria alveolata (Polychaeta Sabellariidae) from southern Sicily (Central Mediterranean) were sampled and analysed through a multidisciplinary approach in order to unravel the construction pattern of arenaceous tubes and explore possible analogies existing between the worm tubes and the agglutinated tests of benthic foraminifera (Protista). Scanning Electron Microscopy and Energy Dispersive Spectroscopy analyses were carried out on entire tubes as well as sectioned ones. Results show that arenaceous tubes are built following a rigorous architectural framework, based on selection and methodical arrangement of the agglutinated grains, and show surprising analogies with the test microstructure previously observed in agglutinated foraminifera. The grain distribution detected in both model species bioconstructions was analysed using a fractal numerical model (Hausdorff fractal dimension). Collected data show that in both organisms the grains were distributed according to a fractal model, indicating that the evolutionary process may have led to finding the same optimal constructive strategy across organisms with an independent evolutionary history, notwithstanding different geometrical scales. Furthermore, in sectioned tubes we observed microplastic fragments agglutinated within the arenaceous wall and in the inter-tube area. This unexpected finding shows that marine animals can be affected by microplastic pollution not only in soft tissues, but also engineered hard structures, and suggests the problem is more pervasive than estimated so far.

The characterization of novel monomeric creatine kinases in the early branching Alveolata species, Perkinsus marinus: Implications for phosphagen kinase evolution.

The genome of the unicellular molluscan parasite Perkinsus marinus contains at least five genes coding for putative creatine kinases (CK), a phosphoryl transfer enzyme which plays a key role in cellular energy transactions. Expression and kinetic analyses of three of the P. marinus CKs revealed them to be true CKs with catalytic properties in the range of typical metazoan CKs. A sequence comparison of the P. marinus CKs with a range of CK dimers and other dimeric phosphoryl transfer enzymes in this family (phosphagen kinases) showed that the P. marinus CKs lacked some of the critical residues involved in dimer stabilization, a trait all previously characterized CKs share. Size exclusion chromatography of all three expressed P. marinus CK constructs indicated they are monomeric, consistent with the observed lack of some critical dimer stabilizing residues. Phylogenetic analyses of the P. marinus CKs and putative dinoflagellate CKs with a broad range of monomeric and dimeric phosphagen kinases revealed that the Perkinsus CKs form a distinct, well-supported clade with dinoflagellate CKs which also lack the dimer stabilizing residues. Analysis of the genomic data for P. marinus showed the presence of putative genes for the two enzymes associated with creatine biosynthesis. CK in higher organisms plays a critical role in energy buffering in cell types displaying high and variable rates of ATP turnover. The presence of multiple CKs and the creatine biosynthetic pathway in P. marinus indicates that this unicellular parasite has the full complement of molecular machinery for CK-mediated energy buffering.

Selection or random picking? Foraminiferal tests in Sabellaria alveolata (Linnaeus, 1767) bioconstructions.

This work investigates whether the honeycomb worm Sabellaria alveolata (Polychaeta) actively selects foraminiferal tests to build its arenaceous bioconstructions and if the tests are chosen based on a defined criterion. To this purpose, both foraminiferal content and structure of communities were compared across samples of bioconstructions and samples of neighbouring sediment collected from two sites of southern Sicily (Central Mediterranean). Results document a higher concentration of foraminiferal tests within the tubes than in sediment, with a clear preference for biconvex and spherical morphologies. We hypothesize that the high proportion of biconvex and spherical tests in the bioconstruction is probably due to the active selection operated by the polychaete combined with the different buoyancy of foraminiferal tests kept in suspension by the wave motion. Among the grains agglutinated in the tubes, we also observed sporadic specimens of Amphistegina lobifera Larsen, a non-indigenous foraminifer native to the Red Sea.

Alveolates (dinoflagellates, ciliates and apicomplexans) and Rhizarians are the most common microbial eukaryotes in temperate Appalachian karst caves.

The purpose of this study was to survey the eukaryotic microbiome of two karst caves in the Valley and Ridge physiographic region of the Appalachian Mountains. Caves are known to harbour eukaryotic microbes but their very low densities and small cell size make them difficult to collect and identify. Microeukaryotes were surveyed using two methodologies, filtering water and submerging glass microscope slides mounted in periphytometers in cave pools. The periphyton sampling yielded 13.5 times more unique amplicon sequence variants (ASVs) than filtered water. The most abundant protist supergroup was Alveolata with large proportions of the ASVs belonging to dinoflagellate, ciliate and apicomplexan clades. The next most abundant were Rhizarians followed by Stramenopiles (diatoms and chrysophytes) and Ameobozoans. Very few of the ASVs, 1.5%, matched curated protist sequences with greater than 99% identity and only 2.5% could be identified from surface plankton samples collected in the same region. The overall composition of the eukaryotic microbiome appears to be a combination of bacterial grazers and parasitic species that could possibly survive underground as well as cells, cysts and spores probably transported from the surface.

Morphological and phylogenetic investigations shed light on evolutionary relationships of the enigmatic genus Copemetopus (Ciliophora, Alveolata), with the proposal of Copemetopus verae sp. nov.

Copemetopus Villeneuve-Brachon, 1940 is a rare, poorly known sapropelic ciliate genus composed of only two valid nominal species. Over time, Copemetopus was taxonomically assigned to Heterotrichea and Armophorea classes, but its phylogenetic affinities remained unknown. Until the present study, there were no molecular data available for Copemetopus representatives. Here, we present the 18S and 28S-rDNA sequences and the phylogenetic position of Copemetopus verae sp. nov., as well as its detailed morphological description based on live observations, protargol impregnation, and scanning electron microscopy. Transmission electron micrographs of the type species C. subsalsus Villeneuve-Brachon, 1940 reveal new morphological traits and a unique somatic ciliature pattern of Copemetopus, composed by short segments of dikinetids with one or two supplementary kinetosomes. The phylogenetic trees recovered Copemetopus as the sister group of the genus Protocruzia, both constituting early-divergent lineages that split first from a common ancestor of Intramacronucleta. Morphological and molecular evidence suggest that Copemetopus is neither a heterotrichean nor an armophorean ciliate, but a distinct clade related to Protocruzia.

Evolutionary Analysis of Dipeptidyl Peptidase I.

Human dipeptidyl peptidase I (DPPI) belongs to the family of papain-like cysteine peptidases. Its distinctive features are the unique exclusion domain which enables the eponymous activity and homotetramerization of DPPI, and its dependence on chloride ions for enzymatic activity. The oligomeric state of DPPI is unique in this family of predominantly monomeric peptidases. However, a distant DPPI ortholog from Plasmodium falciparum has been shown to be monomeric, indicating that the oligomeric state of DPPI varies between lineages. The aim of this work was to study the evolution of DPPI, with particular attention to the structural features that determine its characteristic enzymatic activity and preferences, and to reconstruct the evolution of its oligomerization. We analyzed fifty-seven selected sequences of DPPI and confirmed its presence in three lineages, namely, Amorphea (including animals and Amoebozoa), Alveolates and the metamonad Giardia. The amino acid residues that bind the chloride ion are highly conserved in all species, indicating that the dependence on chloride ions for activity is an evolutionarily conserved feature of DPPI. The number of N-glycosylation sites is significantly increased in animals, particularly vertebrates. Analysis of homology models and subunit contacts suggests that oligomerization is likely restricted to DPPIs in the Amorphea group.

Somatic infraciliature in tintinnid ciliates (Alveolata, Ciliophora, Spirotricha): An ultrastructural comparison.

A recent ultrastructural study on the tintinnid ciliate Schmidingerella meunieri displayed unique types of somatic kinetids. The dikinetids (paired basal bodies) have, besides kinetodesmal fibrils and transverse ribbons, some special features, that is, overlapping postciliary ribbons and three extraordinary microtubular ribbons, which together form a conspicuous network in the ciliated anterior cell portion. The distribution of this feature among tintinnids is studied in chemically fixed and ultrathin-sectioned specimens from six genera and five families collected in European coastal waters. The taxa are scattered across the molecular tree. Actually, the somatic kinetids of these six genera share the special features discovered in S. meunieri. Accordingly, the overlapping postciliary ribbons and the three extraordinary ribbons were already present in the early stages of tintinnid evolution, namely in the last common ancestor of tintinnids with hard loricae. Owing to the lack of ultrastructural data in the basally branching Tintinnidiidae with their soft loricae and in aloricate choreotrichids other than the aberrant strobilidiids, the first appearance of the structures is still uncertain. The related oligotrichids do not possess overlapping postciliary ribbons, but show electron-dense material at the sites where the ribbons I-III originate in tintinnids. None of these features is found in any other spirotrich ciliate.