Trichite features contribute to the revision of the genus Strombidium (Alveolata, Ciliophora, Spirotricha).

Strombidium is a species-rich genus of oligotrichid ciliates mainly inhabiting the marine pelagial. In molecular phylogenies, the genus emerged as non-monophyletic, and cladistic analyses suggest that it is largely characterized by plesiomorphies. A reliable split of the genus and the establishment of new genera necessitate, however, support by novel morphological and/or ultrastructural features. In the present study, the arrangement and ultrastructure of trichites are proposed as taxonomically relevant characters. Strombidium biarmatum Agatha et al., 2005 differs in the trichite pattern from the type species Strombidium sulcatum and most congeners. Aside from the trichites inserting anteriorly to the girdle kinety and generating the typical funnel-shaped complex in the posterior cell portion, the species displays additional trichites between the adoral membranelles even visible in live cells. Here, this exceptional trichite arrangement is detailed based on transmission electron microscopic investigations. In molecular phylogenies, S. biarmatum forms a monophylum with two congeners sharing its trichite arrangement. Therefore, the strombidiid genus Heteropilum nov. gen. is established with S. biarmatum as type species to also include H. paracapitatum (Song et al., 2015) nov. comb. and H. basimorphum (Martin & Montagnes, 1993) nov. comb. Further differences discovered in the trichite ultrastructure support the organelles' taxonomic significance.

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.

A light and electron microscopical study on the resting cyst of the tintinnid Schmidingerella (Alveolata, Ciliophora) including a phylogeny-aware comparison.

Resting cysts protect ciliates against adverse environmental conditions. The morphology and ultrastructure of resting cysts has been described in very few Oligotrichea, a group of mainly marine planktonic ciliates. The present study provides the first ultrastructural data for loricate choreotrichids, applying light and electron microscopy on the cysts of the tintinnid Schmidingerella meunieri (Kofoid and Campbell, 1929) Agatha and Strüder-Kypke, 2012. The morphology of live cysts and the wall ultrastructure of cryofixed cysts were morphometrically analysed. The resting cyst is roughly flask-shaped, broadening to a slightly concave, laterally protruding anterior plate. An emergence pore closed by a skull cap-shaped papula is directed to the bottom of the lorica on the opposite side of the cyst. The cyst wall consists of an ectocyst, mesocyst, and endocyst differing in thickness, structure, and nitrogen concentration as revealed by conventional transmission electron microscopy, electron energy loss spectroscopy, and electron spectroscopic imaging. The cysts of S. meunieri belong to the kinetosome-resorbing type, which also occurs in the majority of hypotrich ciliates. Two main features (flask-shape and presence of an emergence pore) are shared with the closely related aloricate choreotrichids and oligotrichids, distinguishing the Oligotrichea from the hypotrich and the more distantly related euplotid ciliates.

Taxonomy and phylogeny of the freshwater tintinnid Tintinnopsis tubuformis Chiang, 1956 (Ciliophora, Oligotrichea) and a proposed synonymization of T. longa nom. corr. Chiang, 1956.

Tintinnid ciliates are traditionally identified by their loricae; however, increasing evidence indicates that some lorica features (e.g. its length, spiraled structures) are not reliable. The vast majority of tintinnids inhabit the marine pelagial; merely, about thirty species live in freshwater. In the present study, two morphotypes with similar lorica shapes and opening diameters but deviating lorica lengths were isolated from freshwater samples collected at different water temperatures near Chongming Island in the Yangtze Estuary, China. The specimens were studied in vivo and after protargol staining, and their phylogenetic placement was inferred from three ribosomal RNA markers; further, cell division was investigated in the short morphotype. Based on the original descriptions, the longer morphotype is identified as Tintinnopsis longa nom. corr. Chiang, 1956, and the shorter one as Tintinnopsis tubuformis Chiang, 1956. Despite distinct differences in the lorica lengths, the identity of the three molecular markers in both morphotypes suggests conspecificity, which is supported by overlapping ranges in the lorica opening diameters and the length-independent features of the somatic ciliary pattern (e.g. number of kineties). Hence, we synonymized T. longa nom. corr. with T. tubuformis and neotypified the later species.

Molecular signature characters complement taxonomic diagnoses: A bioinformatic approach exemplified by ciliated protists (Ciliophora, Oligotrichea).

Traditionally, taxa following the botanical or zoological codes of nomenclature are diagnosed mainly by morphological characters, although integrative taxonomy advocates including additional features. While many taxonomic studies include DNA sequence analyses, a systematic integration of diagnostic molecular characters (signature characters) is still rare. Here, we suggest a practical guideline for the detection and evaluation of signature characters that provides the means necessary to complement diagnoses and facilitates identifications. The guideline comprises generally applicable criteria exemplified by a case study on an ecologically important group of planktonic protists, the Oligotrichea. The detection of signature characters and their discrete states in multiple sequence alignments is facilitated by the recently developed tool DeSignate. Moreover, we introduce a novel bioinformatic approach to test the influence of different alignment programs on the consistency of signature characters. Our workflow enabled detection of consensus signature characters for most tested taxa and inclusion of such characters in the diagnoses of three orders, eight families, and two genera in the Oligotrichea. The suggested approach is a step towards an integrative taxonomy linking reliable molecular sequence data to organisms' traits.

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.

A comparative ultrastructural study of tintinnid loricae (Alveolata, Ciliophora, Spirotricha) and a hypothesis on their evolution.

Tintinnid ciliates build loricae, whose structure, shape, and size still largely represent the basis for taxonomy and classification, although genetic analyses demonstrated their limited utility for inferring evolutionary relationships. The textures of the lorica walls, however, result from the chemical and physical properties of the forming material, which is supposed to be rather conserved in closely related taxa, viz., congeners and confamilial genera. Within a particular texture, small deviations in the chemical composition might affect the wall's stickiness and accordingly its capability to adhere foreign particles, explaining the intertwining of tintinnids with hyaline and agglutinated loricae in phylogenetic inferences. In a comprehensive comparative study, the lorica textures were electron microscopically and morphometrically analyzed in 21 species from 17 genera and more than nine families together with literature data. Most species were investigated for the first time, and the taxa cover a substantial portion of the molecular genealogy. The phylogeny-aware analysis of the lorica-related features provides a preliminary hypothesis on lorica evolution. Eventually, this conspectus suggests the dominance of hard lorica walls with an alveolar texture and proposes different modes of lorica formation.

The importance of type species and their correct identification: A key example from tintinnid ciliates (Alveolata, Ciliophora, Spirotricha).

Types and the corresponding rules in the International Code of Zoological Nomenclature are crucial for taxonomy and are meant to provide nomenclatural stability. In the case of neotypification, especially diligent taxonomic work is required to retain continuity. In the present communication, we first outline the main principles of typification and neotypification. We then discuss a critical case, using a current example from the marine planktonic tintinnid genus Tintinnopsis Stein, 1867 (Alveolata, Ciliophora). This diverse and ubiquitous genus is nonmonophyletic, but its revision and the erection of new related genera is currently prevented by the uncertain affiliation of its type species.

Symposium on Ciliates in Memory of Denis Lynn.

Denis Lynn (1947-2018) was an outstanding protistologist, applying multiple techniques and data sources and thus pioneering an integrative approach in order to investigate ciliate biology. For example, he recognized the importance of the ultrastructure for inferring ciliate phylogeny, based on which he developed his widely accepted classification scheme for the phylum Ciliophora. In this paper, recent findings regarding the evolution and systematics of both peritrichs and the mainly marine planktonic oligotrichean spirotrichs are discussed and compared with the concepts and hypotheses formulated by Denis Lynn. Additionally, the state of knowledge concerning the diversity of ciliates in bromeliad phytotelmata and amitosis in ciliates is reviewed.

Molecular Data Reveal a Cryptic Diversity in the Genus Urotricha (Alveolata, Ciliophora, Prostomatida), a Key Player in Freshwater Lakes, With Remarks on Morphology, Food Preferences, and Distribution.

Species of the ciliate genus Urotricha are key players in freshwater plankton communities. In the pelagial of lakes, about 20 urotrich species occur throughout an annual cycle, some of which play a pivotal role in aquatic food webs. For example, during the phytoplankton spring bloom, they consume a remarkable proportion of the algal production. In ecological studies, urotrich ciliates are usually merely identified to genus rank and grouped into size classes. This is unsatisfying considering the distinct autecological properties of individual species and their specific spatial and temporal distribution patterns. As a basis for future research, we characterized in detail four common urotrich morphotypes, i.e., specimens identified as U. furcata and tentatively as U. agilis, U. pseudofurcata, and U. castalia, using state-of-the-art methods. We used an integrative polyphasic approach, in which morphological studies (in vivo observation, silver staining methods, scanning electron microscopy) were linked with a molecular approach exploiting four different gene fragments as taxonomic DNA barcodes with different resolution potential (SSU rDNA, ITS-1, ITS-2, hypervariable V4 and V9 regions of the SSU rDNA). We shed light on the diversity of urotrich ciliates as well as on their global distribution patterns, and annual cycles. Additionally, we coupled individual species occurrences and environmental parameters, and subsequently modeled the distribution and occurrence, using logistic regressions. Furthermore, for one strain putatively identified as U. castalia, we ascertained the optimal cultivation media and food preferences. Thereby, our comprehensive view on these important freshwater ciliates that frequently occur in environmental high throughput sequencing datasets worldwide will allow future studies to better exploit protistan plankton data from lakes.

Ultrastructural Studies on a Model Tintinnid - Schmidingerella meunieri (Kofoid & Campbell, 1929) Agatha & Strüder-Kypke, 2012 (Ciliophora). II. The Oral Apparatus.

The ultrastructure of the oral apparatus is supposed to be significant for elucidating more recent common ancestry and might thus provide support for particular groupings of oligotrichean ciliates. The transmission electron microscopical study on mainly cryofixed Schmidingerella meunieri specimens provides the first detailed data for tintinnids and Oligotrichea in general. Ten new characters are included into the cladistic analysis. These features together with the very limited body of literature suggest that substantial changes in the oral ultrastructure correlate only with the formation of a circular adoral zone in choreotrichids. Despite homoplasious morphological and ontogenetic adaptations to the planktonic lifestyle in halteriid hypotrichs and oligotrichids, their oral apparatuses generally retain the plesiomorphic ultrastructure of the Perilemmaphora. The highly complex ultrastructure of the adoral zone is thus able to accomplish an extension in the zone's functionality without obvious changes; only the position of the adoral zone at the apical cell portion together with a globular to obconical cell shape are apparently crucial. Merely, minute apomorphies characterise the Oligotrichea and tintinnids, respectively. Tintinnids with derived somatic ciliary patterns possess distinct microtubular bundles connecting the oral apparatus with the myoneme in the peduncle.

DeSignate: detecting signature characters in gene sequence alignments for taxon diagnoses.

BACKGROUND: Molecular characters have been added in integrative taxonomic approaches in recent years. Nevertheless, taxon diagnoses are still widely restricted to morphological characters. The inclusion of molecular characters into taxon diagnoses is not only hampered by problems, such as their definition and the designation of their positions in a reference alignment, but also by the technical effort. RESULTS: DeSignate is a tool for character-based taxon diagnoses that includes a novel ranking scheme. It detects and classifies individual and combined signature characters (diagnostic molecular characters) based on so-called character state vectors. An intuitive web application guides the user through the analysis process and provides the results at a glance. Further, formal definitions and a uniform terminology of characters are introduced. CONCLUSIONS: DeSignate facilitates the inclusion of diagnostic molecular characters and their positions to complement taxon diagnoses. Compared to previous solutions, the tool simplifies the workflow and improves reproducibility and traceability of the results. The tool is freely available as a web application at (https://designate.dbresearch.uni-salzburg.at/) and is open source (https://github.com/DatabaseGroup/DeSignate/).

Redescription of Dexiotricha colpidiopsis (Kahl, 1926) Jankowski, 1964 (Ciliophora, Oligohymenophorea) from a Hot Spring in Iceland with Identification Key for Dexiotricha species.

We isolated an encysted ciliate from a geothermal field in Iceland. The morphological features of this isolate fit the descriptions of Dexiotricha colpidiopsis Kahl, 1926) Jankowski, 1964 very well. These comprise body shape and size in vivo, the number of somatic kineties, and the positions of macronucleus and contractile vacuole. Using state-of-the-art taxonomic methods, the species is redescribed, including phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) gene as molecular marker. In the phylogenetic analyses, D. colpidiopsis clusters with the three available SSU rRNA gene sequences of congeners, suggesting a monophyly of the genus Dexiotricha. Its closest relative in phylogenetic analyses is D. elliptica, which also shows a high morphological similarity. This is the first record of a Dexiotricha species from a hot spring, indicating a wide temperature tolerance of this species at least in the encysted state. The new findings on D. colpidiopsis are included in a briefly revision of the scuticociliate genus Dexiotricha and an identification key to the species.

Some Ultrastructural Features of the Planktonic Freshwater Ciliate Limnostrombidium viride (Alveolata, Ciliophora, Oligotrichida) and Improved Diagnoses of Oligotrich Taxa.

The first transmission and scanning electron microscopical studies in combination with freeze-fracture technology have disclosed some important morphological and ultrastructural features in the freshwater oligotrichid Limnostrombidium viride. (I) The dikinetids (paired basal bodies) of the girdle kinety have a club-shaped cilium associated only with each left basal body. The electron-dense (paraflagellar) body on one side of its "9×2+2"-axoneme and the regular array of intramembranous particles indicate a sensory, perhaps photoreceptor function of these club-shaped cilia. (II) The stichomonad endoral membrane is proximally covered by a cytoplasmic fold and distally by multiple membranous layers. Thus entirely covered, the endoral is probably no longer involved in food capture; nonetheless, its associated microtubules might stabilise the cytopharynx. (III) Instead of a contractile vacuole, a horizontal ring-canal with supposed osmoregulatory function occurs. (IV) The extrusive nature of the trichites is not only observed in electron micrographs, but the attachment sites of these organelles also display a rosette of "8+1"-particles in the P-face of freeze-fracture replicas typical for ciliate extrusomes. (V) The neoformation organelle, the subsurface tube in which stomatogenesis takes place, shows short basal bodies and normal axonemes about 1 µm long. It is accompanied by numerous membrane vesicles, which might provide membrane material for the outgrowing cilia.

Ultrastructural Studies on a Model Tintinnid - Schmidingerella meunieri (Kofoid and Campbell, 1929) Agatha and Strüder-Kypke, 2012 (Ciliophora). I. Somatic Kinetids with Unique Ultrastructure.

Molecular phylogenies of Oligotrichea currently do not contain all genera and families and display topologies which are often incongruent with morphological findings. In ciliates, the somatic kinetids are rather conserved, i.e., their ultrastructures, particularly the fibrillar associates, often characterise the main groups, except for the choreotrichids. Four different kinetid types are found in protargol-stained choreotrichids and used for reconstructing the taxon's evolution (the "Kinetid Transformation Hypothesis"). Proof for this hypothesis requires transmission electron microscopic studies, which are very rare in the choreotrichids and oligotrichids. Such an approach provides insights into the ultrastructural variability of somatic kinetids in spirotrichs and may also detect apomorphies characterising certain choreotrichid families. In the model tintinnid Schmidingerella meunieri, the ultrastructure of the three kinetid types in the somatic ciliature is studied in cryofixed cells. The data support the "Kinetid Transformation Hypothesis" regarding tintinnids with a ventral kinety. This first detailed study on kinetids in tintinnids and choreotrichids in general reveals totally new kinetid types in ciliates: beyond the three common associates, they are characterised by two or three conspicuous microtubular ribbons extending on the kinetids' left sides. These extraordinary ribbons form together with the overlapping postciliary ribbons a unique network in the cortex of the anterior cell portion. The evolutionary constrains which might have fostered the development of such structures are discussed for the Oligotrichea, the choreotrichids, and tintinnids as their first occurrence is currently uncertain. Additionally, the kinetids in tintinnids, aloricate choreotrichids, oligotrichids, hypotrichs, and euplotids are compared.

Redescription of Antetintinnidium mucicola (Claparède and Lachmann, 1858) nov. gen., nov. comb. (Alveolata, Ciliophora, Tintinnina).

Tintinnid ciliates have traditionally been described and classified exclusively based on their lorica features. Although information on the cell characters is urgently needed for a natural classification, more molecular than cytological data has been accumulated over recent years. Apparently, the tintinnids developed in the marine environment and entered freshwater several times independently. Typical freshwater tintinnids belong to the genera Tintinnidium and Membranicola. The species are comparatively well-known regarding their morphology and characterised by two unusual de novo originating ciliary rows, the ventral organelles. In contrast, the cell features in the marine/brackish Tintinnidium species, specifically their somatic ciliary patterns, are insufficiently known or not known at all. Therefore, the morphology of a common marine/brackish representative, Tintinnidium mucicola, is redescribed based on live observation and protargol-stained material. Furthermore, biogeographical and autecological data of the species are compiled from literature and own records. The phylogenetic relationships of T. mucicola are inferred and the diversity of the family Tintinnidiidae is assessed from 18S rDNA sequences. The study shows that T. mucicola is not only molecularly distinct, but also characterised by many plesiomorphic features, for instance, it does not possess a verifiable homologue to the ventral organelles. Hence, a new genus, Antetintinnidium nov. gen., is established for T. mucicola. The new insights into the diversity of Tintinnidiidae shed light on the early evolution of tintinnids and might provide clues on their adaptions to freshwater.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Redescription of Tintinnopsis everta Kofoid and Campbell 1929 (Alveolata, Ciliophora, Tintinnina) Based on Taxonomic and Genetic Analyses-Discovery of a New Complex Ciliary Pattern.

The about 1,000 species of tintinnid ciliates are identified and classified almost exclusively based on their lorica features, although the shortcomings of this structure are well-known, e.g. causing uncertain species limitations and nonmonophyletic taxa. Hence, the present redescription of Tintinnopsis everta Kofoid and Campbell, 1929 considers not only the lorica characteristics, but focuses on cell and genetic features. The species is redescribed from the North Atlantic and adjacent sea areas, namely the east coast of the USA, using live observation, protargol-stained material, scanning electron microscopy, and genetic analyses. The main stages of cell division are described, and the species' phylogenetic relationships are inferred from morphological data and the small subunit ribosomal RNA gene sequence. The estimates of its biogeographical distribution and autecology are based on a literature survey. The species is characterised by a complex somatic ciliary pattern with a unique position of the posterior kinety and a conspicuously large distance between the somatic ciliary fields and the collar membranelles. The phylogenetic relationships of Tintinnopsis everta vary in the molecular trees depending on the algorithms used and are, therefore, regarded as unresolved. Nevertheless, the new kind of complex somatic ciliary pattern distinctly contributes to a better understanding of the tintinnid biodiversity and evolution and provides features for a future split of the nonmonophyletic genus Tintinnopsis.

Three rDNA Loci-Based Phylogenies of Tintinnid Ciliates (Ciliophora, Spirotrichea, Choreotrichida).

To improve understanding of diversity, phylogeny and evolution in tintinnid ciliates, it is essential to link multiple molecular markers with properly identified and documented morphospecies. Accordingly, 54 tintinnid morphospecies/isolates mainly from the Yellow and East China Seas were collected and analysed. Using single-cell approaches, sequences were obtained for three rDNA loci (18S, ITS1-5.8S-ITS2, D1-D5 region of 28S). Twenty-six tintinnid morphospecies (29 isolates) are documented by micrographs, measurements, morphologically described, and compared with the original species description. Three rDNA loci-based phylogenetic analyses were then performed for these identified isolates. Sequences from 25 unidentified species/isolates were also included in the comparison of the three rDNA loci. Ribosomal DNA genes of the genus Leprotintinnus were analysed for the first time, showing that Leprotintinnus was closely related to Tintinnopsis radix and branched distinctly apart from the family Tintinnidiidae. Four novel clades (VI to IX) of the Tintinnopsis complex emerged in the 18S genealogies. Analyses of the relative variability in the ITS and 28S regions vs. the 18S rDNA showed that the ITS1-5.8S-ITS2 and ITS2 regions well co-varied with the 18S rDNA when the variations of the latter were less than 3%, whereas at difference of less than 1%, no correlation was found between the compared loci. These findings highlight the difficulties in using variable locus-based cut-off divergences in circumscribing tintinnid morphospecies.