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Heat resistance is the ability to survive short, extreme temperature stresses, exceeding the own growth temperature by far. Despite their occurrence in natural substrates and their relevance for the food and healthcare industry, the diversity of fungi with heat resistance abilities is poorly studied. Sampling of boreal forest soils in Canada in combination with a heat-shock treatment (75 °C, 30 min) yielded, among others, four heat resistant, mesophilic fungal isolates. Based on rDNA barcode sequences, the novel isolates were assigned to Basidiomycota. In this study, we use macromorphological and micromorphological observations, cultivation assays and comparative genomics for physiological characterization, interspecific differentiation, and phylogenetic placement of these isolates. A phylogeny of 38 single-copy orthologous genes, an orthology analysis, and septal pore type analysis revealed the isolates as representatives of two new basidiomycetous species of the novel class Peribolosporomycetes, a sister lineage to all other members of Ustilaginomycotina. Further genomic and phenotypic data support two different species (Peribolospora kevripleyi, Peribolospora baueri), which are heat resistant and osmotolerant.

Thwaites Glacier (TG) is thinning and accelerating while sitting on a landward-dipping bed, with an ice shelf that is rapidly disintegrating and losing its ability to buttress ice flow from upstream, and is in deep water that allows warm Circumpolar Deep Water (CDW) to reach its grounding zone. Significant retreat of TG would trigger loss of ice across the region. In recent decades, the mass balance of TG has become increasingly negative, suggesting that unstable retreat may have already begun. The Thwaites Offshore Research (THOR) group has just completed four field deployments aimed at understanding the recent history of TG and neighboring ice, including Pine Island Glacier (PIG). Three cruises on the RVIB N.B. Palmer, combined with sub-ice-shelf sediment coring, provide a suite of new data along the TG and PIG margins. Data include multibeam surveys, 3.5 kHz subbottom profiler, over 100 new sediment cores, and high-resolution seismic profiles. Break-up of floating ice cover in front of TG in 2019 allowed surveying of previously unmapped seafloor. Major calving of PIG in 2020 allowed marine surveying over the locations where sub-ice-shelf cores were collected in the past, allowing direct ties between ice-based and marine work. As of this writing in January 2022, we are entering into the Amundsen Sea for our third marine field season. Sediment cores record the history of grounding-zone retreat and ice interaction with the ocean over timescales from decades to several thousand years. Proxies used to reconstruct ice and ocean histories include sedimentary facies analysis, diatom and foraminiferal assemblage data, and geochemical analyses. Sedimentological analyses show a diverse array of lithofacies attributed to different environmental conditions. Many cores across the region contain laminated mud with sparse gravel and sand, suggesting deposition of meltwater deposits. Downcore 210Pb measurements are used to create age models of the past ~100 years. Combination of ages with facies models, including CT scans, reveals that the progressive detachment of Thwaites from pinning points began in the mid twentieth century, coincident with retreat of PIG (Smith et al., 2017) and with increasing advection of warm water onto the Amundsen shelf (Hillenbrand et al., 2017). Conversely, Cranton Bay, to the northeast of PIG and separated from Pine Island Bay by a shallow sill, appears to be characterized by cold deep water and high productivity, allowing it to serve as an endmember different from the records obtained proximal to the large glacial outlets where CDW is impinging. The satellite record of glacial retreat is inherently short. Observations are accumulating about forcing mechanisms that can impact the stability of ice, such as increased CDW on the Amundsen Sea shelf. However, without the time to observe the response of the ice, discovery of the forcing mechanisms is just half the story. The other half of the story is completed by using the paleo record to see how ice has responded to drivers in the past.

The coastal bathymetry of Thwaites Glacier (TG) is poorly known yet nearshore sea-floor highs have the potential to act as pinning points for floating ice shelves, or to block warm water incursions to the grounding line. In contrast, deeper areas control warm water routing. Here, we present more than 2000 km2 of new multibeam echo-sounder data (MBES) acquired offshore TG during the first cruise of the International Thwaites Glacier Collaboration (ITGC) project on the RV/IB Nathaniel B. Palmer (NBP19-02) in February-March 2019. Beyond TG, the bathymetry is dominated by a >1200 m deep, structurally-controlled trough and discontinuous ridge, on which the Eastern Ice Shelf is pinned. The geometry and composition of the ridge varies spatially with some sea-floor highs having distinctive flat-topped morphologies produced as their tops were planed-off by erosion at the base of the seaward-moving Thwaites Ice Shelf. In addition, submarine landform evidence indicates at least some unconsolidated sediment cover on the highs, as well as in the troughs that separate them. Knowing that this offshore area of ridges and troughs is a former bed for TG, we also used a novel spectral approach and existing ice-flow theory to investigate bed roughness and basal drag over the newly-revealed offshore topography. We show that the sea-floor bathymetry is a good analogue for extant bed areas of TG and that ice-sheet retreat over the sea-floor troughs and ridges would have been affected by high basal drag similar to that acting in the grounding zone today.Comparisons of the new MBES data with existing regional compilations show that high-frequency (finer than 5 km) bathymetric variability beyond Antarctic ice shelves can only be resolved by observations such as MBES and that without these data calculations of the oceanic heat flux may be significantly underestimated. This work supports the findings of recent numerical ice-sheet and ocean modelling studies that recognise the need for accurate and high-resolution bathymetry to determine warm water routing to the grounding zone and, ultimately, for predicting glacier retreat behaviour.

Percolomonads (Heterolobosea) are aquatic heterotrophic flagellates frequently found in saline waters up to hypersaline environments. We isolated and cultivated seven strains of percolomonad flagellates from marine waters and sediments as well as from a hypersaline inland lake in the Atacama Desert. Morphological characterizations, comprising light and scanning electron microscopy, revealed only slight differences between the strains mainly limited to the cell shape, length of flagella, and length of the ventral feeding groove. Phylogenetic analyses of the 18S and 28S rDNA genes showed the formation of three fully supported clades within the Percolomonadida: the Percolomonadidae, the Barbeliidae fam. nov. and the Lulaidae fam. nov. We describe two new families (Barbeliidae fam. nov., Lulaidae fam. nov.), a new genus (Nonamonas gen. nov.), and five new species (Percolomonas adaptabilis sp. nov., Lula levis sp. nov., Barbelia pacifica sp. nov., Nonamonas montiensis gen. et sp. nov., Nonamonas santamariensis gen. et sp. nov.). Salinity experiments showed that P. adaptabilis sp. nov. from the Atlantic was better adapted to high salinities than all other investigated strains. Moreover, comparisons of our cultivation-based approach with environmental sequencing studies showed that P. adaptabilis sp. nov. seems to be globally distributed in marine surface waters while other species seem to be more locally restricted.

The genus Hartaetosiga Carr, Richter and Nitsche, 2017 comprised up to now only three species, H. gracilis (Kent) Carr, Richter, Nitsche, 2017, H. balthica (Wylezich and Karpov) Carr, Richter and Nitsche, 2017 and H. minima (Wylezich and Karpov) Carr, Richter and Nitsche, 2017. Based on distinct molecular data these species were relocated from the strictly freshwater genus Codosiga (Ehrenberg) Bütschli, 1878 to a new genus comprising brackish and marine species. During the cruise MSM82/2 across the Atlantic Ocean in 2019, surface water samples were taken from 15 stations along a transect ranging from 35°S to 23°N. We were able to isolate and cultivate 14 strains of the genus Hartaetosiga. Morphometric data showed no distinct morphological traits allowing for a species delineation, indicating a cryptic species complex within the genus. Based on cultivation, morphological data, and molecular analyses, we recorded H. gracilis for the first time from off-shelf waters of the Atlantic Ocean and could describe a new species, H. australis n. sp. This new species was recorded from sampling stations in the Southern Hemisphere only, which may indicate a potential biogeographic restriction likely caused by the Equatorial Counter Current (ECC), dividing the northern and southern surface waters.

"Spumella"-like flagellates describes similar or even indiscernible colourless non-scaled chrysophytes which are important bacterivores common in different aquatic ecosystems. Recently, phylogenetic analyses revealed a high taxonomic diversity of these flagellates leading to the description of several new genera and species. Our present work on the functional group of pelagic bacterivorous chrysomonads from different water bodies resulted in an extended taxonomic analysis among chrysophytes unveiling yet undescribed genera and species pointing to the high hidden diversity of bacterivores in the pelagial of freshwaters. On the basis of phylogenetic analyses, we describe four new genera Atacamaspumella, Chlorospumella, Pseudapoikia, and Vivaspumella and a new species of the recently described genus Poteriospumella. Beside this, we redescribe the species Ochromonas vasocystis Doflein, 1923 to Poteriospumella vasocystis comb. nov. substantiated on the high sequence similarity with Poteriospumella lacustris Boenigk et Findenig and Poteriospumella maldiviensis nov. sp.

The Global Multi-Resolution Topography (GMRT) Synthesis is a multi-resolution Digital Elevation Model (DEM) developed at the Lamont-Doherty Earth Observatory of Columbia University. The data synthesis is maintained in three projections and is managed with a scalable global architecture and tiling scheme. Primary content assembled into GMRT includes a curated multibeam bathymetry component that is derived from processed swath files and is gridded at ~100m resolution or better. These data are seamlessly assembled with other publicly available gridded data sets, including bathymetry and topography data at a variety of resolutions. GMRT delivers the best resolution data that have been curated for a particular area of interest, and allows users to extract custom grids, images, points and profiles.Most data processing and curation effort for GMRT is focused on cleaning and reviewing ship-based multibeam sonar data to facilitate gridding at their full spatial resolution. In addition to performing standard data cleaning and applying necessary corrections to data, GMRT tools are used to review and assess swath data in the context of the existing data synthesis. This approach ensures that data are fit for purpose and will integrate well with existing content, and is especially well-suited for ensuring the quality of data acquired during transits. GMRT tools and workflows used for data cleaning and assessment have recently been adapted for distributed use to enable the broader community to leverage this approach, streamlining the data pipeline and ensuring high quality processed swath data can be delivered to public archives. This presentation will include a summary of GMRT tools, opportunities, and lessons learned.

The heterotrophic nanoflagellate genus Cafeteria has been found to be ubiquitously distributed in the marine realm. We could isolate and cultivate ten strains morphologically similar to Cafeteria from various types of environment, including the deep sea, brackish waters and also meso- to hypersaline inland waters. Molecular analyses (18S rDNA, 28S rDNA) of newly isolated strains from the marine realm resulted in four more Cafeteria burkhardae strains from the deep North Atlantic Ocean and one new species (C. baltica sp. nov.) isolated from brackish waters of the Baltic Sea. Two strains isolated from the Atacama Desert belong to two new species (C. atacamiensis sp. nov. and C. paulosalfera sp. nov.), one other strain could not yet be assigned. Morphological characterizations of these strains obtained by high resolution microscopy revealed only small differences to already described species. However, molecular analyses showed a clear separation of the different Cafeteria species. We exposed several strains to different salt concentrations (2-150 PSU) to investigate their salinity tolerance. Only the marine strains of C. burkhardae were able to survive at salinities up to 150 PSU, indicating the possibility to inhabit a broader spectrum of habitats including hypersaline environments besides the deep sea with its high hydrostatic pressure.

Heterotrophic protists (unicellular eukaryotes) form a major link from bacteria and algae to higher trophic levels in the sunlit ocean. Their role on the deep seafloor, however, is only fragmentarily understood, despite their potential key function for global carbon cycling. Using the approach of combined DNA metabarcoding and cultivation-based surveys of 11 deep-sea regions, we show that protist communities, mostly overlooked in current deep-sea foodweb models, are highly specific, locally diverse and have little overlap to pelagic communities. Besides traditionally considered foraminiferans, tiny protists including diplonemids, kinetoplastids and ciliates were genetically highly diverse considerably exceeding the diversity of metazoans. Deep-sea protists, including many parasitic species, represent thus one of the most diverse biodiversity compartments of the Earth system, forming an essential link to metazoans., Alexandra Schoenle et al. use DNA metabarcoding and cultivation-based surveys of 11 regions in the Pacific and Atlantic Oceans encompassing the bathyal, abyssal, and hadal zones to identify the role of deep-sea heterotrophic protists relative to other trophic levels. Their results demonstrate that protistan species richness was locally specific and greater than that of metazoans and that measured in pelagic samples, and that deep-sea protist communities are relatively distinct from their pelagic counterparts.

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell–cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testing in vitro (organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapies in vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells in poly-N-isopropylacrylamid (PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Percolomonads are common freshwater, marine and hypersaline tetraflagellated organisms. Current phylogenetic analyses of eukaryotes comprise only two species of this underinvestigated family. Here, we studied the morphology, salinity tolerance and 18S rDNA gene-based phylogeny of seven percolomonad cultures. We describe three new genera and five novel species of Percolomonadida based on phylogenetic distances and morphological characteristics: Barbelia atlantica, B. abyssalis, Lula jakobsenorum, Nakurumonas serrata and Percolomonas doradorae. The new species show features typical for percolomonads, one long flagellum for skidding, three shorter flagella of equal length and a ventral feeding groove. The new species comprise organisms living in marine and athalassic hypersaline waters with salinity ranging from 10 to 150 PSU. Based on these novel taxa, the taxonomy and phylogeny of Percolatea was extended and further resolved.

Multidrug resistant (MDR) bacteria have adapted to most clinical antibiotics and are a growing threat to human health. One promising type of candidates for the everlasting demand of new antibiotic compounds constitute antimicrobial peptides (AMPs). These peptides act against different types of microbes by permeabilizing pathogen cell membranes, whereas being harmless to mammalian cells. Contrarily, another class of membrane-active peptides, namely cell-penetrating peptides (CPPs), is known to translocate in eukaryotic cells without substantially affecting the cell membrane. Since CPPs and AMPs share several physicochemical characteristics, we hypothesized if we can rationally direct the activity of a CPP towards antimicrobial activity. Herein, we describe the screening of a synthetic library, based on the CPP sC18, including structure-based design to identify the active residues within a CPP sequence and to discover novel AMPs with high activity. Peptides with increased hydrophobicity were tested against various bacterial strains, and hits were further optimized leading to four generations of peptides, with the last also comprising fluorinated amino acid building blocks. Interestingly, beside strong antibacterial activities, we also detected activity in cancer cells, while non-cancerous cells remained unharmed. The results highlight our new candidates, particularly those from generation 4, as a valuable and promising source for the development of future therapeutics with antibacterial activity and beyond.

Recent studies suggested that the diversity of microbial eukaryotes in hypersaline environments is widely underestimated. Placidids are a group of heterotrophic stramenopile flagellates that are frequently found in these environments, but up to now only very few species were isolated and fully described, mostly from marine or brackish water sites. In this study, we extend the known diversity of Placididea by three new genera (Allegra, Haloplacidia, and Placilonga) compromising nine new species, isolated from athalassic, mostly hypersaline environments (Allegra dunaii, Allegra atacamiensis, Allegra hypersalina, Haloplacidia cosmopolita, Suigetsumonas keniensis) and marine waters (Placilonga atlantica, Placidia azorensis, Placidia abyssalis, Wobblia pacifica) including a description of their morphology and molecular phylogeny. In total, 36 strains were comparatively analysed. Studies from athalassic waters revealed an especially high number of different genotypes. A multigene analysis based on a ten genes dataset revealed a clear separation into marine, athalassic and brackish water clades. Several representatives were found to cope with hypersaline conditions from 20 to 250 PSU, even up to 284 PSU, suggesting that they may form a halotolerant group.

Environmental sequencing surveys unveil an unexpected magnitude of protist biodiversity and help to understand environmental community structure as well as biogeographical patterns. The interpretation of these data is still hindered by the lack of a verified and reliable reference database, which is the important basis for all analyses. References should rely on detailed and valid taxonomical descriptions including both morphology and autecological properties. In fact, obtaining such data is still a major challenge as cultivation-based approaches are very selective. In the present study, we highlight the potential to resample habitats which showed phylogenetically interesting sequences from environmental molecular surveys. We have been able to reveal a choanoflagellate species with the use of a single cell isolation approach in order to achieve a morphological description to the target sequence. This new species, Enibas thessalia sp. nov. now extends a recently described monospecific genus. In addition, we illustrate a nudiform lorica reproduction of the genus Enibas by observation of living cells. The genus belongs to the family of Acanthoecidae, which comprises five genera. The morphology of the genus Enibas shows a striking resemblance to the genus Stephanoeca, which belongs to the other family of loricate choanoflagellates, the Stephanoecidae, indicating that morphology alone might not reflect phylogenetic relations. We demonstrate that mapping sequences to a taxonomical description of species is a valuable tool to verify the organism behind an environmental amplicon. We emphasize the urgent need of integrative taxonomy matching molecular data with morphological features to verify the outcome of phylogenetic analyses.

Locomotion is considered to be the main form of expression of ciliate behaviour regarding their overall life activity. But how ciliates behave under deep-sea conditions is still unclear. Data on the occurrence of ciliates in the deep sea are scarce and mostly based on molecular studies. We isolated three different ciliates,Aristerostomasp.,Euplotes dominicanusandPseudocohnilembus persalinusfrom two stations located in abyssal depths of the North Atlantic Ocean (≥ 4000 m; 15° 55.89′ N, 68° 53.34′ W; 23° 33.23′ N, 48° 5.04′ W) during the deep-sea expedition with the research vessel R/V Meteor (Cruise M139, 08.07.–08.08.2017). We observed their behaviour directly under high hydrostatic pressures up to 500 bar. The three ciliate species behaved normally up to a pressure of 200 bar, but showed disturbances of the normal behaviour at higher pressures. For all three isolated deep-sea ciliates, additional long-term survival experiments were carried out for 6 days at 200, 350 and 430 bar. Several specimens showed an ability to survive the entire experimental time interval at the highest pressure and to recover from pressure release (returning to their normal movement) indicating their barotolerance. Our results suggest that ciliates are active in the deep sea even in regions deeper than 2000 m and might be an important part of the deep-sea microbial food web.

By measuring the change in soil protist communities, the effect of human land use on grasslands can be monitored to promote sustainable ecosystem functioning. Protists form the active link in the rhizosphere between the plant roots and higher trophic organisms; however, only few morphological species and their ecological values have yet been described in this context. To investigate the communicability between morphological and molecular databases used in the molecular barcoding of protists and in the biomonitoring of grassland soil, the present high-throughput sequencing (HTS) study (N=150) covered the area of central Europe (mesoscale) known to be well studied for ciliated protists. HTS delivered 2,404 unique reads identifying taxa in all major ciliophoran classes but exact reference matches were few. The study identified clear discrepancies between databases for well-studied taxa, where molecular databases contained multiple gene variants for single morphospecies of dominant taxa. Gene variants presented own biogeography - the eukaryotic microdiversity along gradients (e.g., land-use intensity, soil water). It is possible that many of the so called novel phylogenetic lineages and hidden diversity pointed out in environmental surveys could be evidence for the severe lack of molecular data for already known and morphologically described species, present in morphological databases.

Freshwater sponges play a major role in freshwater ecological system as important filter-feeding organisms and bioindicators. There are only few data about their ecological diversity and population genetic structure available, though a deeper knowledge is needed to propose proper conservation and effective management. The aim of this study was to assess data on distribution patterns of freshwater sponges to study the connectivity of genotypes of Ephydatia fluviatilis in a river system. We sampled specimens from River-Sieg system (River Agger and River Sieg, Germany). We hypothesized that strong anthropogenic influence would cause a uniform distribution of population structures. The genetic structure of E. fluviatilis populations was analysed with a set of eleven microsatellite loci from seven locations in River-Sieg system. Besides of E. fluviatilis, three other species co-occurred (Ephydatia mulleri, Spongilla lacustris, Eunapius fragilis). In contrast to our hypothesis, we observed an overall correlation between genetic and geographic distances among populations of this sessile species, which follows a clear isolation-by-distance pattern. A significant microsatellite polymorphism and high levels of genetic divergence between populations (FST) in upstream reaches were present. These results will provide important information for conservation management of populations with limited dispersal ability in connected river systems.

Although the abyssal seafloor represents the most common benthic environment on Earth, eukaryotic microbial life at abyssal depths is still an uncharted territory. This is in striking contrast to their potential importance regarding the material flux and bacteria consumption in the deep sea. Flagellate genotypes determined from sedimentary DNA deep‐sea samples might originate from vital deep‐sea populations or from cysts of organisms sedimented down from surface waters. The latter one may have never been active under deep‐sea conditions. We wanted to analyze the principal ability of cultivable heterotrophic flagellates of different phylogenetic groups (choanoflagellates, ancyromonads, euglenids, kinetoplastids, bicosoecids, chrysomonads, and cercozoans) to survive exposure to high hydrostatic pressure (up to 670 bar). We summarized our own studies and the few available data from literature on pressure tolerances of flagellates isolated from different marine habitats. Our results demonstrated that many different flagellate species isolated from the surface waters and deep-sea sediments survived drastic changes in hydrostatic pressure. Barophilic behavior was also recorded for several species isolated from the deep sea indicating their possible genetic adaptation to high pressures. This is in accordance with records of heterotrophic flagellates present in environmental DNA surveys based on clone libraries established for deep‐sea environments.

Genomic data for less than one quarter of ∼1.8 million named species on earth exist in public databases like GenBank. Little information exists on the estimated one million small sized (1–100 μm) heterotrophic nanoflagellates and ciliates and their taxa-area relationship. We analyzed environmental DNA from 150 geo-referenced grassland plots representing topographical and land-use ranges typical for Central Europe. High through-put barcoding allowed the identification of operational taxonomic units (OTUs) at species level, with high pairwise identity to reference sequences (≥99.7%), but also the identification of sequences at the genus (≥97%) and class (≥80%) taxonomic level. Species richness analyses revealed, on average, 100 genus level OTUs (332 unique individual read (UIR) and 56 class level OTUs per gram of soil sample in the mesoscale (1–1 000 km). Database shortfalls were highlighted by increased uncertain taxonomic lineages at lower resolution (≥80% sequence identity). No single barcode occurred ubiquitously across all sites. Taxa-area relationships indicated that OTUs spread over the entire mesoscale were more similar than in the local scale and increased land-use (fertilization, mowing and grazing) promoted taxa-area separation. Only a small fraction of sequences strictly matched reference library sequences, suggesting a large protistan “dark matter” in soil which warrants further research.

Microbial eukaryotes play an important role in biogeochemical cycles not only in productive surface waters but also in the deep sea. Recent studies based on metagenomics report deep-sea protistan assemblages totally different from continental slopes and shelf waters. To give an overview about the ciliate fauna recorded from the deep sea we summarized the available information on ciliate occurrence in the deep sea. Our literature review revealed that representatives of the major phylogenetic groups of ciliates were recorded from the deep sea (> 1000 m depth): Karyorelictea, Heterotrichea, Spirotrichea (Protohypotrichia, Euplotia, Oligotrichia, Choreotrichia, Hypotrichia), Armophorea (Armophorida), Litostomatea (Haptoria), Conthreep (Phyllopharyngea incl. Cyrtophoria, Chonotrichia, Suctoria; Nassophorea incl. Microthoracida, Synhymeniida, Nassulida; Colpodea incl. Bursariomorphida, Cyrtolophosidida; Prostomatea; Plagiopylea incl. Plagiopylida, Odontostomatida; Oligohymenophorea incl. Peniculia, Scuticociliatia, Hymenostomatia, Apostomatia, Peritrichia, Astomatia). Species occurring in both habitats, deep sea and shallow water, are rarely found to our knowledge to date. This indicates a high deep-sea specific ciliate fauna. Our own studies of similar genotypes (SSU rDNA and cox1 gene) revealed that two small scuticociliate species ( Pseudocohnilembus persalinus and Uronema sp. ) could be isolated from surface as well as deep waters (2687 m, 5276 m, 5719 m) of the Pacific. The adaptation to deep-sea conditions was investigated by exposing the ciliate isolates directly or stepwise to different hydrostatic pressures ranging from 1 to 550 atm at temperatures of 2 °C and 13 °C. Although the results indicated no general barophilic behavior, all four isolated strains survived the highest established pressure. A better survival at 550 atm could be observed for the lower temperature. Among microbial eukaryotes, ciliates should be considered as a diverse and potentially important component of deep-sea microeukaryote communities.

Paramonosiga coloniensis sp. nov., Salpingoeca amphoriscia sp. nov., S. fluviatilis sp. nov. and S. loutrophoria sp. nov. are frequently found craspedid species in the River Rhine which have not yet been described, despite their high abundance. All new species are characterized based on a distinct morphology which is different from all up to now described species and on a molecular level based on transcriptome data. In addition, we give extended redescriptions of S. amphoridium and S. angulosa, based on SSU and LSU rDNA data and morphology. The six-gene phylogenetic analyses place all species into freshwater clades of the craspedids. The separation of the freshwater and marine clades of this group is becoming more distinct with every craspedid sequence added. The River Rhine is one of the largest rivers in Europe but its protist biodiversity is fairly undescribed, especially regarding choanoflagellates. We conclude that the biodiversity of craspedid choanoflagellates is broadly underestimated.

A new Euplotes species, isolated from abyssal depths (>4000 m) of the North Atlantic Ocean, was described based on morphology, ciliary pattern and molecular data. Euplotes dominicanus sp. n. is characterized by a small body size (29–40 × 17–27 μm in vivo), 18–22 adoral membranelles, 10 frontoventral, five transverse and two left marginal cirri and one caudal cirrus, five or six dorsolateral kineties with 7–9 dikinetids in mid-dorsolateral kinety (DK3), and dorsal silverline system of the double-eurystomus type. Phylogenetic analyses inferred from 18S rRNA sequences show that Euplotes dominicanus sp. n. is most closely related to E. curdsi, with a sequence similarity of 97.6 %. Euplotes dominicanus sp. n. was able to survive hydrostatic pressures up to 500 bar indicating its barotolerance. Metabarcoding data demonstrate the presence of E. dominicanus sp. n. in sediments of several deep-sea basins.

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

In general, acanthoecid choanoflagellates have been described to occur exclusively in brackish water to marine habitats. Only recently, two studies have proven their existence in inland waters. One of them has shown, that an acanthoecid species from a small lake (near Apia on the island of Upolu, Samoa) is strictly freshwater adapted, not able to tolerate even brackish water. In this study, we present the first euryoecious acanthoecid species, able to live and reproduce in freshwater as well as under hypersaline conditions. The new species, Enibas tolerabilis gen. et sp. nov. was isolated in 2017 from the Salar de Ascotan in the Altiplano at 3750 m a. s. l., Northern Chile. The salinity at the time of sampling was 6 PSU. A series of autecological experiments have revealed a salinity tolerance from freshwater up to 70 PSU. In our phylogenetic analysis, E. tolerabilis gen. et sp. nov. clustered within the family of Acanthoecidae, forming a well-supported sister clade together with two other, environmental choanoflagellate sequences. We erected a new genus, Enibas gen. nov. and described the morphology, molecular biology and autecology for E. tolerabilis gen. et sp. nov. which has a stephanoecid-like lorica morphology. We emphasize that the definition of the genus Stephanoeca, being polyphyletic, is in urgent need of revision as we showed that this morphology is present in both acanthoecid families.

Acanthoecid choanoflagellates are described and classified according to their distinct siliceous lorica, a unique morphological characteristic within protistan species. Only recently it has become clear that beside morphological and developmental data on the lorica molecular data, mainly based on SSU rDNA, it is necessary to resolve phylogenetic relationships within this group. Previously Didymoeca elongata was thought to be an endemic species from Taiwanese waters. However, this species has now been found in the Caribbean Sea at several sampling sites along with two other species, Diaphanoeca spiralifurca and Polyoeca dichotoma. By sequencing the SSU rDNA and other genes and by adding light and electron microscopical data to the description of these species, the taxonomy and phylogeny of Acanthoecida is being better understood.

Unicellular eukaryotes, also called protists, are potentially fast evolving organisms. The small size of protists, their fast reproduction rate and ability to form cysts as well as their adaptability to extreme conditions allow them to associate to endemic animals, plants, saline lakes and soil even in extremely arid systems. These properties make unicellular eukaryotes ideal model organisms to combine studies on evolutionary processes of very different groups of organisms and across very different time scales comprising even geological ones. The hyperarid Atacama Desert offers a study area unique on Earth, where a predominantly arid climate was present for millions of years. A comprehensive analysis of the diversity of unicellular eukaryotes in different habitats (endemic desert plant phyllosphere, the gut of endemic darkling beetles, isolated hypersaline waters) revealed a dataset distinct and divergent from other regions on Earth. We used standard isolation and cultivation protocols to elucidate divergence patterns for a variety of very different and independent taxonomic groups of protists such as gregarines and ciliates among alveolates, placidids among stramenopiles and choanoflagellates among opisthokonts. The ability to rapidly adapt to extreme environments, which enhance a fast divergence rate at high UV radiation, has only been reported for prokaryotes up to now. The establishment of arid to hyperarid conditions in the Atacama Desert about 20 Ma ago has obviously led to an isolation of protist populations followed by a radiation of species. There are only a few regions on Earth with similar extreme salinity conditions reducing the chance of an exchange between protist populations. Divergence patterns in unicellular eukaryotes in very different phylogenetic groups independently mirror the effect of geological evolution and climate variability during the Neogene.

Cafeteria is one of the most common and ecologically significant genera of heterotrophic nanoflagellates in marine plankton. We could isolate and cultivate 29 strains morphologically similar to Cafeteria obtained from surface waters and the deep sea all over the world's ocean. Morphological characterization obtained by high resolution microscopy revealed only small differences between the strains. Sequencing the type material of the type species C. roenbergensis (CCAP 1900/1) and molecular analyses (18S rDNA, 28S rDNA) of newly isolated strains resulted in a revision and separation of the Cafeteriaceae into two known species (C. roenbergensis, C. mylnikovii) and six new species (C. maldiviensis, C. biegae, C. loberiensis, C. chilensis, C. graefeae, C. burkhardae). Many deposited Cafeteria sequences at GenBank and most of our own sequences clustered within one clade (C. burkhardae) with a p-distance of 5% to strain CCAP 1900/1. Only C. maldiviensis clustered together with the type species C. roenbergensis. While C. burkhardae seems to have a cosmopolitan distribution, the distribution of the other species seems to be more restricted. A strain from the Angola Basin had a p-distance of 10% to Cafeteria species and clustered separately within the Anoecales requiring the erection of a new genus, Bilabrum gen. nov., with B. latius sp. nov. as type species.

With this study we aim to extend the knowledge on the biogeography of craspedid choanoflagellates with additional data from extreme environments. Up to now, very little is known about choanoflagellates from extreme saline environments, as most studies have focused on marine and freshwater habitats. Though previously investigated high saline ice biota communities have indicated a possible adaptation to environments with high salt concentrations. Hypersaline endorheic basins, so-called salt flats or salares from the Atacama Desert in Northern Chile provide an intense environment regarding fluctuating and extreme salinities, which allow for studies on evolutionary adaptations of protists to hypersaline conditions. This study focused on choanoflagellate species isolated from different salt flats, their morphological characteristics using light and electron microscopy, molecular marker genes (SSU and LSU rDNA) and their salinity tolerance. Here, we described four new craspedid choanoflagellate species, highly adapted to the hypersaline environment of the Atacama Desert. This study extends our knowledge on choanoflagellate phylogeny and ecology and can become the basis for further molecular studies to understand the mechanisms of adaptations. Additionally, we emphasize the need of adding additional data such as autecological characteristics to amend species definitions, which is only possible from cultivated strains. This data would support the use of molecular data originating from metagenomic analyses also in an ecological context.

Quantitative aspects of the life cycle of the freshwater shrimpAtyaephyra desmarestiiwere used to investigate its life span at the edge of its distribution area in Central Europe (Upper Rhine) for a period of three years. We estimated the proportion of the population surviving a second hibernation period. The appearance of ovigerous females in late spring was followed by a strong decline in the number of adults, the appearance of a high percentage of juveniles in late summer (0.58 ind./m2), and their subsequent disappearance from the shore in winter. A small semivoltine cohort survived a second winter. The average life span was 12-15 (max. 29) months under laboratory conditions. In molecular studies (COX I), the Upper Rhine and Southern France populations clustered within the North Africa/West-Central Europe species clade. Due to its low productivity, this invasive species is unlikely to cause management problems.

Research on soil microbiota is one of the frontiers in biodiversity research. Bacterivores, such as protozoans, contribute significantly to mineralization processes and key ecosystem functions in soil. Protozoan grazing on rhizobacteria stimulates microbial mineralization processes and thereby indirectly nutrient cycling. Understanding the diversity of protozoans and their fundamental functions is essential for knowledge of nutrient cycling. However, reliable data on protozoan numbers and taxonomic composition in soil are scarce. We compared the occurrence of morphotypes of small cultivable protozoans of eight different grassland and forest sites in Germany. The investigated sites differed both in landscape type and intensity of land use. Additionally, we modified a liquid aliquot method to determine protozoan abundances and community structure on the morphotype level in soil. Protozoan abundances at the investigated sites ranged between 1.7 and 12.7 × 104 ind. g−1 dry weight. The dominant groups were various amoebae (49.9 ± 12.4%) and cercozoans (32.4 ± 13.2%), followed by stramenopiles (7.7 ± 4.2%), euglenozoans (7.5 ± 7.6%), apusomonads (1.5 ± 1.5%) and ciliates (0.5 ± 0.7%). Heterotrophic flagellates and naked amoebae showed similar contributions to total protozoan abundances at all sites. Protozoan abundances were slightly higher and more variable in grassland sites compared to forest sites. Both habitat types showed a surprisingly similar community structure on a rough taxonomic level, but a more diverse pattern at a higher taxonomic resolution.

Choanoflagellates are closely related to metazoans and fungi according to recent phylogenetic studies; therefore the systematics of these organisms is of particular interest. The choanoflagellate morphospecies Codosiga botrytis is the first described choanoflagellate, and is one of the most frequently reported choanoflagellate species. In this study we present phylogenetic and morphological data on eight different strains of Codosiga botrytis . Among these there are five ancient strains; these cultures have been established from up to 43,000 years old cysts from Siberian permafrost. We found that based on the variable V4 region of the small subunit (SSU) of the rDNA, all the investigated freshwater isolates of Codosiga botrytis , together with Sphaeroeca volvox , form a cluster at the base of all other choanoflagellate species. Moreover, the morphospecies described classically as Codosiga botrytis contains at least four different genotypes separated by considerably high genetic distance. All these ‘cryptic species’ have identical general morphology and cell structure. Strains have a similar life cycle with several different life forms and large morphological plasticity. For the first time we were able to establish cultures from cryo-conserved cysts of choanoflagellates. The ancient strains did not differ significantly in partial SSU rDNA from the modern ones. Besides, no biogeographically pattern could be established. This fact and the low genetic distances of some strains from remote locations support the distribution of dormant stages via air.

International audience; Translating the vast amounts of molecular barcodes from global surveys of microbial eukaryotes into ecological insight depends critically on a well-curated reference database with adequate taxonomic coverage. In this respect, the choanoflagellates resemble other eukaryotic lineages: reasonable coverage at higher taxonomic levels, but missing diversity at the species level. The acanthoecid (loricate) choanoflagellates are well-characterized morphologically, with over 115 species described, but less than 10% with any sequence data. Because lorica shape is species-specific, the acanthoecids represent an opportunity to link morphological with molecular data within a lineage of eukaryotes. To match morphospecies to sequences, we sampled the Kattegat and the Isefjord in Denmark in September 2014 and February 2015. We identified 45 morphospecies and sequenced ribosomal DNA of nine previously unsequenced species, roughly doubling the number of acanthoecid species with sequence data, including the first data representing five genera: Bicosta, Calliacantha, Cosmoeca, Crinolina and Pleurasiga. Our phylogenetic analysis is mainly congruent with morphology-based systematics. Five of the newly sequenced species match a previously unidentified barcode from Tara Oceans, providing access to the global distribution of species isolated from Danish waters. One species, Calliacantha natans, is the second most globally abundant choanoflagellate present in Tara Oceans. Our project translating new ribosomal DNA sequences to distributions of described species on a global scale supports the approach linking morphology to molecular barcodes for microbial eukaryote ecology.

The genus Tetrahymena (Ciliophora, Oligohymenophorea) probably represents the best studied ciliate genus. At present, more than forty species have been described. All are colorless, i.e. they do not harbor symbiotic algae, and as aerobes they need at least microaerobic habitats. Here we present the morphological and molecular description of the first green representative, Tetrahymena utriculariae n. sp., living in symbiosis with endosymbiotic algae identified as Micractinium sp. (Chlorophyta). The full life cycle of the ciliate species is documented, including trophonts and theronts, conjugating cells, resting cysts and dividers. This species has been discovered in an exotic habitat, namely in traps of the carnivorous aquatic plant Utricularia reflexa (originating from Okavango Delta, Botswana). Green ciliates live as commensals of the plant in this anoxic habitat. Ciliates are bacterivorous, however symbiosis with algae is needed to satisfy cell metabolism but also to gain oxygen from symbionts. When ciliates are cultivated outside their natural habitat under aerobic conditions and fed with saturating bacterial food, they gradually become aposymbiotic. Based on phylogenetic analyses of 18S rRNA and mitochondrial cox1 genes T. utriculariae forms a sister group to T. thermophila. This article is protected by copyright. All rights reserved.

International audience; It is a trend in loricate choanoflagellate research that our knowledge of species diversity is insufficient in terms of understanding annual successional changes at any specific locality, whereas there is a fairly decent coverage worldwide − at least in more coastal realms − in terms of biodiversity within more narrowly defined time windows. To help address this knowledge gap, we have compiled all available loricate choanoflagellate occurrence data from Danish sampling sites covering an overall time span of close to four decades. The close to 100 samples analysed have a good annual coverage and they encompass in total more than 50 species. We demonstrate clear successional trends among well-defined clusters of species. A large contingent of ‘non-native’ species, which are in a global context largely considered part of the loricate choanoflagellate warm water community, occurred in September 2014 samples from the Baltic Sea entrance, i.e. the Sound between Denmark and Sweden. While the occurrence of these species is likely due to a large inflow of southern Atlantic water, we also discuss whether the findings may instead reflect recent and more permanent climate change-induced alterations to choanoflagellate biodiversity in inner Danish waters.

Heterotrophic flagellates contribute significantly to the matter flux in aquatic and terrestrial ecosystems. Still today their quantification and taxonomic classification bear several problems in field studies, though these methodological problems seem to be increasingly ignored in current ecological studies. Here we describe and test different methods, the live-counting technique, different fixation techniques, cultivation methods like the liquid aliquot method (LAM), and a molecular single cell survey called aliquot PCR (aPCR). All these methods have been tested either using aquatic field samples or cultures of freshwater and marine taxa. Each of the described methods has its advantages and disadvantages, which have to be considered in every single case. With the live-counting technique a detection of living cells up to morphospecies level is possible. Fixation of cells and staining methods are advantageous due to the possible long-term storage and observation of samples. Cultivation methods (LAM) offer the possibility of subsequent molecular analyses, and aPCR tools might complete the deficiency of LAM in terms of the missing detection of non-cultivable flagellates. In summary, we propose a combination of several investigation techniques reducing the gap between the different methodological problems.

The choanoflagellates (Choanoflagellatea) comprise a major group of nanoflagellates, which are ubiquitous in the aquatic environment. Recent molecular phylogenies have shown them to be the sister group to the Metazoa. However, the phylogeny of the choanoflagellates is still far from understood. We present here a 29 taxon, multigene phylogeny that robustly places the root of the choanoflagellates. One of the original nonloricate families, Codonosigidae is shown to be a polyphyletic assemblage nested within the Salpingoecidae. We elaborate on a revised taxonomy that divides Choanoflagellatea into two orders: Craspedida and Acanthoecida. Craspedida is composed of species that possess an organic cell coating and contains the single family Salpingoecidae. Members of the predominantly marine Acanthoecida produce a siliceous lorica in addition to an organic coat and are contained in two families—the Acanthoecidae and Stephanoecidae fam. n. Previous studies of choanoflagellates have been hindered by cases of taxon misidentification as well as the limited resolution of 18S small subunit (SSU) rDNA phylogenies. Unfortunately, cases of misidentification have been heavily repeated in the literature. In an attempt to avoid further confusion, we highlight known instances of misnamed taxa. We also examine the suitability of SSU rDNA sequences alone for choanoflagellate phylogenetics and recommend the use of protein-coding genes, such as hsp90 and tubA, whenever possible.

Microdiaphanosoma arcuatum Wenzel, 1953 is a world-wide distributed ciliate, recorded mainly in soil samples, which we have also identified in ground water samples from South Africa. This ciliate has been frequently overlooked or not determined due to its small size,∼12 μm. The genus Microdiaphanosoma is nowadays included in the class Colpodea, order Bryometopida, family Kreyellidae. The first complete small subunit (SSU) rDNA gene sequence of this ciliate was obtained from a South African isolate. Phylogenetic analysis including available SSU rDNA sequences from another Colpodea species in the GenBank strongly supported the position of M. arcuatum within the order Cyrtolophosidida instead of the order Bryometopida. The analysis also suggested a sister relationship between this species and species from the family Cyrtolophosididae.