Biogeography, autecology, and phylogeny of Percolomonads based on newly described species.

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 dilemma of underestimating freshwater biodiversity: morphological and molecular approaches.

BACKGROUND: Anthropogenic impacts on freshwater habitats are causing a recent biodiversity decline far greater than that documented for most terrestrial ecosystems. However, knowledge and description of freshwater biodiversity is still limited, especially targeting all size classes to uncover the distribution of biodiversity between different trophic levels. We assessed the biodiversity of the Lower Rhine and associated water bodies in the river's flood plain including the river's main channel, oxbows and gravel-pit lakes, spanning from the level of protists up to the level of larger invertebrate predators and herbivores organized in size classes (nano-, micro, meio- and macrofauna). Morphological diversity was determined by morphotypes, while the molecular diversity (amplicon sequencing variants, ASVs) was assessed through eDNA samples with metabarcoding targeting the V9 region of the 18S rDNA. RESULTS: Considering all four investigated size classes, the percentage of shared taxa between both approaches eDNA (ASVs with 80-100% sequence similarity to reference sequences) and morphology (morphotypes), was always below 15% (5.4 ± 3.9%). Even with a more stringent filtering of ASVs (98-100% similarity), the overlap of taxa could only reach up to 43% (18.3 ± 12%). We observed low taxonomic resolution of reference sequences from freshwater organisms in public databases for all size classes, especially for nano-, micro-, and meiofauna, furthermore lacking metainformation if species occur in freshwater, marine or terrestrial ecosystems. CONCLUSIONS: In our study, we provide a combination of morphotype detection and metabarcoding that particularly reveals the diversity in the smaller size classes and furthermore highlights the lack of genetic resources in reference databases for this diversity. Especially for protists (nano- and microfauna), a combination of molecular and morphological approaches is needed to gain the highest possible community resolution. The assessment of freshwater biodiversity needs to account for its sub-structuring in different ecological size classes and across compartments in order to reveal the ecological dimension of diversity and its distribution.

High diversity and isolated distribution of aquatic heterotrophic protists in salars of the Atacama Desert at different salinities.

The species richness of eukaryotes in the hypersaline environment is generally thought to be low. However, recent studies showed a high degree of phylogenetic novelty at these extreme conditions with variable chemical parameters. These findings call for a more thorough look into the species richness of hypersaline environments. In this study, various hypersaline lakes (salars, 1-348 PSU) as well as further aquatic ecosystems of northern Chile were investigated regarding diversity of heterotrophic protists by metabarcoding studies of surface water samples. Investigations of genotypes of 18S rRNA genes showed a unique community composition in nearly each salar and even among different microhabitats within one salar. The genotype distribution showed no clear connection to the composition of main ions at the sampling sites, but protist communities from similar salinity ranges (either hypersaline, hyposaline or mesosaline) clustered together regarding their OTU composition. Salars appeared to be fairly isolated systems with only little exchange of protist communities where evolutionary lineages could separately evolve.

Cafeteria in extreme environments: Investigations on C. burkhardae and three new species from the Atacama Desert and the deep ocean.

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.

High and specific diversity of protists in the deep-sea basins dominated by diplonemids, kinetoplastids, ciliates and foraminiferans.

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.

Protist diversity and function in the dark ocean - Challenging the paradigms of deep-sea ecology with special emphasis on foraminiferans and naked protists.

The dark ocean and the underlying deep seafloor together represent the largest environment on this planet, comprising about 80% of the oceanic volume and covering more than two-thirds of the Earth's surface, as well as hosting a major part of the total biosphere. Emerging evidence suggests that these vast pelagic and benthic habitats play a major role in ocean biogeochemistry and represent an "untapped reservoir" of high genetic and metabolic microbial diversity. Due to its huge volume, the water column of the dark ocean is the largest reservoir of organic carbon in the biosphere and likely plays a major role in the global carbon budget. The dark ocean and the seafloor beneath it are also home to a largely enigmatic food web comprising little-known and sometimes spectacular organisms, mainly prokaryotes and protists. This review considers the globally important role of pelagic and benthic protists across all protistan size classes in the deep-sea realm, with a focus on their taxonomy, diversity, and physiological properties, including their role in deep microbial food webs. We argue that, given the important contribution that protists must make to deep-sea biodiversity and ecosystem processes, they should not be overlooked in biological studies of the deep ocean.

Global comparison of bicosoecid Cafeteria-like flagellates from the deep ocean and surface waters, with reorganization of the family Cafeteriaceae.

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.

A barotolerant ciliate isolated from the abyssal deep sea of the North Atlantic: Euplotes dominicanus sp. n. (Ciliophora, Euplotia).

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.

New phagotrophic euglenids from deep sea and surface waters of the Atlantic Ocean (Keelungia nitschei, Petalomonas acorensis, Ploeotia costaversata).

New phagotrophic euglenoid species from marine surface waters and the deep sea were isolated and described by light and scanning electron microscopy and 18S rDNA sequencing: Keelungia nitschei, Petalomonas acorensis and Ploeotia costaversata. The morphological characteristics of Keelungia nitschei agree with Keelungia pulex besides the slightly truncated anterior front of the cell of our strain. Phylogenetic analysis indicated low sequence similarity between K. nitschei and K. pulex (87.3%). Ploeotia costaversata clustered within the Ploeotia costata clade with a sequence similarity of 96.1% to P. costata strain Tam. Ultrastructural characteristics of our strain revealed helically twisted strips towards both poles of the protoplast. 18S rDNA phylogenies showed that Petalomonas acorensis is related to the clade of Petalomonas cantuscygni/Scytomonas saepesedens with the highest sequence similarity of 81.2% to P. cantuscygni. Six pellicle strips are visible, while two of them reach only the middle of the cell and four (two longitudinal, two helically twisted) join at the posterior front of the cell. Pressure experiments showed that the deep-sea strain K. nitschei was better adapted to high hydrostatic pressures (up to 500 bar) at 4 °C than the two surface water strains. All three strains increased the database (18S rDNA) of the underrepresented group of phagotrophic euglenids.