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This paper reviews current knowledge of the structure, genesis, cytochemistry and putative functions of the haplosporosomes of haplosporidians (Urosporidium, Haplosporidium, Bonamia, Minchinia) and paramyxids (Paramyxa, Paramyxoides, Marteilia, Marteilioides, Paramarteilia), and the sporoplasmosomes of myxozoans (Myxozoa - Malacosporea, Myxosporea). In all 3 groups, these bodies occur in plasmodial trophic stages, disappear at the onset of sporogony, and reappear in the spore. Some haplosporidian haplosporosomes lack the internal membrane regarded as characteristic of these bodies and that phylum. Haplosporidian haplosporogenesis is through the Golgi (spherulosome in the spore), either to form haplosporosomes at the trans-Golgi network, or for the Golgi to produce formative bodies from which membranous vesicles bud, thus acquiring the external membrane. The former method also forms sporoplasmosomes in malacosporeans, while the latter is the common method of haplosporogenesis in paramyxids. Sporoplasmogenesis in myxosporeans is largely unknown. The haplosporosomes of Haplosporidium nelsoni and sporoplasmosomes of malacosporeans are similar in arraying themselves beneath the plasmodial plasma membrane with their internal membranes pointing to the exterior, possibly to secrete their contents to lyse host cells or repel haemocytes. It is concluded that these bodies are probably multifunctional within and between groups, their internal membranes separating different functional compartments, and their origin may be from common ancestors in the Neoproterozoic.

The soils of the McMurdo Dry Valleys (MDV) of Antarctica are established models for understanding fundamental processes in soil ecosystem functioning (e.g. ecological tipping points, community structuring and nutrient cycling) because the extreme physical environment drastically reduces biodiversity and ecological complexity. Understanding the functioning of MDV soils requires in-depth knowledge of the diversity of MDV soil species. Protists, which contribute significantly to soil ecosystem functioning worldwide, remain poorly characterized in the MDV. To better assess the diversity of MDV protists, we performed shotgun metagenomics on 18 sites representing a variety of landscape features and edaphic variables. Our results show MDV soil protists are diverse at both the genus (155 of 281 eukaryote genera) and family (120) levels, but comprise only 6% of eukaryotic reads. Protists are structured by moisture, total N and distance from the local coast and possess limited richness in arid (< 5% moisture) and at high elevation sites, known drivers of communities in the MDV. High relative diversity and broad distribution of protists in our study promotes these organisms as key members of MDV soil microbiomes and the MDV as a useful system for understanding the contribution of soil protists to the structure of soil microbiomes., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

During a histopathological survey of Mytilus galloprovincialis in Galicia (NW Spain), microcells were observed infecting several organs of the symbiont copepod Mytilicola intestinalis. Positive results of PCR assay with specific primers for genus Mikrocytos and a clear signal of in situ hybridization with MACKINI-1 digoxigenin- labelled DNA probe (DIG-ISH) indicated a protozoan parasite of Mikrocytos genus. The ultrastructural study revealed intra and extracellular locations, polymorphic nuclei, intracellular round vesicles in the cytoplasm and absence of mitochondria. The present paper reports the characterization of the Mikrocytos sp. infecting M. intestinalis and proposes a novel species in the genus: Mikrocytos mytilicoli n. sp. A sequence of 18S-28S rDNA was obtained with 95.6% maximum identity (query cover 100%) with Mikrocytos mackini. Phylogenetic analysis showed that M. mytilicoli n. sp. and M. mackini share a common ancestor. However, comparison of the ITS1 rDNA region showed low similarity (75.8%) with M. mackini, which, combined with differences in ultrastructural details, host and geographic location, support the designation of a new species. This is the first description of a microcytid parasite of the genus Mikrocytos from a non-bivalve host., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ventrifissura is a group of poorly studied heterotrophic biflagellates in the phylum Cercozoa. Despite a phylogenetic placement with only weak support and a lack of ultrastructural data, Ventrifissura was assigned to Thecofilosea. In the presented study, we established cultures of two novel species of Ventrifissura (V. oblonga n. sp. and V. velata n. sp.) isolated from coastal marine environments in Japan, and performed light and electron microscopy observations and molecular phylogenetic analysis. Transmission electron microscopy revealed that V. oblonga shares several ultrastructural characteristics with thecofilosean flagellates, including permanently condensed chromosomes, a extracellular theca, and slender extrusomes. Molecular phylogenetic analysis could not resolve the phylogenetic position, but the possibility that Ventrifissura clusters into Ventrifilosa was supported by approximately unbiased tests. Based on both morphological and phylogenetic findings, we concluded that Ventrifissura is a basal lineage of Thecofilosea., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Rigid and persistent shells of microeukaryotes are widely used as bioindicators in ecological and paleontological studies. Drawing conclusions on ecological or evolutionary patterns depends strongly on the right taxonomic assignment of the observed species, however confusion is common. Especially in filose shelled amoebae it is often unclear whether species belong to the Imbricatea or Thecofilosea when only morphological data are collected. Molecular surveys shed light on their evolutionary relationship; based on these we propose a hypothesis how to differentiate doubtful species even light microscopically., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Marine phytomyxids represent often overlooked obligate biotrophic parasites colonizing diatoms, brown algae, and seagrasses. An illustrative example of their enigmatic nature is the phytomyxid infecting the seagrass Halophila stipulacea (a well-known Lessepsian migrant from the Indo-Pacific to the Mediterranean Sea). In the Mediterranean, the occurrence of this phytomyxid was first described in 1995 in the Strait of Messina (southern Italy) and the second time in 2017 in the Aegean coast of Turkey. Here we investigated, using scuba diving, stereomicroscopy, light and scanning electron microscopy, and molecular methods, whether the symbiosis is still present in southern Italy, its distribution in this region and its relation to the previous reports. From the total of 16 localities investigated, the symbiosis has only been found at one site. A seasonal pattern was observed with exceptionally high abundance (> 40% of the leaf petioles colonized) in September 2017, absence of the symbiosis in May/June 2018, and then again high infection rates (~ 30%) in September 2018. In terms of anatomy and morphology as well as resting spore dimensions and arrangement, the symbiosis seems to be identical to the preceding observations in the Mediterranean. According to the phylogenetic analyses of the 18S rRNA gene, the phytomyxid represents the first characterized member of the environmental clade "TAGIRI-5". Our results provide new clues about its on-site ecology (incl. possible dispersal mechanisms), hint that it is rare but established in the Mediterranean, and encourage further research into its distribution, ecophysiology, and taxonomy.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis., Competing Interests: The authors declare no competing interest.

We have compiled a database of functional traits for two widespread and ecologically important groups of protists, Cercozoa and Endomyxa (Rhizaria). The functional traits of microorganisms are crucially important for interpreting results from environmental sequencing surveys. Linking morphological and ecological traits to environmental factors is common practice in studies involving micro- and macroorganisms, but is rarely applied to protists. Our database provides functional and ecologically significant traits linked to morphology, nutrition, locomotion and habitats. We discuss how the use of functional traits may help to unveil underlying ecosystem processes. This database is intended as a common reference for the molecular ecology community and will boost the understanding of ecosystem functions, especially those driven by biological interactions., (© 2019 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. As attempts to establish a culture of Lecythium mutabilis repeatedly failed, it was not yet investigated by molecular means. We isolated single cells of L. mutabilis directly from their habitat and successfully sequenced the V4 region of their SSU rDNA. Phylogenetic analyses showed that L. mutabilis is not directly related to the genus Lecythium and instead branches within the Fiscullidae (Tectofilosida, Thecofilosea). Accordingly, we transfer the species L. mutabilis to a novel genus Omnivora gen. nov., (© 2019 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

The testate amoeba Leptogromia operculata was described by Valkanov in 1970 from marine waters. We re-discovered this species in brackish water along the North Sea in the Netherlands. Based on detailed comparison of morphology and SSU rDNA phylogeny we conclude that this species represents a sister clade to the Euglyphida (Imbricatea, Cercozoa). We further describe a similar species Trivalvularis immunda gen. nov., sp. nov. from freshwater in France and the Netherlands on basis of morphological data. Trivalvularis and Leptogromia share a unique oral apparatus with three valves that can close the aperture of the shell. Due to this unique morphological character and the phylogenetic analysis of L. operculata we place both species in a new family Trivalvulariidae in the new order Trivalvulariida., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Reclamation of anthropogenically impacted environments is a critical issue worldwide. In the oil sands extraction industry of Alberta, reclamation of mining-impacted areas, especially areas affected by tailings waste, is an important aspect of the mining life cycle. A reclamation technique currently under study is water-capping, where tailings are capped by water to create an end-pit lake (EPL). Base Mine Lake (BML) is the first full-scale end-pit lake in the Alberta oil sands region. In this study, we sequenced eukaryotic 18S rRNA genes recovered from 92 samples of Base Mine Lake water in a comprehensive sampling programme covering the ice-free period of 2015. The 565 operational taxonomic units (OTUs) generated revealed a dynamic and diverse community including abundant Microsporidia, Ciliata and Cercozoa, though 41% of OTUs were not classifiable below the phylum level by comparison to 18S rRNA databases. Phylogenetic analysis of five heterotrophic phyla (Cercozoa, Fungi, Ciliata, Amoebozoa and Excavata) revealed substantial novel diversity, with many clusters of OTUs that were more similar to each other than to any reference sequence. All of these groups are entirely or mostly heterotrophic, as a relatively small number of definitively photosynthetic clades were amplified from the BML samples., (© 2019 International Society of Protistologists.)

Vampyrellids (Vampyrellida, Rhizaria) are a major group of predatory amoebae known primarily from freshwater and soil. Environmental sequence data indicate that there is also a considerable diversity of vampyrellids inhabiting marine ecosystems, but their phenotypic traits and ecology remain largely unexplored. We discovered algivorous vampyrellids of the filoflabellate morphotype in coastal habitats in Atlantic Canada, established cultures by single-cell isolation, and characterised three strains using light microscopy, SSU rRNA gene sequencing, feeding experiments and growth experiments at various salinities. These strains exhibit orange, discoid trophozoites with ventral filopodia, moving granules ("membranosomes"), and rolling locomotion, similar to freshwater species previously assigned to Hyalodiscus Hertwig & Lesser, but here moved to Placopus Schulze (due to homonymy with Hyalodiscus Ehrenberg). SSU rRNA gene phylogenies place our strains in two distinct positions within "lineage B3" (here referred to as Placopodidae). Based on these morphological, habitat and molecular data, we describe two new species, Placopus melkoniani sp. nov. and Placopus pusillus sp. nov., both of which feed on chlorophyte flagellates (Tetraselmis, Pyramimonas) and the cryptophyte Chroomonas. They perforate the theca of Tetraselmis to extract the protoplast, and thereby represent the first vampyrellids known to degrade the biochemically exotic cell wall of the Chlorodendrales (Chlorophyta, Viridiplantae)., (© 2018 International Society of Protistologists.)

Paramyxean parasites in the genus Marteilia deteriorate digestive tissues of the host organisms, resulting in mortality of oysters, cockles, and mussels. Most reports of infection by Marteilia spp. are from Europe, while a new species of Marteilia was identified recently in Japan. Here, we report a previously unidentified species in the genus Marteilia from digestive diverticula of Manila clam Ruditapes philippinarum from the south coast of Korea. Prevalence of the parasite was low, 0.5-3.3% in the study sites. We characterized this species using light and transmission electron microscopy (TEM), and analyzed the 18S rDNA sequence. Light microscopy revealed the sporulation process from uninucleated stage to spore in the epithelial tissues of the digestive gland. TEM revealed that the parasites produced four secondary cells containing four tri-cellular spores. An electron-dense haplosporosome-like structure and striated inclusions were evident in the spore and the primary cells, respectively, while refringent granules were rarely observed in the secondary cells. Phylogenetic analyses of the 18S rDNA sequence placed this isolate in the genus Marteilia, although it is not identical to other known species in the genus. Based on morphological and molecular characters, we describe this species as Marteilia tapetis sp. nov., the second Marteilia species reported parasitizing Manila clams in Asian waters., (Copyright © 2019. Published by Elsevier Inc.)

Protists are the most important predators of soil microbes like bacteria and fungi and are highly diverse in terrestrial ecosystems. However, the structure of protistan communities throughout the soil profile is still poorly explored. Here, we used Illumina sequencing to track differences in the relative abundance and diversity of Cercozoa, a major group of protists, at two depths; 10-30 cm (topsoil) and 60-75 cm (subsoil) in an agricultural field in Germany. At the two depths, we also distinguished among three soil compartments: rhizosphere, drilosphere (earthworm burrows) and bulk soil. With increasing depth, we found an overall decline in richness, but we were able to detect subsoil specific phylotypes and contrasting relative abundance patterns between topsoil and subsoil for different clades. We also found that the compartment effect disappeared in the subsoil when compared to the topsoil. More studies are now needed to describe and isolate these possibly subsoil specific phylotypes and better understand their ecology and function., (© FEMS 2019.)

The phylum Cercozoa consists of a diverse assemblage of amoeboid and flagellated protists that forms a major component of the supergroup, Rhizaria. However, despite its size and ubiquity, the phylogeny of the Cercozoa remains unclear as morphological variability between cercozoan species and ambiguity in molecular analyses, including phylogenomic approaches, have produced ambiguous results and raised doubts about the monophyly of the group. Here we sought to resolve these ambiguities using a 161-gene phylogenetic dataset with data from newly available genomes and deeply sequenced transcriptomes, including three new transcriptomes from Aurigamonas solis, Abollifer prolabens, and a novel species, Lapot gusevi n. gen. n. sp. Our phylogenomic analysis strongly supported a monophyletic Cercozoa, and approximately-unbiased tests rejected the paraphyletic topologies observed in previous studies. The transcriptome of L. gusevi represents the first transcriptomic data from the large and recently characterized Aquavolonidae-Treumulida-'Novel Clade 12' group, and phylogenomics supported its position as sister to the cercozoan subphylum, Endomyxa. These results provide insights into the phylogeny of the Cercozoa and the Rhizaria as a whole., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sainouroidea is a molecularly diverse clade of cercozoan flagellates and amoebae in the eukaryotic supergroup Rhizaria. Previous 18S rDNA environmental sequencing of globally collected fecal and soil samples revealed great diversity and high sequence divergence in the Sainouroidea. However, a very limited amount of this diversity has been observed or described. The two described genera of amoebae in this clade are Guttulinopsis, which displays aggregative multicellularity, and Rosculus, which does not. Although the identity of Guttulinopsis is straightforward due to the multicellular fruiting bodies they form, the same is not true for Rosculus, and the actual identity of the original isolate is unclear. Here we isolated amoebae with morphologies like that of Guttulinopsis and Rosculus from many environments and analyzed them using 18S rDNA sequencing, light microscopy, and transmission electron microscopy. We define a molecular species concept for Sainouroidea that resulted in the description of 4 novel genera and 12 novel species of naked amoebae. Aggregative fruiting is restricted to the genus Guttulinopsis, but other than this there is little morphological variation amongst these taxa. Taken together, simple identification of these amoebae is problematic and potentially unresolvable without the 18S rDNA sequence., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

A phaeodarian morphotype, characterized by the feet surrounded with forked pedal spines with anchor-like structures, was collected in the subtropical North Pacific. Considering the morphological and phylogenetic uniqueness, this morphotype is described as Gazelletta kashiwaensis sp. nov. The distribution of this new species is possibly affected by the Kuroshio Current. The feeding behavior of living phaeodarians was first filmed: the present new species floated in the water column stretching "protoplasmic webs" and collected diatoms by repeating the expansion and retraction of "pseudopodium-like tentacles"., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Rhizarian 'Novel Clade 10' (NC10) is frequently detected by 18S rRNA gene sequencing studies in freshwater planktonic samples. We describe a new genus and two species of eukaryovorous biflagellate protists, Aquavolon hoantrani n. gen. n. sp. and A. dientrani n. gen. n. sp., which represent the first morphologically characterized members of NC10, here named Aquavolonida ord. nov. The slightly metabolic cells possess naked heterodynamic flagella, whose kinetosomes lie at a right angle to each other and are connected by at least one fibril. Unlike their closest known relative Tremula longifila, they rotate around their longitudinal axis when swimming and only very rarely glide on surfaces. Screening of a wide range of environmental DNA extractions with lineage-specific PCR primers reveals that Aquavolonida consists of a large radiation of protists, which are most diversified in freshwater planktonic habitats and as yet undetected in marine environments. Earlier-branching lineages in Aquavolonida include less frequently detected organisms from soils and freshwater sediments. The 18S rRNA gene phylogeny suggests that Aquavolonida forms a common evolutionary lineage with tremulids and uncharacterized 'Novel Clade 12', which likely represents one of the deepest lineages in the Rhizaria, separate from Cercozoa (Filosa), Endomyxa, and Retaria., (© 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

Infrakingdom Rhizaria is one of four major subgroups with distinct cell body plans that comprise eukaryotic kingdom Chromista. Unlike other chromists, Rhizaria are mostly heterotrophic flagellates, amoebae or amoeboflagellates, commonly with reticulose (net-like) or filose (thread-like) feeding pseudopodia; uniquely for eukaryotes, cilia have proximal ciliary transition-zone hub-lattices. They comprise predominantly flagellate phylum Cercozoa and reticulopodial phylum Retaria, whose exact phylogenetic relationship has been uncertain. Given even less clear relationships amongst cercozoan classes, we sequenced partial transcriptomes of seven Cercozoa representing five classes and endomyxan retarian Filoreta marina to establish 187-gene multiprotein phylogenies. Ectoreta (retarian infraphyla Foraminifera, Radiozoa) branch within classical Cercozoa as sister to reticulose Endomyxa. This supports recent transfer of subphylum Endomyxa from Cercozoa to Retaria alongside subphylum Ectoreta which embraces classical retarians where capsules or tests subdivide cells into organelle-containing endoplasm and anastomosing pseudopodial net-like ectoplasm. Cercozoa are more homogeneously filose, often with filose pseudopodia and/or posterior ciliary gliding motility: zooflagellate Helkesimastix and amoeboid Guttulinopsis form a strongly supported clade, order Helkesida. Cercomonads are polyphyletic (Cercomonadida sister to glissomonads; Paracercomonadida deeper). Thecofilosea are a clade, whereas Imbricatea may not be; Sarcomonadea may be paraphyletic. Helkesea and Metromonadea are successively deeper outgroups within cercozoan subphylum Monadofilosa; subphylum Reticulofilosa (paraphyletic on site-heterogeneous trees) branches earliest, Granofilosea before Chlorarachnea. Our multiprotein trees confirm that Rhizaria are sisters of infrakingdom Halvaria (Alveolata, Heterokonta) within chromist subkingdom Harosa (= SAR); they further support holophyly of chromist subkingdom Hacrobia, and are consistent with holophyly of Chromista as sister of kingdom Plantae. Site-heterogeneous rDNA trees group Kraken with environmental DNA clade 'eSarcomonad', not Paracercomonadida. Ectoretan fossil dates evidence ultrarapid episodic stem sequence evolution. We discuss early rhizarian cell evolution and multigene tree coevolutionary patterns, gene-paralogue evidence for chromist monophyly, and integrate this with fossil evidence for the age of Rhizaria and eukaryote cells, and revise rhizarian classification.

Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens 'O-type' and 'M-type'. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels (Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within 'M. refringens' are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3' end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5' end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5' end sequence of 28-33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution., Competing Interests: The authors have declared that no competing interests exist.

Protists are among the most diverse and abundant eukaryotes in soil. However, gaps between described and sequenced protist morphospecies still present a pending problem when surveying environmental samples for known species using molecular methods. The number of sequences in the molecular PR 2 database (∼130,000) is limited compared to the species richness expected (>1 million protist species) - limiting the recovery rate. This is important, since high throughput sequencing (HTS) methods are used to find associative patterns between functional traits, taxa and environmental parameters. We performed HTS to survey soil flagellates in 150 grasslands of central Europe, and tested the recovery rate of ten previously isolated and cultivated cercomonad species, among locally found diversity. We recovered sequences for reference soil flagellate species, but also a great number of their phylogenetically evaluated genetic variants, among rare and dominant taxa with presumably own biogeography. This was recorded among dominant (cercozoans, Sandona), rare (apusozoans) and a large hidden diversity of predominantly aquatic protists in soil (choanoflagellates, bicosoecids) often forming novel clades associated with uncultured environmental sequences. Evaluating the reads, instead of the OTUs that individual reads are usually clustered into, we discovered that much of this hidden diversity may be lost due to clustering., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Cercomonads are among the most abundant and diverse groups of heterotrophic flagellates in terrestrial systems and show an affinity to plants. However, we still lack basic knowledge of plant-associated protists. We isolated 75 Cercomonadida strains from the phyllosphere and rhizosphere of plants from three functional groups: grasses (Poa sp.), legumes (Trifolium sp.) and forbs (Plantago sp.), representing 28 OTUs from the genera Cercomonas, Neocercomonas and Paracercomonas. The community composition differed clearly between phyllosphere and rhizosphere, but was not influenced by plant species identity. From these isolates we describe three novel cercomonad species including Neocercomonas epiphylla that was consistently and exclusively isolated from the phyllosphere. For each new species we provide a detailed morphological description as well as an 18S rDNA gene sequence as a distinct marker of species identity. Our data contribute to a better resolution of the systematics of cercomonads and their association with plants, by describing three novel species and adding gene sequences of 10 new cercomonad genotypes and of nine previously described species. In view of the functional importance of cercozoan communities in the phyllosphere and rhizosphere of plants, a more detailed understanding of their composition, function and predator-prey interactions are clearly required., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Quadricilia rotundata is a heterotrophic flagellate with four flagella. However, because this species has no clear morphological characteristics or molecular data affiliating it with any known group, Q. rotundata has been treated as a protist incertae sedis, for a long time. Here, we established a clonal culture of Q. rotundata and sequenced its 18S rDNA sequence. Molecular phylogenetic analysis successfully placed Q. rotundata in an environmental clade within Cercozoa, which contributes to expand the morphological and species diversity within Cercozoa. We also discuss morphological evolution within Cercozoa based on this finding., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation., (© 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.)

The genus Diaphoropodon, Archer 1869, comprises filose amoebae with agglutinated tests made of quartz grains, diatom frustules and other particulate materials. The key trait of the genus is a hyaline theca covered with numerous 5- to 10-μm-long, hairlike rods. Based on SSU rDNA phylogeny, we show that Diaphoropodon groups closely to Lecythium, a testate amoeba genus with a flexible but naked theca. Electron microscopic images reveal that the rods of Diaphoropodon are not perforating the test but lie randomly distributed on the surface of the amoeba. Comparing fairly naked cells from our cultures with cells from the environment leads to the conclusion that these rods play a role in agglutinating the material on the test., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

Chlorotic streak is a global disease of commercial sugarcane (Saccharum spp. hybrids). The disease is transmitted by wet soil, water, as well as in diseased planting material. Although first recognized almost 90 years ago and despite significant research effort, the identity of the causal agent has been elusive. Metagenomic high throughput sequencing (HTS) facilitated the discovery of novel protistan ribosomal and nuclear genes in chlorotic streak-infected sugarcane. These sequences suggest a possible causal agent belonging to the order Cercomonadida (Rhizaria, phylum Cercozoa). An organism with morphological features similar to cercomonads (=Cercomonadida) was isolated into pure axenic culture from internal stalk tissues of infected sugarcane. The isolated organism contained DNA sequences identical to those identified in infected plants by HTS. The DNA sequences and the morphology of the organism did not match any known species. Here we present a new genus and species, Phytocercomonas venanatans, which is associated with chlorotic streak of sugarcane. Amplicon sequencing also supports that P. venanatans is associated with this disease. This is the first reported member from Cercomonadida showing a probable pathogenic association with higher plants.

A rich eukaryotic planktonic community exists in high-mountain lakes despite the diluted, oligotrophic and cold, harsh prevailing conditions. Attempts of an overarching appraisal have been traditionally hampered by observational limitations of small, colorless, and soft eukaryotes. We aimed to uncover the regional eukaryotic biodiversity of a mountain lakes district to obtain general conclusions on diversity patterns, dominance, geographic diversification, and food-web players common to oligotrophic worldwide distributed freshwater systems. An unprecedented survey of 227 high-altitude lakes comprising large environmental gradients was carried out using Illumina massive tag sequencing of the 18S rRNA gene. We observed a large Chrysophyceae dominance in richness, abundance and novelty, and unveiled an unexpected richness in heterotrophic phagotrophs and parasites. In particular, Cercozoa and Chytridiomycota showed diversity features similar to the dominant autotrophic groups. The prominent beta-dispersion shown by parasites suggests highly specific interactions and a relevant role in food webs. Interestingly, the freshwater Pyrenean metacommunity contained more diverse specific populations than its closest marine oligotrophic equivalent, with consistently higher beta-diversity. The relevance of unseen groups opens new perspectives for the better understanding of planktonic food webs. Mountain lakes, with remarkable environmental idiosyncrasies, may be suitable environments for the genetic diversification of microscopic eukaryotic life forms.

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost-effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new-generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%-100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment., (© 2017 John Wiley & Sons Ltd.)

The genus Penardeugenia DEFLANDRE 1958 accommodates a single species which was described as Pamphagus bathybioticus PENARD 1904. Although this species seems to be well characterized in its description, it has never been recorded again. Despite its possession of silica scales, it was assigned to the scale-lacking Chlamydophryidae (Thecofilosea, Cercozoa). We here present morphological data of three isolates. One of them was successfully cultured and enabled the amplification of its SSU rDNA sequence. Molecular analyses revealed the evolutionary origin of Penardeugenia in the scale-bearing flagellate class Thaumatomonadida, which is backed up by morphological similarities of their scales. We consider Penardeugenia to represent a closely related genus to Thaumatomastix, which apparently lost its ability to form flagella. We describe two new species from Germany, P. huelsmannii and P. hausmannii., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Although protists occupy a vast range of habitats and are known to interact with plants among other things via disease suppression, competition or growth stimulation, their contributions to the 'phytobiome' are not well described. To contribute to a more comprehensive picture of the plant holobiont, we examined cercozoan and oomycete taxa living in association with the model plant Arabidopsis thaliana grown in two different soils. Soil, roots, leaves and wooden toothpicks were analysed before and after surface sterilization. Cercozoa were identified using 18S rRNA gene metabarcoding, whereas the Internal Transcribed Spacer 1 was used to determine oomycetes. Subsequent analyses revealed strong spatial structuring of protist communities between compartments, although oomycetes appeared more specialized than Cercozoa. With regards to oomycetes, only members of the Peronosporales and taxa belonging to the genus Globisporangium were identified as shared members of the A. thaliana microbiome. This also applied to cercozoan taxa belonging to the Glissomonadida and Cercomonadida. We identified a strong influence by edaphic factors on the rhizosphere, but not for the phyllosphere. Distinct differences of Cercozoa found preferably in wood or fresh plant material imply specific niche adaptations. Our results highlight the importance of micro-eukaryotes for the plant holobiont., (© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Precise and sufficiently detailed morphological taxonomy is vital in biology, for example in the accurate interpretation of ecological and palaeoecological datasets, especially in polar regions, where biodiversity is poor. Testate amoebae on the Antarctic Peninsula (AP) are well-documented and variations in their population size have recently been interpreted as a proxy for microbial productivity changes in response to recent regional climate change. AP testate amoeba assemblages are dominated by a small number of globally ubiquitous taxa. We examine morphological variation in Corythion spp. across the AP, finding clear evidence supporting the presence of two morphospecies. Corythion constricta (Certes 1889) was identified on the AP for the first time and has potentially been previously misidentified. Furthermore, a southerly trend of decreasing average test size in Corythion dubium (Taránek 1881) along the AP suggests adaptive polymorphism, although the precise drivers of this remain unclear, with analysis hindered by limited environmental data. Further work into morphological variation in Corythion is needed elsewhere, alongside molecular analyses, to evaluate the potential for (pseudo)cryptic diversity within the genus. We advocate a parsimonious taxonomical approach that recognises genetic diversity but also examines and develops accurate morphological divisions and descriptions suitable for light microscopy-based ecological and palaeoecological studies., (Copyright © 2017 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Vampyrellid amoebae (Vampyrellida, Rhizaria) are widespread in freshwater, marine and terrestrial ecosystems and consume a wide range of eukaryotes, e.g. algae, fungi and micrometazoa. Environmental sequences indicate that only a small fraction of their genetic diversity is phenotypically characterised, emphasising the need to further explore unknown vampyrellids and their interactions with prey organisms. This study tests the prey range specificity of three vampyrellid amoebae with 49 strains of three common groups of freshwater algae (Zygnematophyceae, Euglenophyceae and Volvocales), and documents specific interactions by time-lapse microscopy. Two of the amoebae, here introduced as the novel genera Arachnomyxa and Planctomyxa based on morphology and SSU rRNA gene comparisons, display a complementary prey range and consume motile algae, namely Volvocales and Euglenophyceae, respectively. This reveals the existence of specialised 'plankton feeders' in the vampyrellid family Leptophryidae, contrasting with the strikingly broad prey range of Leptophrys vorax. The distinct autecological characteristics found in this group of morphologically rather indistinct amoebae contribute to our knowledge about the vastly understudied vampyrellid amoebae. Furthermore, time-lapse observations suggest that euglenoid movements exerted by the sluggish species of the 'Euglena deses group' as a reaction to vampyrellid contact may serve as an effective defence against microbial predators., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cercozoa are a highly diverse protist phylum in soils and in the phyllosphere of plants. Many families are still poorly described and the vast majority of species are still unknown. Although testate amoebae are among the better-studied protists, only little quantitative information exists on the morphology, phylogeny and ecology of cercozoan Rhogostomidae. We cultured four different strains of Rhogostoma spp. isolated from Arabidopsis leaves, agricultural soil and rhizosphere soil of Ocimum basilicum and Nicotiana sp. We describe Rhogostoma epiphylla sp. nov. and R. cylindrica sp. nov. and present their morphology, studied their food spectra in food range experiments and obtained two SSU rDNA gene sequences resulting in an updated thecofilosean phylogeny. Short generation times, desiccation resistance and the ability to prey on a wide range of algae and yeasts from the phyllosphere were seen as crucial traits for the phyllosphere colonization by Rhogostoma. In contrast, the soil-dwelling R. cylindrica did not feed on eukaryotes in our experiment., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Although testate amoebae have attracted great interest of protistologists for more than a century, some groups, especially those with a hyaline, organic test (=theca) are still poorly known. One of those is the genus Lecythium Hertwig et Lesser, 1874. Only recently Lecythium spp. were characterized by morphological and molecular means, but data on the type species Lecythium hyalinum Hertwig et Lesser, 1874, was still lacking. In this study, we screened for L. hyalinum in freshwater samples of Germany and the Netherlands. Four different isolates of L. hyalinum and one novel species were cultured and characterized by light microscopy. Phylogenetic analyses based on the small ribosomal subunit (SSU) RNA gene show that the genus Lecythium forms two robust clades, one forming a sister group to the Rhizaspididae/Pseudodifflugiidae clade (Tectofilosida), the other branching within 'Novel Clade 4' (Tectofilosida). We untangle this polyphyly by establishing Fisculla gen. nov. and the Fiscullidae fam. nov. for the former of these two clades., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The genus Kraken represents a distinct lineage of filose amoebae within the Cercozoa. Currently a single species, Kraken carinae, has been described. SSU rDNA phylogeny showed an affiliation to the Cercomonadida, branching with weak support at its base, close to Paracercomonas, Metabolomonas, and Brevimastigomonas. Light microscopical analyses showed several unique features of the genus Kraken, but ultrastructure data were lacking. In this study, K. carinae has been studied by electron microscopy, these data conjoined with a two-gene phylogeny were used to give more insight into the evolutionary relationship of the genus Kraken within Cercozoa. The data confirmed the absence of flagella, but also showed novel characteristics, such as the presence of extrusomes, osmiophilic bodies, and mitochondria with flat cristae. Surprising was the presence of single-tier scales which are carried by cell outgrowths, much of what is expected of the last common ancestor of the class Imbricatea. The phylogenetic analyses however confirmed previous results, indicating Kraken as a sister group to Paracercomonas in Sarcomonadea with an increased but still low support of 0.98 PP/63 BP. Based on the unique features of Kraken we establish the Krakenidae fam. nov. that we, due to contradictory results in morphology and phylogeny, assign incertae sedis, Monadofilosa., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The thecate filose amoeba Paulinella chromatophora is a good model organism for understanding plastid organellogenesis because its chromatophore was newly derived from an alpha-cyanobacterium. Paulinella chromatophora was the only known photosynthetic Paulinella species until recent studies that suggested a species level of diversity. Here, we described a new photosynthetic species P. micropora sp. nov. based on morphological and molecular evidence from a newly established strain KR01. The chromatophore genome of P. micropora KR01 was fully determined; the genome was 976,991bp in length, the GC content was 39.9%, and 908 genes were annotated. A pairwise comparison of chromatophore genome sequences between strains KR01 and FK01, representing two different natural populations of P. micropora, showed a 99.85% similarity. Differences between the two strains included single nucleotide polymorphisms (SNPs) in CDSs, which resulted in 357 synonymous and 280 nonsynonymous changes, along with 245 SNPs in non-coding regions. Indels (37) and microinversions (14) were also detected. Species diversity for photosynthetic Paulinella was surveyed using samples collected from around the world. We compared our new species to two photosynthetic species, P. chromatophora and P. longichromatophora. Phylogenetic analyses using four gene markers revealed three distinct lineages of photosynthetic Paulinella species including P. micropora sp. nov., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The genus Hyalodiscus Hertwig and Lesser, 1874 comprises naked freshwater amoebae with a unique set of characters, namely a vibrant orange-red colour, a discoid or fan-shaped morphology, and a characteristic rolling locomotion. Some species feed on the chloroplasts of green algae and were regarded as relatives of Vampyrella Cienkowski, 1865. However, because of striking morphological differences and the lack of molecular data, the exact relationship of Hyalodiscus to vampyrellids is still obscure. Here, I describe Hyalodiscus flabellus sp. nov., a bright orange, fan-shaped amoeba feeding on Oedogonium (Chlorophyceae), which likely is a close relative of the type species H. rubicundus Hertwig and Lesser, 1874. Sequence comparisons of the SSU rRNA gene revealed that H. flabellus belongs to a deep-branching, so far uncharacterised lineage of the order Vampyrellida (Rhizaria), here defined as family Hyalodiscidae POCHE, 1913. Based on these results, the systematic position of the genus Hyalodiscus could be finally clarified, accompanied by the revision of relevant diagnoses and a taxonomic summary. Furthermore, the work reports on endosymbiotic bacteria inhabiting the cytoplasm of H. flabellus, which were identified as "Candidatus Megaira polyxenophila" (Rickettsiales, Alphaproteobacteria) using the full cycle rRNA approach with newly designed FISH probes for this widespread endosymbiotic bacterium., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

For over a century testate amoebae have been a favoured group of interest for protistologists, however there is still an endless amount of unanswered questions. The genus Plagiophrys, Claparède and Lachmann 1859, is still one of the unresolved mysteries as it comprises species with high morphological diversity of which no molecular data are available. To shed light on the phylogeny and taxonomy of Plagiophrys we (a) cultured four isolates of three Plagiophrys morphospecies and provided morphological observations (b) obtained three new SSU RNA gene sequences and conducted phylogenetic analyses of the Thecofilosea and (c) did intensive literature research, showing that Plagiophrys is polyphyletic. We partially untangle this polyphyly by combining several of its species with the genus Rhizaspis, Skuja 1948. Furthermore, we establish Sacciforma gen. nov. to accommodate P. sacciformis as it groups within the formerly known Rhizaspididae, which do not comprise our isolates of Rhizaspis (and therefore were renamed Rhogostomidae) since Rhizaspis it groups with maximum support as a sister-group to the Pseudodifflugiidae., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Although testate amoebae have attracted interest of protistologists for more than 150 years, some groups especially those with a hyaline, organic test (= theca) are still poorly known. One of those is the genus Lecythium (Chlamydophryidae, Tectofilosida, Cercozoa, Rhizaria), first described by Hertwig and Lesser in 1874. Only old, sometimes obscure, species descriptions were available until only recently a new species of Lecythium was described and a small ribosomal subunit RNA gene (SSU) sequence was provided. To shed light on the phylogeny and taxonomy of Lecythium, we (a) cultured six isolates of five Lecythium species and provide morphological as well as ecological observations, (b) obtained six new SSU sequences and conducted phylogenetic analyses of the Tectofilosida, showing that Lecythium splits into terrestrial and freshwater clades, and (c) did an intensive literature research on testate amoebae with a theca and provide an illustrated identification key focusing on Lecythium. For the first time, the presence of cysts in the genus Lecythium is reported and we compared those to the cysts of the presumed closely related Chlamydophrys stercorea. Our results suggest that still many undescribed Lecythium species will be found in terrestrial and freshwater habitats., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Mitochondria exist on a functional and evolutionary continuum that includes anaerobic mitochondrion-related organelles (MROs), such as hydrogenosomes. Hydrogenosomes lack many classical mitochondrial features, including conspicuous cristae, mtDNA, the tricarboxylic acid (TCA) cycle, and ATP synthesis powered by an electron transport chain (ETC); instead, they produce ATP anaerobically, liberating H 2 and CO 2 gas in the process. However, our understanding of the evolutionary transformation from aerobic mitochondria to various MRO types remains incomplete. Here we describe a novel MRO from a cercomonad (Brevimastigomonas motovehiculus n. sp.; Rhizaria). We have sequenced its 30,608-bp mtDNA and characterized organelle function through a combination of transcriptomic, genomic, and cell biological approaches. B. motovehiculus MROs are metabolically versatile, retaining mitochondrial metabolic pathways, such as a TCA cycle and ETC-driven ATP synthesis, but also possessing hydrogenosomal-type pyruvate metabolism and substrate-level phosphorylation. Notably, the B. motovehiculus ETC is degenerate and appears to be losing cytochrome-based electron transport (complexes III and IV). Furthermore, the F 1 F o ATP synthase (complex V) is unique, with the highly conserved Atpα subunit fragmented into four separate pieces. The B. motovehiculus MRO appears to be in the process of losing aerobic metabolic capacities. Our findings shed light on the transition between organelle types, specifically the early stages of mitochondrial adaptation to anaerobiosis., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Cercozoa are abundant free-living soil protozoa and quantitatively important in soil food webs; yet, targeted high-throughput sequencing (HTS) has not yet been applied to this group. Here we describe the development of a targeted assay to explore Cercozoa using HTS, and we apply this assay to measure Cercozoan community response to drought in a Danish climate manipulation experiment (two sites exposed to artificial drought, two unexposed). Based on a comparison of the hypervariable regions of the 18S ribosomal DNA of 193 named Cercozoa, we concluded that the V4 region is the most suitable for group-specific diversity analysis. We then designed a set of highly specific primers (encompassing ~270 bp) for 454 sequencing. The primers captured all major cercozoan groups; and >95% of the obtained sequences were from Cercozoa. From 443 350 high-quality short reads (>300 bp), we recovered 1585 operational taxonomic units defined by >95% V4 sequence similarity. Taxonomic annotation by phylogeny enabled us to assign >95% of our reads to order level and ~85% to genus level despite the presence of a large, hitherto unknown diversity. Over 40% of the annotated sequences were assigned to Glissomonad genera, whereas the most common individually named genus was the euglyphid Trinema. Cercozoan diversity was largely resilient to drought, although we observed a community composition shift towards fewer testate amoebae.

Two heterotrophic flagellate strains were isolated from marine sediment samples off eastern Canada and Korea. These new isolates are indistinguishable by light microscopy from the unclassified protist Cyranomonas australis. The organisms are ovoid-shaped cells, 3.5-6μm long, laterally compressed, and somewhat flexible. They have two unequal flagella, about 1.1-2.5 times body length. Typically, the cells show a gliding motility and do not exhibit any amoeboid form or pseudopodia. 18S rDNA phylogenies clearly indicate that the isolates can be assigned to the taxon Filosa, within Cercozoa. The isolates are closest to an environmental sequence (CYSGM-16; 99% identity). Cyranomonas, CYSGM-16, and uncultured eukaryote RM1-SGM46 form a clade with strong statistical supports, here called novel clade CU (Cyranomonas plus Uncultured eukaryotes). This clade may be sister to the order Marimonadida. The novel clade CU and the Marimonadida have been detected only in marine habitats. Our findings suggest that C. australis may not belong to any previously described family within Filosa and Cercozoa., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Paramyxida is an order of rhizarian protists that parasitise marine molluscs, annelids and crustaceans. They include notifiable pathogens (Marteilia spp.) of bivalves and other taxa of economic significance for shellfish production. The diversity of paramyxids is poorly known, particularly outside of commercially important hosts, and their phylogenetic position is unclear due to their extremely divergent 18S rDNA sequences. However, novel paramyxean lineages are increasingly being detected in a wide range of invertebrate hosts, and interest in the group is growing, marked by the first 'Paramyxean Working Group' Meeting held in Spain in February 2015. We review the diversity, host affiliations, and geographical ranges of all known paramyxids, present a comprehensive phylogeny of the order and clarify its taxonomy. Our phylogenetic analyses confirm the separate status of four genera: Paramarteilia, Marteilioides, Paramyxa and Marteilia. Further, as including M. granula in Marteilia would make the genus paraphyletic we suggest transferring this species to a new genus, Eomarteilia. We present sequence data for Paramyxa nephtys comb. n., a parasite of polychaete worms, providing morphological data for a clade of otherwise environmental sequences, sister to Marteilioides. Light and electron microscopy analyses show strong similarities with both Paramyxa and Paramyxoides, and we further discuss the validity of those two genera. We provide histological and electron microscopic data for Paramarteilia orchestiae, the type species of that genus originally described from the amphipod Orchestia; in situ hybridisation shows that Paramarteilia also infects crab species. We present, to our knowledge, the first known results of a paramyxid-specific environmental DNA survey of environmental (filtered water, sediment, etc.) and organismally-derived samples, revealing new lineages and showing that paramyxids are associated with a wider range of hosts and habitat types than previously known. On the basis of our new phylogeny we propose phylogenetic hypotheses for evolution of lifecycle and infectivity traits observed in different paramyxid genera., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

The largest biological surface on earth is formed by plant leaves. These leaf surfaces are colonized by a specialized suite of leaf-inhabiting microorganisms, recently termed "phyllosphere microbiome". Microbial prey, however, attract microbial predators. Protists in particular have been shown to structure bacterial communities on plant surfaces, but virtually nothing is known about the community composition of protists on leaves. Using newly designed specific primers targeting the 18S rDNA gene of Cercozoa, we investigated the species richness of this common protist group on leaves of four Brassicaceae species from two different locations in a cloning-based approach. The generated sequences revealed a broad diversity of leaf-associated Cercozoa, mostly bacterial feeders, but also including known plant pathogens and a taxon of potential endophytes that were recently described as algal predators in freshwater systems. This initial study shows that protists must be regarded as an integral part of the microbial diversity in the phyllosphere of plants., (© 2016 The Authors. The Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.)

The term 'filose amoebae' describes a highly polyphyletic assemblage of protists whose phylogenetic placement can be unpredictable based on gross morphology alone. We isolated six filose amoebae from soils of two European countries and describe a new genus and species of naked filose amoebae, Kraken carinae gen. nov. sp. nov. We provide a morphological description based on light microscopy and small subunit rRNA gene sequences (SSU rDNA). In culture, Kraken carinae strains were very slow-moving and preyed on bacteria using a network of filopodia. Phylogenetic analyses of SSU sequences reveal that Kraken are core (filosan) Cercozoa, branching weakly at the base of the cercomonad radiation, most closely related to Paracercomonas, Metabolomonas, and Brevimastigomonas. Some Kraken sequences are >99% similar to an environmental sequence obtained from a freshwater lake in Antarctica, indicating that Kraken is not exclusively soil dwelling, but also inhabits freshwater habitats., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The case study targeted to determine the aetiology of nodular gill disease (NGD) of farmed rainbow trout. The methods included microscopical examination of gill material in fresh, culturing of isolated organisms, histology, transmission electron microscopy and molecular biology identification. The results revealed an intravital colonization of fish gills by the testate amoeba Rhogostoma minus Belar, 1921. Rhogostoma infection was found in all fish examined microscopically (15/15); in contrast, naked amoebae related to fully developed NGD lesions were found in minority of these fish (5/15). They belonged to four genera, Acanthamoeba, Vermamoeba, Naegleria and Vannella. Results presented in this study contribute to the mosaic of findings that contrary to amoebic gill disease of marine fish turn attention to the possibility of the heterogeneous, multi-amoeba-species and multifactorial aetiology of NGD., (© 2015 John Wiley & Sons Ltd.)

Testate amoebae have been frequently studied by protistologists, but still little information is available on some groups like the Chlamydophryidae. These amoebae are difficult to culture and therefore quantitative information on their morphology, phylogeny and ecology is scarce. We isolated and cultured a small testate amoeba from an agricultural field at Müncheberg near Berlin, Germany. Morphological analyses revealed it to be a new species of the genus Lecythium. We describe Lecythium terrestris sp. nov. and present its morphology, mycophagous and algivorous feeding habits and its ability to form cell aggregates by fusion. Using small-subunit ribosomal RNA gene phylogeny, we could confirm the phylogenetic position of the genus Lecythium among the Cercozoa where it groups closely to Pseudodifflugiidae (Tectofilosida)., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Protozoan parasites of the genus Marteilia have been detected in marine bivalves and other invertebrates around the world, associated in some cases with mass mortalities. The present paper reports the characterization of the Marteilia sp. protozoan infecting the digestive gland of the Grooved Razor Shell clam (Solen marginatus) from Galicia (NW Spain), proposing a novel species in the genus: Marteilia octospora n. sp. Morphological and molecular techniques were used for the description of this parasite. Tissue imprints were essential in the study to confirm the presence of 8 spores per sporangium, a number never reported in other species from this genus. An ultrastructural study revealed that the size and number of dense granules, free in the mature sporangia, were quite different from granules in other Marteilia spp. Another morphological difference is the absence of a layer of concentric membranes found surrounding the mature spore in other species. In addition, concentric membranous structures observed in the different stages of the parasite have never been mentioned in other species of genus Marteilia. Moreover, molecular analysis of the rDNA intergenic spacer (IGS) and the internal transcribed spacer (ITS-1) showed differences with the sequences available for other Marteilia spp., (Copyright © 2016 Elsevier Inc. All rights reserved.)