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BackgroundParasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such asSphaerospora molnarireplicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of aS. molnarigenome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identifyS. molnariproteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets.ResultsAn assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25–30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development.ConclusionsIn silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study ofS. molnari’s protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change.

BackgroundLineage-specific gene expansions represent one of the driving forces in the evolutionary dynamics of unique phylum traits. The obligate parasitic group of Cnidaria, Myxozoa, are evolutionarily altered and highly reduced organisms with a simple body plan including cnidarian-specific organelles, polar capsules (a type of nematocyst). A prominent constituent of nematocysts, linking Myxozoa and Cnidaria, represents a group of structural proteins, minicollagens. Despite recent advances in the identification of minicollagens in Myxozoa, the evolutionary history and diversity of minicollagens in Myxozoa and Cnidaria remain elusive. ResultsWe present newly generated transcriptomes of two myxozoan species with a novel pipeline for filtering of closely related contaminant species in RNA-seq data. Mining of the set of our transcriptomes and published -omics data confirmed the existence of myxozoan Ncol-4, once only reported, and we discovered a novel non-canonical type of minicollagen, Ncol-5, which is exclusive to Myxozoa. Phylogenetic analyses support a close relationship of myxozoan Ncol-1,2,3 with minicollagens of Polypodium hydriforme, however, suggest rather independent evolution in the case of two myxozoan minicollagens Ncol-4 and Ncol-5. Additional genome- and transcriptome-wide searches of cnidarian minicollagens expand the dataset to clarify better minicollagen evolutionary trajectories. ConclusionsThe development of a new approach for the handling of contaminated next-generation data by closely related species represents a useful tool for future applications not only in the field of myxozoan research. This pipeline of data processing allowed us to expand the dataset and study the evolution and diversity of minicollagen gene family in Myxozoa and Cnidaria. We identified a novel type of minicollagen in Myxozoa (Ncol-5). We suggest that a high number of minicollagen paralogs in some cnidarians is a result of several recent large gene multiplication events. We revealed a close localization of minicollagens Ncol-1 and Ncol-4 in myxozoan genomes suggesting their common evolutionary history. The unique gene structure of myxozoan Ncol-5 may indicate a putative specific function in the myxozoan polar capsule or tubule. The range of myxozoan minicollagens proves that the myxozoan minicollagen gene repertoire is similar to other cnidarians and is not simplified by the parasitic lifestyle.

International audience; It is very rare to meet protistologists who are not passionate about their study subject. The vast majority of people, however, never get the chance to hear about the work of these researchers. Although every researcher working on protists is likely to be aware of this situation, efforts made and tools employed for dissemination of knowledge are rarely documented. Following a proposal by the Italian Society of Protistology, a workshop at the 2019 VIII European Congress of Protistology in Rome, Italy, was dedicated to protistological knowledge dissemination. Through the many interventions, we discovered the diversity of efforts to reveal the protistan world to the general public, including museum exhibitions and activities, public understanding of science events, citizen science projects, specific book publications, the use of protists in teaching at all levels from primary school children to university undergraduate students, and to a global audience via social media. The participation of the workshop delegates in the discussions indicated that presentations on the wonderful world of protists to the public not only increase the visibility and accessibility of protistology research, but are also very important for the scientific community. Here we report on some of the key aspects of the presentations given in the dissemination workshop.

Molecular data have considerably contributed to building the taxonomy of protists. Recently, the systematics of Hyalospheniidae (Amoebozoa; Tubulinea; Arcellinida) has been widely revised, with implications extending to ecological, biogeographical and evolutionary investigations. Certain taxa, however, still have an uncertain phylogenetic position, including the common and conspicuous species Nebela militaris. A phylogenetic reconstruction of the Hyalospheniidae using partial sequences of the mitochondrial Cytochrome Oxidase Subunit 1 (COI) gene shows that N. militaris does not belong to genus Nebela, but should be placed in its own genus. The morphological singularities (strongly curved pseudostome and a marked notch in lateral view) and phylogenetic placement of our isolates motivated the creation of a new genus: Alabasta gen. nov. Based on their morphology, we include in this genus Nebela kivuense and Nebela longicollis. We discuss the position of genus Alabasta within Hyalospheniidae, and the species that could integrate this new genus based on their morphological characteristics.

Amoeboid protists are extremely abundant and diverse in natural systems where they often play outstanding ecological roles. They can be found in almost all major eukaryotic divisions, and genomic approaches are bringing major changes in our perception of their deep evolutionary relationships. At fine taxonomic levels, the generalization of barcoding is revealing a considerable and unsuspected specific diversity that can be appreciated with careful morphometric analyses based on light and electron microscopic observations. We provide examples on the difficulties and advances in amoeboid protists systematics in a selection of groups that were presented at the VIIIth ECOP/ISOP meeting in Rome, 2019. We conclude that, in all studied groups, important taxonomical rearrangements will certainly take place in the next few years, and systematics must be adapted to incorporate these changes. Notably, nomenclature should be flexible enough to integrate many new high level taxa, and a unified policy must be adopted to species description and to the establishment of types.

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species. Fil: Kosakyan, Anush. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Alama Bermejo, Gema. Universidad Nacional del Comahue. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". - Provincia de Río Negro. Ministerio de Agricultura, Ganadería y Pesca. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Centro Nacional Patagónico. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni"; Argentina. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. State University of Oregon; Estados Unidos Fil: Bartošová-Sojková, Pavla. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Born-Torrijos, Ana. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Šíma, Radek. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa Fil: Nenarokova, Anna. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. University of South Bohemia; República Checa Fil: Eszterbauer, Edit. Hungarian Academy Of Sciences; Hungría Fil: Bartholomew, Jerri. State University of Oregon; Estados Unidos Fil: Holzer, Astrid Sybylle. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa

We describe here a new species of sphagnicolous testate amoeba found abundantly in the forested part of the Le Cachot peatland (Jura Mountains, Neuchâtel, Switzerland) based on microscopical observations (LM, SEM). The new species, called Nebela gimlii was placed in a phylogenetic tree based on mitochondrial cytochrome oxidase sequences (COI), and branched robustly within the N. collaris complex next to the morphologically similar N. guttata and N. tincta. It is however genetically clearly distinct from these two species, and differs morphologically from them by its smaller size and stouter shape of the shell. This new species completes the phylogeny of the Nebela collaris species complex, with now eight species described, mostly from peatlands and acidic forest litter, and further demonstrates the existence of an unknown diversity within testate amoebae. Improving the taxonomy of testate amoebae in peatlands and clarifying the ecology of newly discovered species should make these organisms even more valuable as bioindicator and for palaeoecological reconstruction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Life was microbial for the majority of Earth's history, but as very few microbial lineages leave a fossil record, the Precambrian evolution of life remains shrouded in mystery. Shelled (testate) amoebae stand out as an exception with rich documented diversity in the Neoproterozoic as vase-shaped microfossils (VSMs). While there is general consensus that most of these can be attributed to the Arcellinida lineage in Amoebozoa, it is still unclear whether they can be used as key fossils for interpretation of early eukaryotic evolution. Here, we present a well-resolved phylogenomic reconstruction based on 250 genes, obtained using single-cell transcriptomic techniques from a representative selection of 19 Arcellinid testate amoeba taxa. The robust phylogenetic framework enables deeper interpretations of evolution in this lineage and demanded an updated classification of the group. Additionally, we performed reconstruction of ancestral morphologies, yielding hypothetical ancestors remarkably similar to existing Neoproterozoic VSMs. We demonstrate that major lineages of testate amoebae were already diversified before the Sturtian glaciation (720 mya), supporting the hypothesis that massive eukaryotic diversification took place in the early Neoproterozoic and congruent with the interpretation that VSM are arcellinid testate amoebae.Arcellinid testate amoebae are some of the earliest eukaryotic microbes. Lahr et al. show that these amoebae have seven major deep phylogenetic lineages, probably diverged as early as 730 mya. Diversification of these microbes in the mid-Neoproterozoic was probably happening alongside the gradual oxygenation of oceans.

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

Quadrulella (Amoebozoa, Arcellinida, Hyalospheniidae) is a genus of testate amoebae with unmistakable morphology, which secretes characteristic square plates to reinforce the test. They are mainly known from fens and freshwater habitats and have never been documented in deserts. We describe a new species, Quadrulella texcalense, from biological soil crusts in the intertropical desert of Tehuacán (state of Puebla, Mexico). Quadrulella texcalense occurred only at altitudes between 2140 and 2221 m.a.s.l., together with the bryophyte genera Pseudocrossidium, Weissia, Bryum, Didymodon, Neohyophyla and Aloina. The soil was extremely dry (moisture of 1.97–2.6%), which contrasts sharply with previous reports for the Quadrulella genus. Single cell mitochondrial cytochrome oxidase I (COI) barcoding of thirteen isolated cells showed an important morphological variability despite having all the same COI barcode sequence. Quadrulella texcalense was placed in a tree containing other Hyalsopheniidae, including a newly barcoded South African species, Q. elegans. Q. texcalense unambiguously branched within genus Quadrulella in a compact clade but with a long branch, suggesting accelerated evolution due to a transition towards a new environment and/or under-sampling.

Nebela collaris (Ehrenberg 1848) Leidy, 1879 Updated description. See Kosakyan et al. (2013, p. 12). Neotype. Kosakyan et al., 2013; Fig. 3., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on page 619, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712, {"references":["Leidy, J., 1879. Fresh-water Rhizopods of North America. U. S. Geol. Surv. Terr. 12, 1 - 324.","Kosakyan, A., Gomaa, F., Mitchell, E. A. D., Heger, T. J., Lara, E., 2013. Using DNA-barcoding for sorting out protist species complexes: a case study of the N ebela tincta-collaris-bohemica group (Amoebozoa; Arcellinida, Hyalospheniidae). Eur. J. Protistol. 49, 222 - 237."]}

Arcellinida Kent 1880 Hyalospheniidae (Schulze) Kosakyan et Lara 2012

Quadrulella madibai sp. nov. Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara Description. Test ovoid or pyriform, slightly elongated, with a rounded posterior end, laterally compressed towards the aperture. Test colourless, composed of square plates, regularly arranged in rows. The plates are smaller near the aperture (5 ��� 6 ��m) then gradually become larger (9 ��� 11 ��m) toward the posterior end of the test. Dimensions (based on five individuals) of the test: L = 83 ��� 91 ��m, B = 37 ��� 45 ��m. Aperture 21 ��� 23 ��m wide, strongly curved and bordered by a thin organic lip. Differential diagnosis. See diagnosis of Q. symmetrica. Type locality. Pitse mire, a valley peatland in a landscape of upland grassland in the Welgevonden Game Park adjacent to Marakele national park, South Africa; 24 �� 24 I 29.3 II S; 0 27 �� 48 I 43.8 II E. Holotype. Fig. 4b. Etymology. This species was named to honour Nelson Mandela, the South African revolutionary and later president, who was called Madiba by his friends in reference to the name of his clan., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on page 620, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712

Mrabella gen. nov. Kosakyan, Lahr, Mulot, Meis- terfeld, Mitchell and Lara Description. Test elongated-pyriform, laterally compressed, with the lateral sides gradually tapering toward the pseudostome. Lateral margins distinctly compressed giving the impression of a thick and wide hollow keel. Because of this, the test is elongated elliptical with the pointed end in the profile. Test colourless, composed of quadrangular shell plates similar to those in Quadrulella species. Differential diagnosis. See diagnosis of the genus Quadrulella above. Type species. Mrabella subcarinata comb. nov. (Gauthier-Li��vre, 1957) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara. Included taxa. Q. subcarinata Gauthier-Li��vre, 1957 and the similar taxa Q. plicata Hoogenraad et de Groot 1940. Etymology. The name of the genus derives from the Swahili word ���mraba��� (square)., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on pages 620-621, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712, {"references":["Gauthier-Lievre, L., 1957. Additions aux Nebela d'Afrique. Bull. Soc. Hist. Nat. l'Afrique du Nord 48, 494 - 523."]}

Cornutheca gen. nov. Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara Description. Test elongated-pyriform, with a distinct neck, lateral margins tapering towards the aperture. Two lateral horns, pointing towards the posterior part of the test, either free or connected to the main part of the test by a lateral keel surrounding the posterior part of the test. Test hyaline or slightly yellowish, composed of circular to elongated shell plates probably recycled from euglyphid testate amoeba prey. Differential diagnosis. Members of this genus differ from other hyalospheniid genera in the presence of two lateral horns. Type species. Cornutheca ansata (Leidy, 1879) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara comb. nov. Included taxa. We include in this genus all Nebela taxa bearing two lateral horns, such as N. ansata, N. saccifera and N. hippocrepis. Etymology. The name of the genus derives from the Latin words ���cornus��� and ���theca���, meaning horns and shell, the traits that are characteristic for the members of the genus., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on page 622, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712, {"references":["Leidy, J., 1879. Fresh-water Rhizopods of North America. U. S. Geol. Surv. Terr. 12, 1 - 324."]}

G. galeata Penard, 1890 comb. nov. Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara Description. Test elongated in broad lateral view, laterally compressed, with the lateral sides gradually tapering toward the pseudostome. Lateral margins distinctly compressed giving the impression of a thick lateral keel. Because of this, the test is elongated elliptical with a pointed aboral end in profile (narrow lateral view). Two small lateral pores are present, one on each side of the test (sometimes hard to observe) almost at the point where the keel begins. Test colourless, reinforced with siliceous plates seemingly recycled from predated organisms. Test relatively large, L = 180 ��� 283 ��m, B = 94 ��� 190 ��m. Pseudostome linear or slightly curved in broad lateral view, surrounded by a thick organic collar, 26 ��� 51 ��m wide. Type. Plate VI, figs 78 ��� 84 (Penard, 1890)., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on page 621, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712, {"references":["Penard, E, 1890. Etudes sur les Rhizopodes d'eau douce: Mem. Soc. Phys. Hist. Nat. Geneve 31, 1 - 230."]}

Planocarina gen. nov. Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara Description. Test elongated-pyriform, with a distinct neck, lateral margins tapering towards the aperture. A flat keel surrounds the entire posterior part of the test. Test hyaline or slightly yellowish, composed of circular to elongated shell plates probably recycled from euglyphid testate amoeba prey. Differential diagnosis. See diagnosis of Gibbocarina. Type species. Planocarina carinata (Archer 1867) Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara comb. nov. [syn.: Nebela carinata (Archer) Leidy, 1879]. Included taxa. We include in this genus all Nebela species with a flat keel, such as: N. carinata, N. marginata, N. maxima and N. spumosa, but excluding those with a lateral horn (i.e. the genus Cornutheca). Etymology. The name of the genus derives from the Latin words ���planus��� and ���carina���, meaning flat keel, a characteristic trait for the members of the genus., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on pages 621-622, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712, {"references":["Leidy, J., 1879. Fresh-water Rhizopods of North America. U. S. Geol. Surv. Terr. 12, 1 - 324."]}

Species diversity in most protistan groups has been underestimated. Many morpho-species are in fact complexes that require detailed morphometric studies to be discriminated. However, which traits can be used for species descriptions remains in many cases unclear. The testate amoeba genus Quadrulella produces self-secreted, siliceous plates with a very characteristic square shape-such plates were assumed to be synapomorphic of the genus. Here we demonstrate that Quadrulella symmetrica (the most common Holarctic species) is not monophyletic. Square plate size and arrangement, test size and general shape are efficient criteria for species discrimination. Based on morphology and sequence data, we describe Quadrulella variabilis sp. nov. from Switzerland, and Quadrulella madibai sp. nov. from South Africa, and confirm the validity of Q. alata. The former species Q. subcarinata does not belong to the genus Quadrulella. We therefore transfer this species to the new genus Mrabella gen. nov. Our results show that hyalosphenids presenting siliceous square shell plates do not form a monophyletic clade. Several possible hypotheses about the origins of square plates are discussed. Additionally, this comprehensive phylogenetic analysis of the family Hyalospheniidae confirms that the genus Nebela is paraphyletic and needs to be divided into genera based on general shell shape: Nebela sensu stricto, Longinebela gen. nov., Planocarina gen. nov., Gibbocarina gen. nov., Cornutheca gen. nov. and Mrabella gen. nov.

Quadrulella variabilis sp. nov. Kosakyan, Lahr, Mulot, Meisterfeld, Mitchell and Lara Description. Test ovoid or pyriform, with a rounded posterior end, laterally compressed towards the pseudostome, very similar to that in Q. symmetrica, sometimes with an elongated neck. Test colourless, composed of square plates, regularly arranged in rows. The plates are smaller near the aperture (3 ��� 4 ��m), then gradually become larger (up to 7 ��� 9 ��m) toward the posterior end of the test. Dimensions of the test (based on the seven sequenced individuals): length = 66 ��� 69 (96) ��m, breadth = 35 ��� 40.5 (45) ��m. Aperture 17 ��� 18.5 ��m wide, often curved and bordered by a thin organic lip. Differential diagnosis. See diagnosis of Q. symmetrica. Type locality. The tests were collected from Sphagnum mosses in Le Cachot bog, Vall��e de la Br��vine, Canton of Neuch��tel, Switzerland (47.5 �� N, 6.4 �� E). Holotype. Fig. 2, paratype Fig. 7 d. Etymology. The species has members with a variable neck length. Notes. One other possibility is to describe the entire clade as Q. longicollis. However, given that we have only one Q. longicollis sequence (which may not correspond to the typical Q. longicollis, see Discussion), DNA of which was extracted from more than one cell, we would need additional data to understand whether the elongated neck is a result of phenotypic plasticity or a genetically fixed character., Published as part of Anush Kosakyan, Daniel J. G. Lahr, Matthieu Mulot, Ralf Meisterfeld, Edward A. D. Mitchell & Enrique Lara, 2016, Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes, pp. 606-623 in Cladistics 32 on page 620, DOI: 10.1111/cla.12167, http://zenodo.org/record/238712

Nebela gimlii n.sp. Singer and Lara. The test is wide pyriform or drop-shaped, laterally compressed, with a protruding narrow neck. Two lateral pores are present ca. 1 / 4 from the distance from the pseudostome to the fundus of the test (Fig. 1A and B). A variable number of small pores can be seen on SEM images in apparently random positions from near the aperture to the fundus of the test (Fig. 2A, B, E and F). The test is colourless or slightly brownish, composed of small particles (likely obtained from preys, e.g. euglyphid testate amoebae), which are covered by a thin layer of organic cement. The aperture is oval in frontal view, slightly curved in lateral view, surrounded by a thin organic collar characteristic of family Hyalospheniidae (Kosakyan et al., 2012). Dimensions based on 14 individuals: length: 67.7–77.6 μm (mean = 72.8 μm), breadth: 49.7–61.64 μm (mean = 53.9 μm), width of aperture: 17.5–21 μm (mean = 19 μm). Illustrations and morphological data of the new species are given in Figs. 1 and 2 and Tables 1 and 2. Ecology Nebela gimlii was found exclusively in the relatively dry Sphagnum mosses in the forested part of a peat bog (influenced by lateral drainage) and the drained peatland margin a few meters from the base of old peat extraction walls. These habitats are characterised by low pH, moderate moisture and low nutrient content. Hapantotype The tests were collected from a Sphagnum sample in a peatland in Le Cachot, Jura Mountains, Switzerland (47 º00' 15.23 ''N, 6 º 39 ' 52.83 ''E). One SEM stub with several specimens is deposited at the Natural History Museum of Neuchâtel (Ref. Nr.: UniNe-EM- 5). COI gene sequences of Nebela gimlii el 3 1 (499 bp) and Nebela gimlii el 3 4 (499 bp) were deposited in GenBank (Nebela gimlii el 3 1 KP083297 and, respectively, Nebela gimlii el 3 4 KP083298). Etymology The name of this species refers to the name of Gimli, one of the dwarfs in J.R.R. Tolkien’s masterpiece “The Lord of the Rings”, because of its small size (the smallest known member of the Nebela collaris complex) and stout shape. In addition, it has been found abundantly in a forest, and Gimli was unique among his kind to have been travelling in the woods. gimlii and N. guttata. Min – minimum, Max – maximum, n – number of individuals examined (measurements in μm). * Data from Kosakyan et al. (2013). Note Nebela gimlii can be confused with Nebela guttata, but clearly differs in the wider shape of the shell and the smaller dimensions of the test (Table 2). Both species are closely related and differ by 3.7 % on the considered COI fragment. The two sequences of N. gimlii obtained from two different cells share 99.8 % of similarity.

Environmental DNA surveys are used for screening eukaryotic diversity. However, it is unclear how quantitative this approach is and to what extent results from environmental DNA studies can be used for ecological studies requiring quantitative data. Mitochondrial cytochrome oxidase (COI) is used for species-level taxonomic studies of testate amoebae and should allow assessing the community composition from environmental samples, thus bypassing biases due to morphological identification. We tested this using a COI clone library approach and focusing on the Nebela collaris complex. Comparisons with direct microscopy counts showed that the COI clone library diversity data matched the morphologically identified taxa, and that community composition estimates using the two approaches were similar. However, this correlation was improved when microscopy counts were corrected for biovolume. Higher correlation with biovolume-corrected community data suggests that COI clone library data matches the ratio of mitochondria and that within closely-related taxa the density of mitochondria per unit biovolume is approximately constant. Further developments of this metabarcoding approach including quantifying the mitochondrial density among closely-related taxa, experiments on other taxonomic groups and using high throughput sequencing should make if possible to quantitatively estimate community composition of different groups, which would be invaluable for microbial food webs studies.

Endosymbiosis is a central and much studied process in the evolution of eukaryotes. While plastid evolution in eukaryotic algae has been extensively studied, much less is known about the evolution of mixotrophy in amoeboid protists, which has been found in three of the five super groups of Eukaryotes. We identified the green endosymbionts in four obligate mixotrophic testate amoeba species belonging to three major eukaryotic clades, Hyalosphenia papilio and Heleopera sphagni (Amoebozoa: Arcellinida), Placocista spinosa (Rhizaria: Euglyphida), and Archerella flavum (Stramenopiles: Labyrinthulomycetes) based on rbcL (ribulose-1,5-diphosphate carboxylase/oxygenase large subunit) gene sequences. We further investigated whether there were different phylotypes of algal endosymbionts within single H. papilio cells and the degree of host-symbiont specificity by amplifying two genes: COI (mitochondrial cytochrome oxydase subunit 1) from the testate amoeba host, and rbcL from the endosymbiont. Results show that all studied endosymbionts belong to genus Chlorella sensu stricto, closely related to Paramecium bursaria Chlorella symbionts, some lichen symbionts and also several free-living algae. Most rbcL gene sequences derived from symbionts from all testate amoeba species were almost identical (at most 3 silent nucleotides difference out of 780 bp) and were assigned to a new Trebouxiophyceae taxon we named TACS (Testate Amoeba Chlorella Symbionts). This “one alga fits all mixotrophic testate amoeba” pattern suggests that photosynthetic symbionts have pre-adaptations to endosymbiosis and colonise diverse hosts from a free-living stage.

Species identification by means of morphology is often problematic in protists. Nebela tincta–collaris–bohemica (Arcellinida) is a species complex of small to medium-sized (ca.100 μm) testate amoebae common in peat bogs and forest soils. The taxonomic validity of characters used to define species within this group is debated and causes confusion in studies of biogeography, and applications in palaeoecology. We examined the relationship between morphological and genetic diversity within this species complex by combined analyses of light microscopy imaging and Cytochrome Oxidase Subunit 1(COI) sequences obtained from the same individual amoeba cells. Our goals were (1) to clarify the taxonomy and the phylogenetic relationships within this group, and (2) to evaluate if individual genotypes corresponded to specific morphotypes and the extent of phenotypic plasticity. We show here that small variations in test morphology that have been often overlooked by traditional taxonomy correspond to distinct haplotypes. We therefore revise the taxonomy of the group. We redefine Nebela tincta (Leidy) Kosakyan et Lara and N. collaris (Ehrenberg 1848) Kosakyan et Gomaa, change N. tincta var. rotunda Penard to N. rotunda (Penard 1890), describe three new species: N. guttata n. sp. Kosakyan et Lara, N. pechorensis n. sp. Kosakyan et Mitchell, and N. aliciae n. sp. Mitchell et Lara.

We used Cytochrome Oxidase Subunit 1 (COI) to assess the phylogenetic relationships and taxonomy of Nebela sensu stricto and similar taxa (Nebela group, Arcellinida) in order to clarify the taxonomic validity of morphological characters. The COI data not only successfully separated all studied morphospecies but also revealed the existence of several potential cryptic species. The taxonomic implications of the results are: (1) Genus Nebela is paraphyletic and will need to be split into at least two monophyletic assemblages when taxon sampling is further expanded. (2) Genus Quadrulella, one of the few arcellinid genera building its shell from self-secreted siliceous elements, and the mixotrophic Hyalosphenia papilio branch within the Nebela group in agreement with the general morphology of their shell and the presence of an organic rim around the aperture (synapomorphy for Hyalospheniidae). We thus synonymise Hyalospheniidae and Nebelidae. Hyalospheniidae takes precedence and now includes Hyalosphenia, Quadrulella (previously in the Lesquereusiidae) and all Nebelidae with the exception of Argynnia and Physochila. Leptochlamys is Arcellinida incertae sedis. We describe a new genus Padaungiella Lara et Todorov and a new species Nebela meisterfeldi n. sp. Heger et Mitchell and revise the taxonomic position (and rank) of several taxa. These results show that the traditional morphology-based taxonomy underestimates the diversity within the Nebela group, and that phylogenetic relationships are best inferred from shell shape rather than from the material used to build the shell.

Untangling the relationships between morphology and phylogeny is key to building a reliable taxonomy, but is especially challenging for protists, where the existence of cryptic or pseudocryptic species makes finding relevant discriminant traits difficult. Here we use Hyalosphenia papilio (a testate amoeba) as a model species to investigate the contribution of phylogeny and phenotypic plasticity in its morphology. We study the response of H. papilio morphology (shape and pores number) to environmental variables in (i) a manipulative experiment with controlled conditions (water level), (ii) an observational study of a within-site natural ecological gradient (water level), and (iii) an observational study across 37 European peatlands (climate). We showed that H. papilio morphology is correlated to environmental conditions (climate and water depth) as well as geography, while no relationship between morphology and phylogeny was brought to light. The relative contribution of genetic inheritance and phenotypic plasticity in shaping morphology varies depending on the taxonomic group and the trait under consideration. Thus, our data call for a reassessment of taxonomy based on morphology alone. This clearly calls for a substantial increase in taxonomic research on these globally still under-studied organisms leading to a reassessment of estimates of global microbial eukaryotic diversity.