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Abstract 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Molecular phylogeny is an indispensable tool for assessing evolutionary relationships among protists. The most commonly used marker is the small subunit ribosomal RNA gene, a conserved gene present in many copies in the nuclear genomes. However, this marker is not variable enough at a fine-level taxonomic scale, and intra-genomic polymorphism has already been reported. Finding a marker that could be useful at both deep and fine taxonomic resolution levels seemed like a utopic dream. We designed Amoebozoa-specific primers to amplify a region including partial sequences of two subunits of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase gene (NAD9/NAD7). We applied them to arcellinids belonging to distantly related genera ( Arcella , Difflugia , Netzelia and Hyalosphenia ) and to Arcellinid-rich environmental samples to obtain additional Amoebozoa sequences. Tree topology was congruent with previous phylogenies, all nodes being highly supported, suggesting that this marker is well-suited for deep phylogenies in Arcellinida and perhaps Amoebozoa. Furthermore, it enabled discrimination of close-related taxa. This short genetic marker (ca. 250 bp) can therefore be used at different taxonomic levels, due to a fast-varying intergenic region presenting either a small intergenic sequence or an overlap, depending on the species. [ABSTRACT FROM AUTHOR]

Cryptotephra (particles <125 μm) is a key record for monitoring past and current volcanic activity. However, its extraction from the host sediment and analysis is often long and difficult because of its small size. Finding a simple method to extract cryptotephra from environmental samples would therefore make its analysis much easier. We hypothesized that arcellinid testate amoebae may hold such a potential. These free-living shelled protists are among the earliest microorganisms to colonize volcanic tephra, and build their shell by agglutinating minerals from their environment. We analyzed by X-ray Spectrometry the mineral signature of tephra from the 2011 Puyehue-Cordon Caulle Volcanic Complex (Chile) eruption ash fallout and compared it to that of the shells of 51 individual testate amoebae (three individuals from each of 17 species) from 13 samples collected at different distances from the active vent. The mineral composition of particles within shells closely matched that of similar size class particles from their environment. The capacity of testate amoebae to randomly use mineral grains from their environment makes it possible to use their shells to assess the mineral composition of cryptotephra from soil, peat or sediment samples. Testate amoebae therefore represent the microbial world's version of Cinderella's helping pigeons. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Planktonic arcellinid testate amoebae exhibit a broad-range of morphological variability but it is currently unclear to what extent this variability represents phenotypic plasticity or if it is genetically determined. We investigated the morphology and phylogenetic relationships of three endemic east-asian Difflugia taxa 1) the vase-shaped D. mulanensis , 2) and a spinose and a spineless morphotypes of D. tuberspinifera using scanning electron microscopy and two ribosomal genetic markers (SSU rDNA and ITS sequences). Our phylogenetic analyses shows that all three taxa are genetically distinct and closely related to D. achlora and Netzelia oviformis . The genetic variations between the spineless and spinose morphotypes of D. tuberspinifera were low at the SSU rRNA level (0.4%), but ten times higher at the ITS level (4.5-6%). Our data suggest that the two forms of D. tuberspinifera are sufficiently differentiated in terms of morphology and genetic characteristics to constitute two separate entities and that the presence of spines does not result from phenotypic plasticity due to environmental selective pressure. However further observational and experimental data are needed to determine if these two forms constitute different biological species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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.100μm) 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

The systematics of lobose testate amoebae (Arcellinida), a diverse group of shelled free-living unicellular eukaryotes, is still mostly based on morphological criteria such as shell shape and composition. Few molecular phylogenetic studies have been performed on these organisms to date, and their phylogeny suffers from typical under-sampling artefacts, resulting in a still mostly unresolved tree. In order to clarify the phylogenetic relationships among arcellinid testate amoebae at the inter-generic and inter-specific level, and to evaluate the validity of the criteria used for taxonomy, we amplified and sequenced the SSU rRNA gene of nine taxa - Difflugia bacillariarum, D. hiraethogii, D. acuminata, D. lanceolata, D. achlora, Bullinularia gracilis, Netzelia oviformis, Physochila griseola and Cryptodifflugia oviformis. Our results, combined with existing data demonstrate the following: 1) Most arcellinids are divided into two major clades, 2) the genus Difflugia is not monophyletic, and the genera Netzelia and Arcella are closely related, and 3) Cryptodifflugia branches at the base of the Arcellinida clade. These results contradict the traditional taxonomy based on shell composition, and emphasize the importance of general shell shape in the taxonomy of arcellinid testate amoebae. [Copyright &y& Elsevier]

The mitochondrial Cytochrome Oxidase Subunit 1 gene (COI) has been promoted as an ideal “DNA barcode” for animal species and other groups of eukaryotes. However, the utility of the COI marker for species level discrimination and for phylogenetic analyses has yet to be tested within the Rhizaria. Accordingly, we analysed mitochondrial COI gene sequences and nuclear small subunit rDNA (SSU) sequences from several morphospecies of euglyphid testate amoebae (Cercozoa, Rhizaria) in order to evaluate the utility of these DNA markers for species discrimination and phylogenetic reconstructions. Sequences were obtained from eleven populations belonging to six Cyphoderia morphospecies that were isolated from field samples in North America and Europe. Mean inter-population COI sequence dissimilarities were on average 2.9 times greater than in the SSU, while the intra-population sequence dissimilarities were higher in the SSU (0-0.95%) than in the COI (0%); this suggests that the COI fragment is valuable for discriminating Cyphoderiidae isolates. Our study also demonstrated that COI sequences are useful for inferring phylogenetic relationships among Cyphoderiidae isolates. COI and SSU tree topologies were very similar even though the COI fragment used in these analyses (500bp) was much shorter than the SSU sequences (1600bp). Altogether, these results demonstrate the utility of the COI as a potential taxonomic DNA barcode for assessing cyphoderiid species diversity and for inferring phylogenetic relationships within the group. [ABSTRACT FROM AUTHOR]

Culture-independent molecular methods based on the amplification, cloning and sequencing of small-subunit ribosomal RNA genes (SSU rDNAs) are powerful tools to study the diversity of microorganisms. Despite so, the eukaryotic microbial diversity of many ecosystems, including peatlands has not yet received much attention. We analysed the eukaryotic diversity by molecular surveys in water from the centre of a pristine Sphagnum -dominated peatland in the Jura Mountains of Switzerland during a complete seasonal cycle. The clone libraries constructed from five different temporal samplings revealed a high diversity of protists with representatives of all major eukaryotic phyla. In addition, four sequence types could not be assigned to any known high-level eukaryotic taxon but branched together with a rather good statistic support, raising the possibility of a novel, deep branching eukaryotic clade. The analysis of seasonal patterns of phylotypes showed a clear change in the eukaryotic communities between the warm period (late spring and summer) and the cold period (autumn and winter). Chrysophytes dominated the samples in the cold period while testate amoebae (Arcellinida and Euglyphida) and a few other groups peaked in summer. A few phylotypes (such as a cryptomonad and a perkinsid) were abundant at given sampling times and then almost disappeared, suggesting bloom-like dynamics. [ABSTRACT FROM AUTHOR]

To date only five partial and two complete SSU rRNA gene sequences are available for the lobose testate amoebae (Arcellinida). Consequently, the phylogenetic relationships among taxa and the definition of species are still largely dependant on morphological characters of uncertain value, which causes confusion in the phylogeny, taxonomy and the debate on cosmopolitanism of free-living protists. Here we present a SSU rRNA-based phylogeny of the Hyalospheniidae including the most common species. Similar to the filose testate amoebae of the order Euglyphida the most basal clades have a terminal aperture; the ventral position of the pseudostome appears to be a derived character. Family Hyalospheniidae appears paraphyletic and is separated into three clades: (1) Heleopera sphagni, (2) Heleopera rosea and Argynnia dentistoma and (3) the rest of the species from genera Apodera, Hyalosphenia, Porosia and Nebela. Our data support the validity of morphological characters used to define species among the Hyalospheniidae and even suggest that taxa described as varieties may deserve the rank of species (e.g. N. penardiana var. minor). Finally our results suggest that the genera Hyalosphenia and Nebela are paraphyletic, and that Porosia bigibbosa branches inside the main Nebela clade. [Copyright &y& Elsevier]

Testate amoebae are a diverse group of shelled protists frequently used as model organisms in microbial biogeography. Relatively few species have been reported for the Southern Hemisphere, however, it remains unclear whether this lower diversity is real or an artifact of under-sampling or misidentifications, which would reduce their potential to address macroecological questions. We evaluated testate amoebae diversity from the full range of habitats occurring within two Tierra del Fuego peatlands and compared it with the reported diversity for the area and from the Northern Hemisphere peatlands. We recorded 87 species, of which 69 are new for the region and 45 of them probably new to science and likely to have restricted geographical distributions. Combined with previous studies, the total diversity of testate amoebae only from Tierra del Fuego peatlands now reaches 119, as compared with 183 reported from all Northern Hemisphere peatlands. Our results demonstrate that the number of Gondwanian and Neotropical endemic testate amoeba may be substantially higher than currently known. Previous reports of Holarctic taxa in Tierra del Fuego may result from forcing the identification of morphotypes to the descriptions in the most common literature (force-fitting) South American species into species common in literature from other regions. [ABSTRACT FROM AUTHOR]

Abstract: Testate amoebae (Arcellinida and Euglyphida) and diatoms (Bacillariophyta) respond to different ecological gradients. These protists are useful tools for biomonitoring and paleoecological studies in aquatic and terrestrial ecosystems. However, little is known about the responses of these microeukaryotes to soil management practices. We analyzed the testate amoeba and diatom communities from the DOK-trial (D: biodynamic, O: bio-organic, K: german ‘‘konventionell’’ integrated conventional) agricultural experiment at Therwil, Switzerland. Soil samples were collected from biodynamic and conventional plots and subsequently incubated for four months in a growth chamber. The diatom diversity tended to be higher in the biodynamic than in the two conventional systems. Redundancy analysis (RDA) suggested that diatom community structure differed between organic and the two conventional systems. Testate amoeba abundance was about five times higher in biodynamic than in conventional systems (P < 0.05) but no significant differences in diversity were reported between treatments. Altogether, these data suggest that diatoms and testate amoebae are sensitive to farming systems. As direct analyses of soil samples are time-consuming, molecular tools would be very useful for further development of the use of protists in bioindication. [Copyright &y& Elsevier]

Abstract: Among the non-pollen micro-fossils commonly encountered in Quaternary sediment samples prepared for pollen analysis are many shells of testate amoebae. Testate amoebae are eukaryotic micro-organisms which are increasingly used in ecological and palaeoecological studies, particularly as indicators of hydrological change in Sphagnum-dominated peatlands. In this study we address the extent to which testate amoebae are used in palynological research, the key challenges to more widespread use, and the extent to which ecological information is retained in the testate amoeba assemblages of standard palynological slides. To achieve this we review the literature on the use of testate amoebae in palynology, compare testate amoeba records produced by palynological and water-based preparation methods and carry out simulations using previously-derived datasets. Our results show that testate amoebae are widely encountered in Quaternary palynological studies, primarily in peatlands, but the information which they can provide is undermined by limited taxonomic knowledge. Many taxa are destroyed in pollen preparations, but for taxa that are retained patterns of abundance parallel those determined using water-based preparation methods. Although the loss of sensitive taxa limits the ecological information contained in testate amoeba assemblages the information preserved is likely to be useful in a multiproxy approach to palaeoenvironmental reconstruction. To help improve taxonomic awareness and encourage the use of testate amoebae in palynology we present a basic introduction to testate amoeba taxonomy and a guide to the taxonomic literature. [Copyright &y& Elsevier]

Summary 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. Highlights • Arcellinida testate amoebae are comprised of seven major lineages • Reconstructed hypothetical ancestral states are congruent with Tonian fossils • Combined analysis of phylogenies and fossils suggest divergence as early as 730 mya 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. [ABSTRACT FROM AUTHOR]