Deconstructing Difflugia: The tangled evolution of lobose testate amoebae shells (Amoebozoa: Arcellinida) illustrates the importance of convergent evolution in protist phylogeny.

Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.

Anthropogenic Stressors in Upland Rivers: Aquatic Macrophyte Responses. A Case Study from Bulgaria.

Upland rivers across Europe still exhibit undisturbed conditions and represent a treasure that we cannot afford to lose. We hypothesize that the combination of pristine and modified conditions could demonstrate biological responses along the stressor gradients. Thus, the response of aquatic macrophyte communities to anthropogenic stressors along upland rivers in Bulgaria was studied. Six stressors were selected out of 36 parameters grouped into hydromorphological, chemical variables and combined drivers (catchment land use). The stressors strongly affected species richness on the basis of biological type (bryophytes vs. vascular plants) and ecomorphological type (hydrophytes vs. helophytes). Hydrological alteration expressed by the change of the river's base flow and altered riparian habitats has led to a suppression of bryophytes and a dominance of riverbank plant communities. Seventy-five percent of mountain sites were lacking bryophytes, and the vegetation at semi-mountainous sites was dominated by vascular plants. It can be concluded that hydropeaking, organic and inorganic pollution, and discontinuous urban structures caused important modifications in the aquatic macrophyte assemblages. Macrophyte abundance and the biological and ecomorphological type of aquatic macrophytes reflect multi-stressor effects in upland rivers.

Phylogenetic divergence within the Arcellinida (Amoebozoa) is congruent with test size and metabolism type.

Arcellinida (lobose testate amoebae) are abundant and diverse in many ecosystems, especially in moist to aquatic environments. Molecular phylogeny has shown that overall test morphology (e.g., spherical or elongate) is generally conserved in Arcellinida lineages, but the taxonomic value of other traits (e.g., size, ornamentation, mixotrophy/heterotrophy metabolism type) has not been systematically evaluated. Morphological and physiological traits that correspond to genetic differences likely represent adaptive traits of ecological significance. We combined high-resolution phylogenetics (NAD9-NAD7 genes) and advanced morphometrics to assess the phylogenetic signal of morphological traits of a group of elongate Difflugia species (Arcellinida). The phylogenetic analyses revealed two clades which could be reliably separated by test size and the presence/absence of mixotrophy. Differences in test size may reflect trophic level, with smaller organisms occupying lower trophic levels. In addition to having larger tests, elongate mixotrophic Difflugia are characterised by wide, flat bases and an inflation of the lower two thirds of their test. These morphological traits may provide additional volume for endosymbionts and/or increased surface area to aid light transmission. Our results showcase greater diversity within the elongate Difflugia and highlight morphological traits of ecological and evolutionary significance.

Checklist of Sphagnum-dwelling testate amoebae in Bulgaria.

BACKGROUND: Until now, a complete checklist of Sphagnum-dwelling testate amoebae in Bulgaria has never been published. Records for species diversity and distribution in the country were scattered in many faunistic and ecological publications. The aim of the present study is to summarise all data for the species distribution at the level of country by reviewing the existing literature and by additional data obtained in our research over the past two years. NEW INFORMATION: The checklist comprises 171 species, classified into 43 genera, 20 families, three orders, three classes and three phyla. We present data for 16 new Sphagnum-dwelling testate amoebae in Bulgaria and new distribution data for 134 species. Of them, 99 species are recorded from Stara Planina Mt., for which there was no available data to date. Additionally are recorded 69 new species for Pirin Mt., 21 for Vitosha Mt. and 18 for Rila Mt. Thirty six species are synonymised according to the latest taxonomic changes. Two misidentified taxa (Euglypha brachiata Penard, 1902 and Difflugia compressa var. africana Gauthier-Lièvre et Thomas, 1958) are transferred into valid species E. acanthophora and Zivkovicia compressa, respectively. Three of the recorded species have not been included in the checklist, because they are currently not refering to testate amoebae (Cochliopodium bilimbosum (Auerbach 1856) and Cochliopodium echinatum Korotneef, 1879 are gymnamoebae (naked amoebae) and Microgromia elegantula (Penard 1904) = Paralieberkuehnia elegantula (Penard 1904) is freshwater foraminifera).

A contribution to the phylogeny of agglutinating Arcellinida (Amoebozoa) based on SSU rRNA gene sequences.

Arcellinid testate amoebae include a wide variety of amoeboid organisms whose test (shell) varies in shape, composition and size. A decade ago, we initiated molecular phylogenetic analyses based on SSU rRNA gene sequences and a taxonomic revision of Arcellinida. However, many lineages within Arcellinida still lack molecular data, and the phylogeny of this group is largely incomplete. In this study, we obtained SSU rRNA gene sequences from seven taxa, of which six have agglutinated shell (Difflugia oblonga, D. labiosa, D. gramen, Mediolus corona, Netzelia wailesi, and N. tuberculata), and one has an entirely proteinaceous shell (Arcella intermedia). All species but Difflugia oblonga branched within the recently erected suborder Sphaerothecina, confirming the synapomorphic value of an oviform or discoid shell. Thus, we propose that species with an oviform or discoid shell currently classified within genus Difflugia must be transferred to other genera, thus continuing the process of taxonomic revision of genus Difflugia, the largest Arcellinida genus. We therefore transferred the current and the previously sequenced oviform Difflugia spp. to Netzelia spp., based on the shared globular/oviform shell shape and their monophyly. Another species, D. labiosa, formed an independent lineage that branched as a sister clade to Arcella spp.; based on the shell morphology and their phylogenetic position, we considered D. labiosa as incertae sedis.

NAD9/NAD7 (mitochondrial nicotinamide adenine dinucleotide dehydrogenase gene)-A new "Holy Grail" phylogenetic and DNA-barcoding marker for Arcellinida (Amoebozoa)?

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. 250bp) 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.

Morphological and molecular diversification of Asian endemic Difflugia tuberspinifera (Amoebozoa, Arcellinida): a case of fast morphological evolution in protists?

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.

On the morphology, biometry and biogeography of Lamtopyxiscallistoma (Amoebozoa: Arcellinida).

The ultra-structure of the shell and the morphometric variability of soil inhabiting testate amoeba Lamtopyxiscallistoma from Madagascar were studied by using light- and scanning electron microscopy. The biometrical characteristic of the species was made on the basis of 75 specimens measured. In addition to the diameter of the shell, six other shell characters were described biometrically for the first time. The analysis of the variation coefficients shows that the studied population of L.callistoma is comparatively homogeneous and almost all measured characters are weakly to moderate variable (CV less than 10%). Scanning electron microscopy (SEM) studies on the shell ultra-morphology show that it has a smooth apertural surface with a thick layer of porous and fibrous organic cement and a rough dorsal surface composed of bigger and angular pieces of quartz. The shell wall has a thickness of about 5-6 µm and is composed of three layers. Unlike the previously accepted opinion that species is characterized by the presence of four teeth, this study shows that population of L.callistoma from Madagascar is comprised of both, specimens with four teeth and specimens with three teeth, in ratio of about 60% to 40%. Taking into account the restricted geographical distribution, large sizes and characteristic apertural morphology of L.callistoma it is assumed that this species, like some bryophilic 'Nebelas' with circumaustral distribution (e.g. Apoderavas, Alocoderacockayni, Certesellacertesi, Certesellamartiali, etc.), can be used as an example that in free-living microbial eukaryotes 'not everything is everywhere'.

SSU rRNA phylogeny of Arcellinida (Amoebozoa) reveals that the largest Arcellinid genus, Difflugia Leclerc 1815, is not monophyletic.

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.

COI barcoding of Nebelid testate amoebae (Amoebozoa: Arcellinida): extensive cryptic diversity and redefinition of the Hyalospheniidae Schultze.

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.

Comparing potential COI and SSU rDNA barcodes for assessing the diversity and phylogenetic relationships of cyphoderiid testate amoebae (Rhizaria: Euglyphida).

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 sixCyphoderiamorphospecies 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.

Molecular phylogeny of euglyphid testate amoebae (Cercozoa: Euglyphida) suggests transitions between marine supralittoral and freshwater/terrestrial environments are infrequent.

Marine and freshwater ecosystems are fundamentally different regarding many biotic and abiotic factors. The physiological adaptations required for an organism to pass the salinity barrier are considerable. Many eukaryotic lineages are restricted to either freshwater or marine environments. Molecular phylogenetic analyses generally demonstrate that freshwater species and marine species segregate into different sub-clades, indicating that transitions between these two environments occur only rarely in the course of evolution. It is, however, unclear if the transitions between freshwater and environments characterized by highly variable salinities, such as the marine supralittoral zone, are also infrequent. Here, we use testate amoebae within the Euglyphida to assess the phylogenetic interrelationships between marine supralittoral and freshwater taxa. Euglyphid testate amoebae are mainly present in freshwater habitats but also occur in marine supralittoral environments. Accordingly, we generated and analyzed partial SSU rRNA gene sequences from 49 new marine/supralittoral and freshwater Cyphoderiidae sequences, 20 sequences of the Paulinellidae, Trinematidae, Assulinidae, and Euglyphidae families as well as 21 GenBank sequences of unidentified taxa derived from environmental PCR surveys. Both the molecular and morphological data suggest that the diversity of Cyphoderiidae is strongly underestimated. The results of our phylogenetic analyses demonstrated that marine supralittoral and freshwater euglyphid testate amoeba species are segregated into distinct sub-clades, suggesting that transitions between these two habitats occurred only infrequently.

Morphology, biometry, and taxonomy of freshwater and marine interstitial cyphoderia (cercozoa: euglyphida).

Good taxonomy is essential for ecological, biogeographical, and evolutionary studies of any group of organisms. Therefore, we performed detailed light- and scanning electron microscopy investigations on the shell ultrastructure and biometric analyses of the morphometric variability of five freshwater and marine interstitial testate amoebae of the genus Cyphoderia (C. trochus var. amphoralis, C. ampulla, C. margaritacea var. major, C. compressa, and C. littoralis), isolated from different populations in Bulgaria and Switzerland. Our aims were (1) to clarify the morphological characteristics of these taxa, and (2) to compare the morphology of a given taxon (C. ampulla) among different locations in Bulgaria and Switzerland as a first step towards an assessment of the geographical variation within a supposedly cosmopolitan taxon. Four of the studied taxa are characterized by a well-expressed main-size class and by a small size range of all the characters and can be defined as size-monomorphic species. Based on these results, the following systematic changes are proposed: C. major (Penard, 1891) n. comb. (Syn.: C. margaritacea var. major (Penard, 1891) and C. amphoralis (Wailes & Penard, 1911) n. comb. (Syn.: C. trochus var. amphoralis (Wailes & Penard, 1911)). However, we also show significant morphological variability between the Swiss and Bulgarian populations of C. ampulla, suggesting the possible existence of more than one taxon within this species. Further studies are required to assess (1) if these two morphologically different taxa represent individual species, (2) if so, if more species exist, and if this diversity is due to limited distribution ranges (endemism) or if several closely related taxa occur together in different geographical areas.