Phylogenetic analyses of small subunit ribosomal RNA coding regions reveal a monophyletic lineage of euglyphid testate amoebae (Order Euglyphida)

The Testaceafilosia includes amoebae with filopodia and with a proteinaceous, agglutinated or siliceous test. To explore the deeper phylogeny of this group, we sequenced the small subunit ribosomal RNA coding region of 13 species, including the first sequence of an amoeba with an agglutinated test, Pseudodifflugia sp. Phylogenetic analyses using maximum parsimony and maximum likelihood methods as well as neighbor joining method yielded the following results: the order Euglyphida forms a monophyletic lineage with the sarcomonads as sister group. The next related taxa are the Chlorarachnea and the unidentified filose strain N-Por. In agreement with the previous studies the Phytomyxea branch off at the base of this lineage. The Monadofilosa (Testaceafilosia and Sarcomonadea) appear monophyletic. The Testaceafilosia are polyphyletic, because Pseudodifflugia sp. is positioned as the sister taxon to the sarcomonads. Within the order Euglyphida Paulinella branches off first, together with Cyphoderia followed by Tracheleuglypha. In maximum likelihood and neighbor joining analyses, the genus Euglypha is monophyletic. The branching pattern within the order Euglyphida reflects the evolution of shell morphology from simple to complex built test.     

The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa

Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida.     

Description of Phaeobola aeris gen. nov., sp. nov (Rhizaria,Cercozoa, Euglyphida) Sheds Light on Euglyphida’s Dark Matter

The majority of Euglyphida species are characterised by shells with imbricated silica scales. Environmental surveys indicate a large unexplored diversity and recent efforts hinted at a certain diversity of yet undescribed, inconspicuous, scale-lacking Euglyphida. Here we describe Phaeobola aeris gen. nov., sp. nov. that shows a variety of morphological characters typical for the Euglyphida but lacks silica scales-instead, this species bears an agglutinated test. Neither its morphology nor phylogenetic placement allows its assignment to any currently described family. We erected the yet monospecific genus Phaeobola gen. nov., which with yet available data remain Euglyphida incertae sedis.     

Relationships among testate amoebae (Protozoa), vegetation and water chemistry in five Sphagnum-dominated peatlands in Europe

To study the relationships between groups of organisms and the degree to which these relationships are consistent across major climatic gradients, we analysed the testate amoeba ( Protozoa ) communities, vegetation and water chemistry of one peatland in five countries: Switzerland, The Netherlands, Great Britain, Sweden and Finland, as part of the BERI (Bog Ecosystem Research Initiative) project. The relationships between the different data sets and subsets were investigated by means of detrended correspondence analysis, canonical correspondence analysis and Mantel permutation tests. The comparison of data on vegetation and testate amoebae showed that inter-site differences are more pronounced for the vegetation than for the testate amoebae species assemblage. Testate amoebae are a useful tool in multi-site studies and in environmental monitoring of peatlands because: (1) the number of species in Sphagnum -dominated peatlands is much higher than for mosses or vascular plants; (2) most peatland species are cosmopolitan in their distributions and therefore less affected than plants by biogeographical distribution patterns, thus differences in testate amoeba assemblages can be interpreted primarily in terms of ecology; (3) they are closely related to the ecological characteristics of the exact spot where they live, therefore they can be used to analyse small-scale gradients that play a major role in the functioning of peatland ecosystems. This study revealed the existence of small-scale vertical gradients within the vegetation and life-form niche separation in response to water chemistry. The deep-rooted plants such as Carex spp. and Eriophorum spp. are related to the chemistry of water sampled at or near the ground water table, whereas the mosses are not. Testate amoebae were shown to be ecologically more closely related to the chemistry of water sampled at or near the water table level and to the mosses than to the deep-rooted plants.     

Morphology, Biometry, and Taxonomy of Freshwater and Marine Interstitial Cyphoderia (Cercozoa: Euglyphida)

ABSTRACT. 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.     

The curse of taxonomic uncertainty in biogeographical studies of free-living terrestrial protists: a case study of testate amoebae from Amsterdam Island

Aim A current debate in microbial biogeography contrasts two views concerning the distribution of free‐living microorganisms. The first view assumes a ubiquitous distribution, while the second assumes that at least some species have limited geographical distributions. We tested for limited geographical distributions by identifying testate amoebae morphospecies from an extremely remote oceanic island where the potential for endemism is high. Location Amsterdam Island, Indian Ocean. Methods Sixty moss and water samples collected from the top of the volcano to the lowland were investigated for their testate amoeba content. Due to taxonomic uncertainties among the Argynnia (Nebela) dentistoma species complex (including A. antarctica), we also performed light and scanning electron microscopy investigations on the shell ultrastructure and biometric analyses on several specimens of this taxon. Results We identified a total of 43 testate amoeba taxa belonging to 15 genera. Only four testate amoeba taxa had previously been recorded on this island. Testate amoeba communities of Amsterdam Island are dominated by cosmopolitan ubiquitous euglyphid taxa such as Trinema lineare, Assulina muscorum and Corythion dubium. The length and width ranges for Argynnia dentistoma on Amsterdam Island overlap with other records of this species and of A. antarctica, suggesting that A. antarctica is not a distinct taxon. Main conclusions Although Amsterdam Island is among the most remote islands in the world, an extensive inventory of testate amoeba morphospecies provided no clear evidence for endemism. On the one hand, our detailed morphometric analysis of the A. dentistoma complex revealed that A. antarctica, a morphospecies previously suggested to display endemism, cannot be confidently distinguished from the cosmopolitan morphospecies A. dentistoma. On the other hand, five morphotaxa could not be identified with certainty and might represent new species, potentially with limited distribution. These examples illustrate how taxonomic uncertainties undermine biogeographical studies of testate amoebae. In order to allow better interpretation of morphology‐based testate amoeba distribution data, an assessment of genetic diversity among and within morphotaxa in relation to geographical distance for some common testate amoebae should be given high priority.     

Ribosomal RNA genes challenge the monophyly of the Hyalospheniidae (Amoebozoa: Arcellinida)

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.     

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.     

Evaluating the use of dominant microbial consumers (testate amoebae) as indicators of blanket peatland restoration

Peatlands represent globally-important ecosystems and carbon stores. However, large areas of peatland have been drained for agriculture, or peat has been harvested for use as fuel or in horticulture. Increasingly, these landscapes are being restored through ditch blocking and rewetting primarily to improve biodiversity and promote peat accumulation. To date we have little knowledge of how these interventions influence the microbial communities in peatlands. We compared the responses of dominant microbial consumers (testate amoebae) to drainage ditch restoration relative to unblocked ditches in a UK upland blanket peatland (Migneint, North Wales). Two techniques were used for restoration: (i) dammed ditches with re-profiling; and (ii) dammed ditches with pools of open water behind each dam. Testate communities in the inter-ditch areas changed markedly over time and between treatments illustrating the potential of this group of organisms as indicators of blanket peatland restoration status. However, the responses of testate amoebae to peat rewetting associated with restoration were partially obscured by inter-annual variability in weather conditions through the course of the experiment. Although there was considerable variability in the response of testate amoebae communities to peatland drain blocking, there were clearly more pronounced changes in samples from the dammed and reprofiled treatments including an increase in diversity, and the appearance of unambiguous wet-indicator species in relatively high abundances (including Amphitrema stenostoma, Archerella flavum, Arcella discoides type, Difflugia bacillifera and Difflugia bacillarium). This reflects a shift towards overall wetter conditions across the site and the creation of new habitats. However, water-table was not a significant control on testate amoebae in this case, suggesting a poor relationship between water table and surface moisture in this sloping blanket peatland. Our findings highlight the potential of testate amoebae as bioindicators of peatland restoration success; however, there is a need for caution as mechanisms driving change in the microbial communities may be more complex than first assumed. Several factors need to be taken into account when implementing biomonitoring studies in peatlands including: (i) the natural variability of the peatland ecosystem under changing weather conditions; (ii) any disturbance connected with the restoration procedures; and (iii) the timescales over which the ecosystem responds to the management intervention. Our results also suggest an indicator species approach based on population dynamics may be more appropriate for biomonitoring peatland restoration than examining changes at the community level.     

Biodiversity of testate amoebae (Arcellinida and Euglyphida) in different habitats of a lake in the Upper Paraná River floodplain

This study aimed to investigate the testate amoebae (Arcellinida and Eugliphida) species diversity in plankton, macrophytes and aquatic sediment samples from a shallow lake of the Upper Paraná River floodplain. Samples were carried out from April 2007 to March 2008. We recorded 89 taxa, belonging to 10 families. Eighty-two taxa were found in the aquatic sediment, 71 in the macrophytes and 53 in the plankton. Highest values of alpha diversity were observed in the aquatic sediment. Although the plankton had the highest number of accidental species, accessory and constant species were also observed in this habitat. Most of the species classified as constant for the plankton belonged to the genus Arcella. Most of the constant species in the macrophytes and aquatic sediment belonged to the genus Difflugia. This study supports the idea that the presence of these protists in the plankton should not be attributed only to stochastic processes because (i) the species diversity recorded in this habitat was remarkably high in relation to the total biodiversity of the lake, and (ii) we also recorded frequent and constant species in the plankton.     

Assessing the ecological value of small testate amoebae (<45 μm) in New Zealand peatlands

Methodological advances are essential for robust ecological research. Quantitative reconstructions of environmental conditions using testate amoebae rely on sound taxonomy. While the taxonomy of large species is relatively well resolved, this is not the case for most small taxa (typically <45 μm long). In New Zealand, peatlands contain a diversity of both cosmopolitan and characteristic large southern endemic taxa, but also have a high abundance of small taxa. The latter are often lumped into morphotypes reducing their value as ecological indicators. In this study, we demonstrate how (a) lumping small taxa versus splitting them into unique types, and (b) including or excluding them from community analysis influenced their ecological inference. We assessed testate amoeba composition in six peat bogs from New Zealand, three that were moderately-to-highly impacted, and three that were non-impacted. Environmental variables were measured at each sampling site and the surface testate amoeba community patterns and community-environment relationships compared. We found a clear division between impacted and non-impacted sites. Several distinct small taxa were more strongly related to water-table depth and conductivity, while the larger taxa were more correlated to pH. These results show that improved taxonomic resolution of small taxa can provide more informed environmental assessment.     

Rediscovery of Nebela ansata (Amoebozoa: Arcellinida) in eastern North America: biogeographical implications

Aim The question whether free‐living protists are generally cosmopolitan is currently a matter of debate. In this study we investigate the geographical distribution of a distinctive testate amoeba species, Nebela ansata, and use our data to assess the potential for highly restricted distribution patterns in some protist species. Location Global. Methods We analysed (1) 3400 testate amoeba publications from North America and other continents, (2) unpublished slides of the Penard Collection of the Natural History Museum, London, UK, and (3) 104 Sphagnum samples from eastern North America. Non‐metric multidimensional scaling (NMDS) was used to visualize the similarities in testate amoeba community composition among 1012 North American samples, including two communities that contained N. ansata. Results We rediscovered N. ansata at a site in New Jersey located close to its type locality, and in Nova Scotia. We also report the existence of an apparently unpublished museum specimen originally collected from New Jersey. Our extensive literature survey confirmed the presence of this species only in the temperate part of eastern North America. The NMDS revealed that communities with N. ansata were less similar to each other than to communities from other parts of North America, suggesting that favourable habitats for N. ansata occur in other Sphagnum‐dominated peatlands, a habitat type that has been extensively sampled in North America and elsewhere. Main conclusions These data provide an unusually convincing case of a free‐living microorganism with a very limited distribution range in the temperate part of eastern North America. The remarkably restricted distribution of N. ansata highlights the extent of our ignorance about the natural history of free‐living microorganisms, and raises questions about the lack of attention to microbial diversity in conservation biology.     

Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Description of the marine predator Sericomyxa perlucida gen. et sp. nov., a cultivated representative of the deepest branching lineage of vampyrellid amoebae (Vampyrellida,Rhizaria)

The vampyrellids (Vampyrellida, Rhizaria) are naked amoebae of considerable genetic diversity. Three families have been well-defined (Vampyrellidae, Leptophryidae, and Placopodidae), but most vampyrellid lineages detected by environmental sequencing are poorly known or completely uncharacterized. In the brackish sediment of Lake Bras D’Or, Nova Scotia, Canada, we discovered an amoeba with a vampyrellid-like life history that was morphologically dissimilar from previously known vampyrellid taxa. We established a culture of this amoeba, studied its feeding behavior and prey range specificity, and characterized it with molecular phylogenetic methods and light and electron microscopy. The amoeba was a generalist predator (i.e. eukaryotroph), devouring a range of marine microalgae, with a strong affinity for some benthic diatoms and Chroomonas. Interestingly, the amoeba varied its feeding strategy depending on the prey species. Small diatoms were engulfed whole, while larger species were fed on through extraction with an invading pseudopodium. The SSU rRNA gene phylogenies robustly placed the amoeba in the most basal, poorly described lineage (“clade C”) of the Vampyrellida. Based on the phylogenetic position and the distinct morphology of the studied amoeba, we here describe it as Sericomyxa perlucida gen. et sp. nov., and establish the new vampyrellid family Sericomyxidae for “clade C.”