A Comparative Characterization of the Mitochondrial Genomes of Paramoeba aparasomata and Neoparamoeba pemaquidensis (Amoebozoa, Paramoebidae).

Marine amebae of the genus Paramoeba (Amoebozoa, Dactylopodida) normally contain a eukaryotic endosymbiont known as Perkinsela-like organism (PLO). This is one of the characters to distinguish the genera Neoparamoeba and Paramoeba from other Dactylopodida. It is known that the PLO may be lost, but PLO-free strains of paramoebians were never available for molecular studies. Recently, we have described the first species of the genus Paramoeba which has no parasome-Paramoeba aparasomata. In this study, we present a mitochondrial genome of this species, compare it with that of Neoparamoeba pemaquidensis, and analyze the evolutionary dynamics of gene sequences and gene order rearrangements between these species. The mitochondrial genome of P. aparasomata is 46,254 bp long and contains a set of 31 protein-coding genes, 19 tRNAs, two rRNA genes, and 7 open reading frames. Our results suggest that these two mitochondrial genomes within the genus Paramoeba have rather similar organization and gene order, base composition, codon usage, the composition and structure of noncoding, and overlapping regions.

Dactylomonas gen. nov., a Novel Lineage of Heterolobosean Flagellates with Unique Ultrastructure, Closely Related to the Amoeba Selenaion koniopes Park, De Jonckheere & Simpson, 2012.

We report the discovery of a new genus of heterolobosean flagellates, Dactylomonas gen. nov., with two species, D. venusta sp. nov. and D. crassa sp. nov. Phylogenetic analysis of the SSU rRNA gene showed that Dactylomonas is closely related to the amoeba Selenaion, the deepest-branching lineage of Tetramitia. Dactylomonads possess two flagella, and ultrastructural studies revealed an unexpected organization of the flagellar apparatus, which resembled Pharyngomonada (the second lineage of Heterolobosea) instead of Tetramitia: basal bodies were orthogonal to each other and a putative root R1 was present in the mastigont. On the other hand, Dactylomonas displayed several features uncommon in Heterolobosea: a microtubular corset, a distinctive rostrum supported by the main part of the right microtubular root, a finger-like projection on the proximal part of the recurrent flagellum, and absence of a ventral groove. In addition, Dactylomonas is anaerobic and seems to have lost mitochondrial cristae. Dactylomonas and Selenaion are accommodated in the family Selenaionidae fam. nov. and order Selenionida ord. nov. The taxonomy of Tetramitia is partially revised, and the family Neovahlkampfiidae fam. nov. is established.

Elucidating Waterborne Pathogen Presence and Aiding Source Apportionment in an Impaired Stream.

Fecal indicator bacteria (FIB) are the basis for water quality regulations and are considered proxies for waterborne pathogens when conducting human health risk assessments. The direct detection of pathogens in water and simultaneous identification of the source of fecal contamination are possible with microarrays, circumventing the drawbacks to FIB approaches. A multigene target microarray was used to assess the prevalence of waterborne pathogens in a fecally impaired mixed-use watershed. The results indicate that fecal coliforms have improved substantially in the watershed since its listing as a 303(d) impaired stream in 2002 and are now near United States recreational water criterion standards. However, waterborne pathogens are still prevalent in the watershed, as viruses (bocavirus, hepatitis E and A viruses, norovirus, and enterovirus G), bacteria (Campylobacter spp., Clostridium spp., enterohemorrhagic and enterotoxigenic Escherichia coli, uropathogenic E. coli, Enterococcus faecalis, Helicobacter spp., Salmonella spp., and Vibrio spp.), and eukaryotes (Acanthamoeba spp., Entamoeba histolytica, and Naegleria fowleri) were detected. A comparison of the stream microbial ecology with that of sewage, cattle, and swine fecal samples revealed that human sources of fecal contamination dominate in the watershed. The methodology presented is applicable to a wide range of impaired streams for the identification of human health risk due to waterborne pathogens and for the identification of areas for remediation efforts.IMPORTANCE The direct detection of waterborne pathogens in water overcomes many of the limitations of the fecal indicator paradigm. Furthermore, the identification of the source of fecal impairment aids in identifying areas for remediation efforts. Multitarget gene microarrays are shown to simultaneously identify waterborne pathogens and aid in determining the sources of impairment, enabling further focused investigations. This study shows the use of this methodology in a historically impaired watershed in which total maximum daily load reductions have been successfully implemented to reduce risk. The results suggest that while the fecal indicators have been reduced more than 96% and are nearing recreational water criterion levels, pathogens are still detectable in the watershed. Microbial source tracking results show that additional remediation efforts are needed to reduce the impact of human sewage in the watershed.

Genetic Determinism vs. Phenotypic Plasticity in Protist Morphology.

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.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Leptomyxa valladaresi n. sp. (Amoebozoa, Tubulinea, Leptomyxida), from Mount Teide, Tenerife, Spain.

Leptomyxa valladaresi was isolated from soil in a pine forest on the southern flank of Mt Teide in Tenerife, Spain. It feeds on bacteria and on a range of other amoebae, and it was possible to establish bi-axenic cultures with L. valladaresi and Acanthamoeba. It is easily propagated on a E. coli also. 18S rDNA gene sequence analysis suggests that it is most closely related to Leptomyxa variabilis, however this amoeba differs in important detail. L. valladaresi is primarily mononucleate whereas L. variabilis is multinucleate. L. valladaresi is a larger amoeba and although the cysts are similar in size, there is no sign of the pore-like structures described in L. variabilis cysts. L. valladaresi can adopt a rapid monopodal and tubular morphology similar to that described for L. neglecta and Rhizamoeba matisi, and is never reticulated as larger L. variabilis individuals tend to be. The mean generation time was found to be 18 h, in line with amoebae of this size. Like other members of the genus, L. valladaresi is reported to harbour intracellular, presumably endosymbiotic bacteria, and a Delftia sp has been identified by 16S PCR a bacterium which is also known to grow within Acanthamoeba. The availability of this easily cultured species will help to characterize of this little studied genus and family and their relationship with bacteria, both prey and symbionts.

Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran.

The present study tested 80 samples of municipal, geothermal and recreational water samples for the occurrence of waterborne free living amoebae (FLA) including Acanthamoeba, Balamuthia mandrillaris, Vahlkampfiids and Vermamoeba in Semnan province, North half of Iran. Four sets of primers including JDP1,2 primers, ITS1,2 primers (Vahlkampfiids), 16S rRNABal primers (Balamuthia mandrillaris) and NA1,2 primers (Vermamoeba) were used to confirm the morphological identification. From the 80 water samples tested in the present study, 16 (20%) were positive for the outgrowth of free living amoebae based on the morphological page key. Out of the 34 municipal water samples, 7 (20.6%) were positive for outgrowth of Free living amoeba, belonging to Vermamoeba, Naegleria and Acanthamoeba using molecular tools. Three out of the six investigated hot springs were also contaminated with Naegleria spp. Sequencing of the ITS1,2 region of the Vahlkampfiid isolates revealed the highest homology with N. gruberi (2 isolates), N. australiensis (1 isolate) and N. pagei (3 isolates). This is the first report of N. gruberi in the country. Using morphological and molecular analysis, Balamuthia mandrillaris was undetected in all the water samples. The present study further confirmed the occurrence of potentially pathogenic waterborne free living amoebae in habitats with high human activity. It is of utmost importance that more studies are conducted to evaluate the niches of B. mandrillaris and N. fowleri in Iran and worldwide. Such investigations regarding the relevance of FLA as a hazard to humans, should be brought to the notice of the health authorities.

Nebela jiuhuensis nov. sp. (Amoebozoa; Arcellinida; Hyalospheniidae): A New Member of the Nebela saccifera - equicalceus - ansata Group Described from Sphagnum Peatlands in South-Central China.

Hyalospheniids are among the most common and conspicuous testate amoebae in high-latitude peatlands and forest humus. These testate amoebae were widely studied as bioindicators and are increasingly used as models in microbial biogeography. However, data on their diversity and ecology are still very unevenly distributed geographically: notably, data are lacking for low-latitude peatlands. We describe here a new species, Nebela jiuhuensis, from peatlands near the Middle Yangtze River reach of south-central China with characteristic morphology. The test (shell) has hollow horn-like lateral extensions also found in N. saccifera, N. equicalceus (=N. hippocrepis), and N. ansata, three large species restricted mostly to Sphagnum peatlands of Eastern North America. Mitochondrial cytochrome oxidase (COI) data confirm that N. jiuhuensis is closely related to the morphologically very similar North American species N. saccifera and more distantly to N. ansata within the N. penardiana group. These species are all found in wet mosses growing in poor fens. Earlier reports of morphologically similar specimens found in South Korea peatlands suggest that N. jiuhuensis may be distributed in comparable peatlands in Eastern Asia (China and Korea). The discovery of such a conspicuous new species in Chinese peatlands suggests that many new testate amoebae species are yet to be discovered, including potential regional endemics. Furthermore, human activities (e.g., drainage, agriculture, and pollution) have reduced the known habitat of N. jiuhuensis, which can thus be considered as locally endangered. We, therefore, suggest that this very conspicuous micro-organism with a probably limited geographical distribution and specific habitat requirement should be considered as a flagship species for microbial biogeography as well as local environmental conservation and management.

Ptolemeba n. gen., a novel genus of hartmannellid amoebae (Tubulinea, Amoebozoa); with an emphasis on the taxonomy of Saccamoeba.

Hartmannellid amoebae are an unnatural assemblage of amoeboid organisms that are morphologically difficult to discern from one another. In molecular phylogenetic trees of the nuclear-encoded small subunit rDNA, they occupy at least five lineages within Tubulinea, a well-supported clade in Amoebozoa. The polyphyletic nature of the hartmannellids has led to many taxonomic problems, in particular paraphyletic genera. Recent taxonomic revisions have alleviated some of the problems. However, the genus Saccamoeba is paraphyletic and is still in need of revision as it currently occupies two distinct lineages. Here, we report a new clade on the tree of Tubulinea, which we infer represents a novel genus that we name Ptolemeba n. gen. This genus subsumes a clade of hartmannellid amoebae that were previously considered in the genus Saccamoeba, but whose mitochondrial morphology is distinct from Saccamoeba. In accordance with previous research, we formalize the clade as distinct from Saccamoeba. Transmission electron microscopy of our isolates illustrate that both molecularly discrete species can be further differentiated by their unique mitochondrial cristal morphology.

Multiplex Real-Time Polymerase Chain Reaction Assay To Detect Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris in Different Water Sources.

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea).

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.

New data on the fine structure of Deuteramoeba mycophaga CCAP 1586/1 (Amoebozoa, Tubulinea).

The genus Deuteramoeba is one of the six amoebae genera belonging to the best-known amoeba family - Amoebidae (Amoebozoa, Tubulinea), containing such a popular species as Amoeba proteus. However, members of other genera of the family Amoebidae are much less known, and most of the studies of their morphology and ultrastructure date back to the 1970s and 1980s. Since these "classical" species are believed to be "well studied", their morphology and fine structure rarely become a subject of re-investigation. The absence of modern morphological data may be critical when molecular data of the type strain are not available, and the only way to identify a species is by morphological comparison. For this paper, we performed an ultrastructural study of the strain CCAP 1586/1 - the type strain of the species Deuteramoeba mycophaga. Our study revealed new details of the nuclear structure, including a peripheral layer of filaments and a heterogeneous nucleolus, and provided new data on the cytoplasmic inclusions of this species. We performed a whole-genome amplification of the DNA from a single amoeba cell followed by NGS sequencing and searched for genetic evidence for the presence of a putative nuclear parasite detected in 2017, but found no evidence for the presence of Opisthosporidia.

Polychaos centronucleolus n. sp. - a new terrestrial species of the genus Polychaos (Amoebozoa, Tubulinea) with nontypical nuclear structure.

A new species of the "proteus-type" naked amoebae (large cells with discrete tubular pseudopodia) was isolated from tree bark sample of a birch tree in the surrounding of Kislovodsk town, Russia and named Polychaos centronucleolus n. sp. (Amoebozoa, Tubulinea). Amoebae of this species have a filamentous cell coat and a nucleus with a central compact nucleolus. This type of nucleolar organization has not been previously known for the genus Polychaos. A sequence of the 18S rRNA gene of this strain was obtained using whole genome amplification of DNA from the single amoeba cell, followed by NGS sequencing. The analysis of molecular data robustly groups this species with Polychaos annulatum within the family Hartmannellidae. Our results, together with the results of our previous studies, show that the taxonomic assignment of "proteus-type" amoebae species is becoming increasingly complex, and the taxonomic characters that can be used to classify these organisms are becoming more shadowed.

Description and phylogenetic relationships of Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. (Amoebozoa, Arcellinida).

Spumochlamys perforata n. sp. and Spumochlamys bryora n. sp. were isolated and described from dry epiphytic moss. The morphology and ultrastructure of both species clearly demonstrate that they belong to the genus Spumochlamys (family Microchlamyiidae). They differ from its only described member, Spumochlamys iliensis (as well as from species of Microchlamys), in the relief of the dorsal surface of the test, revealed by scanning electron microscopy, which can represent a good characteristic for species identification. They also differ in the structure of the dorsal part of the test wall (especially S. perforata). Small subunit ribosomal DNA-based molecular phylogenetic analyses show that Spumochlamys is a deeply branching lineage of the Arcellinida, without any close affinities. Actin gene sequence analysis places this genus within the Tubulinea, close to two other arcellinid lineages but without forming a monophyletic group with them. These data together strongly suggest that the lack of resolution in the arcellinid molecular phylogenies is due to serious undersampling of taxa, a limited number of sequence data, and high divergence rates in most of the species.

Phylogenomics and Morphological Reconstruction of Arcellinida Testate Amoebae Highlight Diversity of Microbial Eukaryotes in the Neoproterozoic.

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.

Balamuthia mandrillaris, agent of amebic encephalitis: detection of serum antibodies and antigenic similarity of isolates by enzyme immunoassay.

We report the development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Balamuthia mandrillaris, a free-living ameba that is an etiologic agent of granulomatous amebic encephalitis (GAE). As part of the California Encephalitis Project (CEP), we have tested serum and cerebrospinal fluid (CSF) samples from a subgroup of 130 hospitalized encephalitis patients (out of approximately 430 samples) over a 16-month period. Case criteria were based on clinical, laboratory, and occupational/recreational histories. All serum samples initially underwent screening by immunofluorescent antibody (IFA) staining with results ranging from no detectable ameba antibodies to titers of 1:256. In addition to the 130 samples tested prospectively, sera and/or CSF from 11 previously confirmed cases of balamuthiasis, six healthy individuals, and earlier CEP submissions with high IFA antibody titers were also tested retrospectively. Among the 130 samples, two cases of balamuthiasis were identified by ELISA and confirmed by the polymerase chain reaction (PCR). The availability of sera from human and animal cases and from varied geographic areas allowed comparisons of serologic similarities of the different Balamuthia strains and human sera. All sera, whether from human or other mammals, reacted with all strains of Balamuthia, as they did with Balamuthia amebae from different geographic areas. Enzyme-linked immunosorbent assay results were consistent with the IFA results. Differences between readings were likely due to cross-reactivity between Balamuthia antigens and unidentified antibodies in serum.

Correct identification of species makes the amoebozoan rRNA tree congruent with morphology for the order Leptomyxida Page 1987; with description of Acramoeba dendroida n. g., n. sp., originally misidentified as 'Gephyramoeba sp.'.

Morphological identification of protists remains an expert task, especially for little known and poorly described species. Culture collections normally accept organisms under the name provided by depositors and are not responsible for identification. Uncritical acceptance of these names by molecular phylogeneticists may result in serious errors of interpretation of phylogenetic trees based on DNA sequences, making them appear more incongruent with morphology than they really are. Several cases of misidentification in a major culture collection have recently been reported. Here we provide evidence for misidentifications of two more gymnamoebae. The first concerns "Gephyramoeba sp." ATCC 50654; it is not Gephyramoeba, a leptomyxid with lobose pseudopods, but a hitherto undescribed branching amoeba with fine, filamentous subpseudopods named here Acramoeba dendroida gen. et sp. nov. We also sequenced 18S rRNA of Page's strain of Rhizamoeba saxonica (CCAP 1570/2) and show that it is the most deeply branching leptomyxid and is not phylogenetically close to 'Rhizamoeba saxonica' ATCC 50742, which was misidentified. Correcting these misidentifications improves the congruence between morphological diversity of Amoebozoa and their rRNA-based phylogenies, both for Leptomyxida and for the Acramoeba part of the tree. On morphological grounds we transfer Gephyramoebidae from Varipodida back to Leptomyxida and remove Flamella from Leptomyxida; sequences are needed to confirm these two revisions.

Neoparamoeba perurans is a cosmopolitan aetiological agent of amoebic gill disease.

Previously we described a new member of the Neoparamoeba genus, N. perurans, and showed that it is an agent of amoebic gill disease (AGD) of Atlantic salmon Salmo salar cultured in southeast Tasmania, Australia. Given the broad distribution of cases of AGD, we were interested in extending our studies to epizootics in farmed fish from other sites around the world. Oligonucleotide probes that hybridise with the 18S rRNA of N. perurans, N. branchiphila or N. pemaquidensis were used to examine archival samples of AGD in Tasmania as well as samples obtained from 4 host fish species cultured across 6 countries. In archival samples, N. perurans was the only detectable amoeba, confirming that it has been the predominant aetiological agent of AGD in Tasmania since epizootics were first reported. N. perurans was also the exclusive agent of AGD in 4 host species across 6 countries. Together, these data show that N. perurans is a cosmopolitan agent of AGD and, therefore, of significance to the global mariculture industry.

En garde! Redefinition of Nebela militaris (Arcellinida, Hyalospheniidae) and erection of Alabasta gen. nov.

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.

Evaluation of Morphological Characteristics to Delineate Taxa of the Genus Trigonopyxis (Amoebozoa, Arcellinida).

Morphological features are often the only characteristics suitable for identification of taxa in testate amoebae, especially in ecological and palaeoecological studies. However, whereas the morphology of some species is rather stable it may vary considerably in others. Within the order Arcellinida the genus Trigonopyxis with the type species Trigonopyxis arcula is morphologically highly variable. To identify reliable characteristics for morphology-based taxon delineation we investigated variations in shell size, pseudostome diameter and pseudostome form in T. arcula from three different sites of the Ecuadorian Andes, where these characteristics vary even more than previously described. Further, we investigated if morphological characteristics in Trigonopyxis varied with changes in environmental factors. We studied 951 shells of Trigonopyxis collected along an altitudinal gradient with varying abiotic factors. We established a method for characterization of the pseudostome form, which lead to five different morphotypes. Our results suggest that shell size alone is not an appropriate character for taxon delineation but can be used as an indicator for changes in environmental conditions. In contrast, the pseudostome form might be used for taxon delineation, but likely also varies considerably within taxa. Overall, the study provides an overview of the morphological variability of the genus Trigonopyxis.