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The ultra-structure of the shell and the morphometric variability of soil inhabiting testate amoeba Lamtopyxis callistoma 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. Apodera vas, Alocodera cockayni, Certesella certesi, Certesella martiali, etc.), can be used as an example that in free-living microbial eukaryotes ‘not everything is everywhere’.

Arcellinida is ascending in importance in protistology, but description of their diversity still presents multiple challenges. Furthermore, applicable tools for surveillance of these organisms are still in developing stages. Importantly, a good database that sets a correspondence between molecular barcodes and species morphology is lacking. Cytochrome oxidase (COI) has been suggested as the most relevant marker for species discrimination in Arcellinida. However, some major groups of Arcellinida are still lacking a COI sequence. Here we expand the database of COI marker sequences for Arcellinids, using single-cell PCR, transcriptomics, and database scavenging. In the present work, we added 24 new Arcellinida COI sequences to the database, covering all unsampled infra- and suborders. Additionally, we added six new SSUrRNA sequences and described four new species using morphological, morphometrical, and molecular evidence: Heleopera steppica, Centropyxis blatta, Arcella uspiensis, and Cylindrifflugia periurbana. This new database will provide a new starting point to address new research questions from shell evolution, biogeography, and systematics of arcellinids., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Transcriptome sequencing has become a method of choice for evolutionary studies in microbial eukaryotes due to low cost and minimal sample requirements. Transcriptome data has been extensively used in phylogenomic studies to infer ancient evolutionary histories. However, its utility in studying cryptic species diversity is not well explored. An empirical investigation was conducted to test the applicability of transcriptome data in resolving two major types of discordances at lower taxonomic levels. These include cases where species have the same morphology but different genetics (cryptic species) and species of different morphologies but have the same genetics. We built a species comparison bioinformatic pipeline that takes into account the nature of transcriptome data in amoeboid microbes exemplifying such discordances. Result: Our analyses of known or suspected cryptic species yielded consistent results regardless of the methods of culturing, RNA collection or sequencing. Over 95% of the single copy genes analyzed in samples of the same species sequenced using different methods and cryptic species had intra- and interspecific divergences below 2%. Only a minority of groups (2.91–4.87%) had high distances exceeding 2% in these taxa, which was likely caused by low data quality. This pattern was also observed in suspected genetically similar species with different morphologies. Transcriptome data consistently delineated all taxa above species level, including cryptically diverse species. Using our approach we were able to resolve cryptic species problems, uncover misidentification and discover new species. We also identified several potential barcode markers with varying evolutionary rates that can be used in lineages with different evolutionary histories. Conclusion: Our findings demonstrate that transcriptome data is appropriate for understanding cryptic species diversity in microbial eukaryotes. [ABSTRACT FROM AUTHOR]

Sex and reproduction are often treated as a single phenomenon in animals and plants, as in these organisms reproduction implies mixis and meiosis. In contrast, sex and reproduction are independent biological phenomena that may or may not be linked in the majority of other eukaryotes. Current evidence supports a eukaryotic ancestor bearing a mating type system and meiosis, which is a process exclusive to eukaryotes. Even though sex is ancestral, the literature regarding life cycles of amoeboid lineages depicts them as asexual organisms. Why would loss of sex be common in amoebae, if it is rarely lost, if ever, in plants and animals, as well as in fungi? One way to approach the question of meiosis in the "asexuals" is to evaluate the patterns of occurrence of genes for the proteins involved in syngamy and meiosis. We have applied a comparative genomic approach to study the occurrence of the machinery for plasmogamy, karyogamy, and meiosis in Amoebozoa, a major amoeboid supergroup. Our results support a putative occurrence of syngamy and meiotic processes in all major amoebozoan lineages. We conclude that most amoebozoans may perform mixis, recombination, and ploidy reduction through canonical meiotic processes. The present evidence indicates the possibility of sexual cycles in many lineages traditionally held as asexual. [ABSTRACT FROM AUTHOR]

Mitochondrial genome sequence of Vannella croatica (Amoebozoa, Discosea, Vannellida) was obtained using pulse‐field gel electrophoretic isolation of the circular mitochondrial DNA, followed by the next‐generation sequencing. The mitochondrial DNA of this species has the length of 28,933 bp and contains 12 protein‐coding genes, two ribosomal RNAs, and 16 transfer RNAs. Vannella croatica mitochondrial genome is relatively short compared to other known amoebozoan mitochondrial genomes but is rather gene‐rich and contains significant number of open reading frames. [ABSTRACT FROM AUTHOR]

Abstract Two marine members of the genus Vexillifera Schaeffer, 1926 (Amoebozoa, Dactylopodida) are described. Vexillifera abyssalis n. sp. originates from an abyssal sample of the Western Atlantic 4.5 km deep, which is the first unambiguous record of a deep-sea Vexillifera. The second species, V. kereti n. sp. was isolated from the soft bottom sediments of the White Sea (depth 106 m). An analysis of available data on the genus Vexillifera shows that it comprises many different species, yet they are very unevenly studied. The majority of species have only been described using light microscopy, and their phylogenetic relationships with other amoebae are unclear. However, available small-subunit (SSU) rRNA gene sequences of Vexillifera spp. form a robust, yet very heterogeneous clade in the phylogenetic tree. These species demonstrate a wide range of morphological and ultrastructural characters and originate from diverse habitats, suggesting that Vexillifera may need to be subdivided into several genera in the future. In addition to the described species, we sequenced the COI gene of original CCAP strains of Vexillifera bacillipedes , V. minutissima and Pseudoparamoeba pagei , thereby performing a phylogenetic reconstruction of the Dactylopodida based on a decent taxonomic sampling. [ABSTRACT FROM AUTHOR]

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]

A new species of myxomycete, Lepidoderma cristatosporum , is described from Australia. This species is represented by material collected on subantarctic Macquarie Island. Photographs of fruiting bodies and morphological features as observed under light microscopy are provided, along with micrographs of spore ornamentation as observed by scanning electron microscopy. In addition, the new species is compared with other taxa belonging to the genera Lepidoderma and Diderma , especially with the type material of Lepidoderma crustaceum , a very similar species, and Diderma ochraceum and D. sauteri , two species also muscicolous, for which microphotographs are provided. [ABSTRACT FROM AUTHOR]

Testate amoebae are unicellular organisms characterized by a shell-like test. Due to their potential use as bioindicators (and paleoindicators), these organisms have been increasingly studied in the last decade, particularly in temperate latitudes. This study's objective was two-fold: to identify the testate amoeba communities sampled from 29 water bodies in Mexico and to determine if their presence and distribution also made them suitable bioindicators for tropical latitudes. A total of 40 taxa were recorded within 12 genera, and six significant variables -oxygen, pH, depth, temperature, conductivity, and total alkalinity - that explained testate amoeba distribution within and among the water bodies were identified through a canonical correspondence analysis. The Q-mode clusters rendered five assemblages, each named after their respective dominant species: 1) Centropyxis aculeata strain "aculeata" assemblage, 2) Difflugia oblonga strain "bryophila" assemblage, 3) diverse assemblage, 4) Cucurbitella tricuspis assemblage, and 5) Difflugia protaeiformis strain "acuminata" assemblage. We found that Cucurbitella tricuspis and the Difflugia protaeiformis strain "acuminata" have similar ecological preferences to those reported previously for temperate lakes, with the former identified as an indicator of eutrophic environments and the latter as an indicator of low oxygen levels. On the other hand, Centropyxis aculeata strain "aculeata" and Arcella vulgaris seem to indicate adverse conditions, but the source of this environmental stress apparently differs from that reported in temperate latitudes. Although this stress source could not be identified in all cases, our study nonetheless demonstrates that testate amoebae in the water bodies of central Mexico could reveal the presence of environmental stress. [ABSTRACT FROM AUTHOR]

Myxomycetes is one of the largest groups of protists belonging to Amoebozoa, with ca 1,000 species recognised and more than 4,000 names in use. Historically, myxomycetes were considered fungi or protozoans which, however, fell under the provisions of the former International Code of Botanical Nomenclature (ICBN), currently the International Code of Nomenclature for algae, fungi, and plants (ICN). Attempts to apply the International Code of Zoological Nomenclature (ICZN) to myxomycetes were rare and inconsistent; thus, we argue that Myxomycetes is not a truly ambiregnal group (i.e. one falling under both Codes). Recently, nomenclatural novelties within Myxomycetes have been proposed using ICZN rules, and the application of zoological orthography to myxomycete higher-level taxa in the recent amoebozoan phylogenies is increasingly common. We summarise the consequences of application of either ICN or ICZN to Myxomycetes. In our opinion, nomenclatural stability within Myxomycetes is best served by strict application of ICN. Either treating myxomycetes as falling under ICZN or considering them an ambiregnal group would cause serious nomenclatural instability, mainly owing to the incompatibility of the two Codes as to the date of the starting point of nomenclature and to the appearance of numerous homonyms. [ABSTRACT FROM AUTHOR]

The molecular phylogeny of Flabellula Schaeffer, 1926 has been updated by analysing 18S rRNA and actin gene sequences of 19 new strains collected and characterised by the authors over the past ten years. The genus Flabellula Schaeffer, 1926 (Amoebozoa: Leptomyxida) is a taxon in which species delineation based on morphological data by themselves is insufficient or even misleading. The description of two novel species, F . schaefferi n. sp. and F . sawyeri n. sp., is justified by the congruence of morphological data with 18S rRNA and actin gene sequence phylogenies, in-silico secondary structure prediction of the V2 region in the 18S rRNA, and by recognition of species-specific sequential motifs within this region. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A new species, Phalansterium arcticum sp. n., was isolated from an 8580-year-old Arctic permafrost layer. This organism typically lives as a sedentary uniflagellated cell enclosed in a thin flexible mucilaginous sheath, but can form naked swimming cells and amoeboid cells with eruptive pseudopodia accompanied with the formation of short, filopodia-like projections. In an SSU rDNA phylogenetic tree, it robustly groups with other species of this genus. Along with a description of the species, we also add new details to the description of the cell division of Phalansterium and the feeding process in this organism. [ABSTRACT FROM AUTHOR]

Vannella simplex (Amoebozoa, Discosea, Vannellida) is one of the commonest freshwater free-living lobose amoebae, known from many locations worldwide. In the present study, we describe the complete mitochondrial genome of this species. The circular mitochondrial DNA of V. simplex has 34,145öbp in length and contains 27 protein-coding genes, 2 ribosomal RNAs, 16 transfer RNAs and 4 open reading frames. Mitochondiral genome of V. simplex is one of the most gene compact due to overlapping genes and reduced intergenic space. It has much in common with its closest relative, mitochondrial genome of V. croatica GenBank number MF508648. In the same time, both of them show considerable differences in length and in gene order from the next close relative – that of Neoparamoeba pemaquidensis KX611830 (deposited as Paramoeba ) and even more – from other sequenced amoebozoan mitochondrial genomes. The present study confirms the opinion that the level of synteny between the mitochondrial genomes across the entire Amoebozoa clade is low. More or less considerable similarity yet was found only between members of the same clade of the genera or family level, but hardly — among more distant lineages. [ABSTRACT FROM AUTHOR]

Establishment of multicellularity represents a major transition in eukaryote evolution. A subgroup of Amoebozoa, the dictyosteliids, has evolved a relatively simple aggregative multicellular stage resulting in a fruiting body supported by a stalk. Protosteloid amoeba, which are scattered throughout the amoebozoan tree, differ by producing only one or few single stalked spores. Thus, one obvious difference in the developmental cycle of protosteliids and dictyosteliids seems to be the establishment of multicellularity. To separate spore development from multicellular interactions, we compared the genome and transcriptome of a Protostelium species (Protostelium aurantium var. fungivorum) with those of social and solitary members of the Amoebozoa. During fruiting body formation nearly 4,000 genes, corresponding to specific pathways required for differentiation processes, are upregulated. A comparison with genes involved in the development of dictyosteliids revealed conservation of >500 genes, but most of them are also present in Acanthamoeba castellanii for which fruiting bodies have not been documented. Moreover, expression regulation of those genes differs between P. aurantium and Dictyostelium discoideum. Within Amoebozoa differentiation to fruiting bodies is common, but our current genome analysis suggests that protosteliids and dictyosteliids used different routes to achieve this. Most remarkable is both the large repertoire and diversity between species in genes that mediate environmental sensing and signal processing. This likely reflects an immense adaptability of the single cell stage to varying environmental conditions. We surmise that this signaling repertoire provided sufficient building blocks to accommodate the relatively simple demands for cell-cell communication in the early multicellular forms. [ABSTRACT FROM AUTHOR]

The species Leptomyxa flabellata was described by Goodey in 1915 and re-isolated by Pussard and Pons in 1976. It seems that it was never seen (or never recognized) again since that time. The strain designated as “ Leptomyxa flabellata CCAP 1546/2” was studied by Cann in 1984, however the quality of the electron microscopic images of that time was poor. Based on the cyst structure and size characters, Page in 1988 suggested that this strain is not co-specific with Goodey’s Leptomyxa flabellata , but represents a species ‘Ripidomyxa’ australiensis Chakraborty and Pussard, 1985, nowadays known as Rhizamoeba australiensis . In the present paper light- and electron-microscopic images of CCAP 1546/2 strain, which is now lost, are provided. Based on the morphological evidences it is suggested to establish it in a rank of a new species, Leptomyxa ambigua n. sp. Neither “true” L. flabellata Goodey, 1915 nor original R. australiensis Chakraborty et Pussard, 1985 are nowadays represented in the culture collections, and no original type material is available on both these species. [ABSTRACT FROM AUTHOR]

This work is a morphological study of the nivicolous species within the genus Lepidoderma and, wherever possible, the type material has been studied. Both macro- and microphotographs are provided. The ornamentation of the spores was studied under SEM with the critical point technique, so we can provide a plate with the most distinctive features of each species. They are compared with Diderma fallax, because the latter sometimes shows lime scales on the peridium. At the end of the paper we present a dichotomous key for the genus Lepidoderma, including as well the nonnivicolous species. [ABSTRACT FROM AUTHOR]

Physarum pauciverrucosum, a new species which has profusely fructified on barks from Populus deltoides and Ulmus pumila in moist chamber, is described. Samples come from Juárez city urban area. Photographs of its most relevant macro and microscopic features are provided. [ABSTRACT FROM AUTHOR]

Four taxa of nivicolous myxomycetes not previously known to occur in the Himalayan Mountains of northwestern India are reported herein. Information is provided on these taxa (Didymium dubium, Lamproderma ovoideum, L. ovoideum var. cucumer and Trichia alpina), which were recorded as a result of a survey carried out in May 2006. Important morphological features are illustrated for the first three taxa, and the occurrence of nivicolous myxomycetes in the snowbank habitats of northwestern India is discussed. [ABSTRACT FROM AUTHOR]

Comatricha calderaensis, Didymium dubium, D. nivicola, and Physarum vernum are described herein, they are the first records of nivicolous he first records of nivicolous Myxomycetes from the Canary Islands. Both LM and Both LM and SEM microphotographs are provided to illustrate the distinguishing features of these species. [ABSTRACT FROM AUTHOR]

This is a review of over 400 published research papers on free-living, non-testate amoebae during the approximate last half century (1965-2017) particularly focusing on three topics: Biogeography, Ecology, and Physiology. These topics were identified because of the substantial attention given to them during the course of the last half century, and due to their potential importance in issues of local and global expanse, such as: aquatic and terrestrial stability of habitats, ecosystem processes, biogeochemistry and climate change, and the role of eukaryotic microbes generally in ecosystem services. Moreover, there are close epistemological and thematic ties among the three topics, making a synthesis of the published research more systematic and productive. The number of reviewed publications for each of the three individual topics is presented to illustrate the trends in publication frequencies during the historical period of analysis. Overall, the number of total publications reviewed varied somewhat between 1965 and early 2000 (generally less than 10 per year), but increased to well over 10 per year after 2000. The number of Biogeography and Ecology studies identified in the online citations increased substantially after the mid 1990s, while studies focusing on Physiology were relatively more abundant in the first decade (1965-1974) and less were identified in the 1985 to 2004 period. Citations to the literature are listed in tables for each of the three topics for convenience in retrieving references to specific aspects, and representative examples of the cited research in the tables are reviewed in the text under subheads dedicated to each of the three topics. Biogeographic studies largely focused on the geographic distribution and localized patterns of occurrence of amoebae, with more recent studies incorporating more attention to likely correlates with environmental and biotic factors in the distribution and community composition of amoebae. Ecological studies reviewed in the later decades tended to focus more on community dynamics, the effects of environmental variables on communities (including climate-related topics), a trend toward more physiological ecology studies, combined field-based and experimental studies, and incorporation of newer methodologies such as molecular genetics. In general, physiology studies in the first decades of the review tended to focus on topics of cell physiology such as basic biochemistry, enzyme assays, mechanisms of cell division and development, encystment, and motility. Later studies examined broader topics such as osmoregulation, nutrition, fine structure evidence of cellular changes during the life cycle (including encystment and excystment), and issues related to asexual and sexual reproduction, with increasing substantial evidence of evolutionary patterns and phylogenetics based on molecular evidence. A final section on Conclusions and Recommendations summarizes the findings and presents some potentially productive approaches to future research studies on Amoebozoa within the three designated topics of analysis. [ABSTRACT FROM AUTHOR]

Amoebae of the genus Mayorella are widespread in marine, freshwater and soil habitats. These amoebae have relatively low number of morphological characters allowing species differentiation, so the number of valid species remains rather small. Representatives of the genus Mayorella are hard to maintain in culture, the very most of them are polyphagous and carnivorous. To live and multiply they require a variety of food objects, including other protists. Thus they are difficult objects for molecular studies. Only two sequences of Mayorella were available until recently in GenBank. For the present study we isolated eight strains of Mayorella from different locations worldwide, documented them with the light microscopy and obtained complete or partial sequences of their 18S rRNA gene. Results show that all members of the genus Mayorella form a robust clade within the order Dermamoebida (Amoebozoa: Discosea). The vast majority of our strains probably represent new species; this evidences that diversity of the genus Mayorella in natural habitats is high and that this genus is rather species-rich, comprising no less than 11 species. Our results show that nowadays sequence data are necessary for reliable identification of Mayorella species. [ABSTRACT FROM AUTHOR]

Background: The existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences. Results: When compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones. Conclusion: The results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance. [ABSTRACT FROM AUTHOR]

Physarum spectabile is reported from the Central Forest State Nature Biosphere Reserve (Nelidovsky district, Tver region), new to Russia. The short description of ecology and distribution of this rare species is provided. The morphology of the fruit bodies (sporocarps) of the species was examined by light microscopy and scanning electron microscopy (SEM), and images of relevant details are included. [ABSTRACT FROM AUTHOR]

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii , instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 μm in diameter, are the smallest so far described in genus Vannella . Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in Dictyostelium and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms. Using these vectors, precise genome modifications can be achieved within 2–3 weeks, beginning with the design of the target sequence. Finally, we discussed future perspectives on the use of CRISPR/Cas9-mediated genome editing in Dictyostelium.

A new species of Didymium (Myxomycetes), D. azorellae, isolated from plant debris in a moist chamber, collected during studies of cold arid areas of Argentina and Peru, is described. It can be distinguished by its small size, the tightly packed layer of lime crystals on the peridium, the very scant, or absent, capillitium, and the unique spore ornamentation, especially by scanning electron microscopy. The species developed on dead leaves of cushion plants growing in the extremely harsh environments of the central Andean puna at almost 5000 m elevation and the Andino- Patagonian steppe. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. In order to confirm the identity of the new species described in this paper, a molecular study was conducted based on partial sequences of both the 18S rRNA and the elongation factor 1-alpha gene. Phylogenetic analysis including two specimens from different countries of the newly described species, Didymium azorellae, strongly supports the grouping of these specimens as a separate clade from the rest of the analyzed species. [ABSTRACT FROM AUTHOR]

A new species of Macbrideola (Myxomycetes) from Mexico. Bol. Soc. Micol. Madrid 41: 37–41. Macbrideola deserticola, a new species which has profusely fructified on barks from several plants in moist chamber is described. Samples come from the central plains of the Chihuahuan and Sonoran deserts. Photographs of its most relevant macro and microscopic features are provided. [ABSTRACT FROM AUTHOR]

A new species of Hemitrichia in succulent plants. Bol. Soc. Micol. Madrid 41: 31–35. Hemitrichia succulenticola is described as a new species to science based on abundant material collected on remains of Opuntia spp. from Spain and Mexico. This new species can be distinguished from other members of the genus by brown to orange brown globose sporocarps, a capillitium network of tubular threads with few free ends and big warty spores. Images of diagnostic morphological features are provided. [ABSTRACT FROM AUTHOR]

A new species of Didymium from Spain. Bol. Soc. Micol. Madrid 41: 17–22. A new folicolous species, Didymium radiaticolumellum is described from Spain. The new species has been collected on leaves of Quercus pyrenaica. Macro and microphotographs of fruiting bodies and morphological features as observed under magnifying glass and light microscope (LM) are provided, along with micrographs of spore ornamentation as observed by scanning electron microscope (SEM). [ABSTRACT FROM AUTHOR]

A new species of Didymium (Myxomycetes), confused with other species with similar morphology. Bol. Soc. Micol. Madrid 41: 23–30. A new species, Didymium aggregatum, collected on wheat straw (Poaceae) wastes, is described from Chihuahua (Mexico). LM and SEM photographs illustrate the most representative characters, and D. aggregatum is compared with D. obducens, D. fulvum and D. crustaceum, species with similar morphology. [ABSTRACT FROM AUTHOR]

Two new species, Ripella decalvata and R . tribonemae (Amoebozoa, Vannellida), are described and the diversity of known strains assigned to the genus analyzed. Ripella spp. are closely similar to each other in the light microscopic characters and sequences of small-subunit (SSU) ribosomal RNA gene, but differences in the cell coat structure and cytochrome oxidase (COI) gene sequences are more prominent. SSU rRNA in R . platypodia CCAP1589/2, R . decalvata and R . tribonemae demonstrates an unusual pattern of intragenomic variation. Sequencing of multiple molecular clones of this gene produced numerous sequence variants in a number of specific sites. These sites were usually terminal parts of several variable helices in all studied strains. Analysis of all known Ripella strains shows that SSU rRNA sites differing between strains of different origin are mainly restricted to these areas of the gene. There are only two sites, which differ between strains, but not within genomes. This intragenomic variability of the SSU rRNA gene, seemingly characteristic of all Ripella spp., was never reported to be so extensive in Amoebozoa. The data obtained show another example of complex organization of rRNA gene cluster in protists and emphasize caution needed when interpreting the metagenomic data based on this marker. [ABSTRACT FROM AUTHOR]

Morphological and molecular characterizations of three newly isolated tubulinean strains and re-examination of five strains formerly considered representatives of Saccamoeba and one strain formerly considered as Trichamoeba resulted in ( a ) the determination of strain BA02, isolated from a dripping rock ledge in Skansbukta (Billefjorden, Svalbard), as a new representative of Ptolemeba bulliensis Brown et al., 2014; ( b ) identification of strain ATCC ® 50249™, deposited in the American Type Culture Collection as Trichamoeba , as the same species ( P . bulliensis ); ( c ) characterization of the new strain POHL into the Saccamoeba clade as a member closely related to S . lacustris ; and ( d ) changing the generic residence of three strains formerly considered as representatives of Saccamoeba (strain PV67 to the P . bulliensis clade, and W187G and DP7 into the sister group of Ptolemeba noxubium Brown et al., 2014) whereas two other strains (MSED6, NTSHR) retain their original Saccamoeba clade position. Within the individual clades, the ultrastructure (especially the inner architecture of mitochondria) is congruent and thus of superior taxonomic value to that of light microscopic (morphometric) features. [ABSTRACT FROM AUTHOR]

Longamoebia is one of the most morphologically diverse member of Amoebozoa. It includes the human pathogen Acanthamoeba , which causes minor skin and serious eye infections as well as fatal central nervous system complications. The taxonomy and phylogeny of Longamoebia is poorly understood partly due to the growing number of molecular studies that report unsuspected affiliations of lineages with extremely different morphotypes in the group. A recent molecular study questioned the monophyly of Longamoebia. In this study, we conducted a more comprehensive phylogenomic analysis including all of putative members of Longamoebia to assess its monophyly. We conducted extensive analyses to see effects of outgroup choice, missing data, and gene and taxon sampling on resulting phylogenies. We also collected morphological characters derived from the cytoskeleton using immunocytochemistry to assess homologies of pseudopodia at a finer scale. Our phylogenomic analysis yielded a well-resolved tree of Amoebozoa and highly supported novel relationships. Discosea is recovered as a monophyletic group with all of its known taxonomic orders. However, its within-group relationships dramatically differed from those originally proposed. Our study strongly demonstrates that Longamoebia sensu Smirnov et al. (2011) is not monophyletic and an invalid taxon. Thecamoebida forms a strongly supported sister group relationship with clade Flabellinea (Dactylopodida and Vannellida), while Dermamoebida ( Mayorella + Dermamoeba ) form an independent branch basal to other members of Discosea. The remaining groups including members of Centramoebida form a consistently well-supported clade that was shown to form a sister group relationship with Himatismenida. This robust clade shares the unique cytoskeletal features of coiled cytoplasmic microtubule network and F-actin characters. Our analyses demonstrated that placement of unstable taxa in large-scale analysis with varying levels of missing data might be compromised by some confounding factors such as outgroup choice and gene and taxon sampling. [ABSTRACT FROM AUTHOR]