Molecular phylogenetic analyses support the validity of Ceratiomyxa porioides (Amoebozoa, Eumycetozoa) at species level.

The frequently encountered macroscopic slime molds of the genus Ceratiomyxa have long been recognized by mycologists and protistologists for hundreds of years. These organisms are amoebozoan amoebae that live and grow inside and on the surface of decaying wood. When conditions are favorable, they form subaerial sporulating structures called fruiting bodies which take on a variety of forms. These forms are typically some arrangement of column and/or branches, but one is uniquely poroid, forming folds instead. Originally, this poroid morphology was designated as its own species. However, it was not always clear what significance fruiting body morphology held in determining species. Currently, Ceratiomyxa fruticulosa var. porioides, the poroid form, is considered a taxonomic variety of Ceratiomyxa fruticulosa based on morphological designation alone. Despite its long history of observation and study, the genus Ceratiomyxa has been paid little molecular attention to alleviate these morphological issues. We have obtained the first transcriptomes of the taxon C. fruticulosa var. porioides and found single gene phylogenetic and multigene phylogenomic support to separate it from C. fruticulosa. This provides molecular evidence that fruiting body morphology does correspond to species level diversity. Therefore, we formally raise Ceratiomyxa porioides to species level.

Identification of new amoebae strains in rainbow trout (Oncorhynchus mykiss, Walbaum) farms affected by nodular gill disease (NGD) in Northeastern Italy.

Nodular gill disease (NGD) is an emerging condition associated with amoeba trophozoites in freshwater salmonid farms. However, unambiguous identification of the pathogens still must be achieved. This study aimed to identify the amoeba species involved in periodic NGD outbreaks in two rainbow trout (Oncorhynchus mykiss) farms in Northeastern Italy. During four episodes (February-April 2023), 88 fish were euthanized, and their gills were evaluated by macroscopic, microscopic and histopathological examination. The macroscopic and microscopic severity of the lesions and the degree of amoebae infestation were scored and statistically evaluated. One gill arch from each animal was put on non-nutrient agar (NNA) Petri dishes for amoeba isolation, cultivation and subsequent identification with SSU rDNA sequencing. Histopathology confirmed moderate to severe lesions consistent with NGD and mild to moderate amoeba infestation. The presence of amoebae was significantly correlated with lesion severity. Light microscopy of cultured amoebae strains and SSU rDNA analysis revealed the presence of a previously characterized amoeba Naegleria sp. strain GERK and several new strains: two strains from Hartmannelidae, three vannelid amoebae from the genus Ripella and cercozoan amoeba Rosculus. Despite the uncertainty in NGD etiopathogenesis and amoebae pathogenic role, identifying known and new amoebae leans towards a possible multi-aetiological origin.

Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.

Living in the cracks: Two novel genera of Variosea (Amoebozoa) discovered on an urban sidewalk.

Biological soil crusts represent a rich habitat for diverse and complex eukaryotic microbial communities. A unique but extremely common habitat is the urban sidewalk and its cracks that collect detritus. While these habitats are ubiquitous across the globe, little to no work has been conducted to characterize protists found there. Amoeboid protists are major predators of bacteria and other microbial eukaryotes in these microhabitats and therefore play a substantial ecological role. From sidewalk crack soil crusts, we have isolated three naked amoebae with finely tapered subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a holistic approach including light, electron, and fluorescence microscopy as well as phylogenetics using the ribosomal small subunit rRNA gene and phylogenomics using 230 nuclear genes, we find that these amoeboid organisms fail to match any previously described eukaryote genus. However, we determined the amoebae belong to the amoebozoan lineage Variosea based on phylogenetics. The molecular analyses place our isolates in two novel genera forming a grade at the base of the variosean group Protosteliida. These three novel varioseans among two novel genera and species are herein named "Kanabo kenzan" and "Parakanabo toge."

From Argentine Abyssal Plain to farmed turbot in Spain: A ubiquitous amoeba species Vannella robusta sp. nov. (Amoebozoa, Vannellida).

A strain with the characters of the genus Vannella was isolated from the water layer immediately above the deep-sea sediment collected in the south-western Atlantic Ocean, ca. 4.6 km deep. Small-subunit ribosomal RNA (SSU rRNA) and cytochrome c oxidase (Cox1) gene phylogenetic analyses showed that the new strain branches within the clade of previously isolated unnamed Vannella strains from different marine fish and invertebrate hosts. Although the SSU rRNA gene sequences of these strains show variability within 2% of all nucleotide positions without any regular pattern, the available Cox1 gene sequences from within this clade are identical. Given the morphological homogeneity of the revealed clade, all of its strains can be assigned under the same species name, and the variation of their SSU rRNA is comparable to its intragenomic variation, as shown by molecular cloning of the PCR amplicons. High variability of the SSU rRNA gene sequences within and between independently isolated morphologically identical strains in combination with highly conserved Cox1 gene sequences may be a feature in some clades of Vannella, but is not a general rule for this genus, as SSU rRNA genes conserved between different morphospecies occur in several other clades within Vannella.

A morphological and molecular reinvestigation of Janickina pigmentifera (Grassi, 1881) Chatton 1953 - an amoebozoan parasite of arrow-worms (Chaetognatha).

Amoebozoan parasites of arrow-worms (Chaetognatha) were isolated from their hosts living in plankton of the Bay of Villefranche (Mediterranean Sea). Based on the light microscopic characters, the amoebae were identified as Janickina pigmentifera (Grassi, 1881) by their limax locomotive form and due to the presence of the intracellular symbiont, Perkinsela amoebae, surrounded by a layer of pigment granules. Sequences of the 18S rRNA gene of both J. pigmentifera and its symbiont were obtained for the first time. The molecular phylogenetic analyses of 18S rRNA gene placed J. pigmentifera within the genus Neoparamoeba, a taxon also characterized by the presence of a symbiont, known as Perkinsela amoebae-like organism (PLO). The 18S rRNA gene sequence of P. amoebae from J. pigmentifera grouped with the sequences of 18S rRNA genes of PLOs from Neoparamoeba branchiphila and Neoparamoeba invadens. The first photo documentation of the light microscopic features of J. pigmentifera, such as locomotive form, the morphology of the nucleus and P. amoebae have been provided. The new results support the affinity of J. pigmentifera with the family Paramoebidae suggested previously based on the presence of PLO. In contrast to Janickina, typical members of Paramoebidae (Neoparamoeba and Paramoeba) have a flattened, dactylopodial locomotive form. This discrepancy in morphology can be explained by the obligate parasitic lifestyle of Janickina.

New Approach to the Ultrastructure of the Capillitium in the Order Trichiales (Myxomycetes, Amoebozoa) and its Phylogenetic Implications.

Myxomycetes constitute one of the major lineages within the supergroup Amoebozoa. At the end of their life cycles, most myxomycetes produce spore-bearing fruiting bodies, in which additional structures develop, like the capillitium, a system of sterile filaments intermingled with the spores. The capillitium is a relevant structure in the taxonomy of the order Trichiales, the target group in this study. However, the introduction of molecular phylogenies in Myxomycetes systematics is challenging our comprehension of this structure. We studied the capillitium of 25 species representing nine Trichiales genera, with both scanning and transmission electron microscopy. In this order, the capillitium showed higher diversity than so far recognized. Thus, we distinguished and described five capillitium types and two subtypes based on the presence or absence of a lumen and the wall ultrastructure. These types followed the evolutionary history reported in recent phylogenies, although not all of them defined monophyletic groups. Besides, the spiral ornamentation, which most taxonomists considered to have appeared once, occurred in three different capillitium types. The ultrastructural approaches in Myxomycetes systematics enable the reconsideration of their morphological features in the new phylogenetic scenario.

Vannella primoblina n. sp. - an unusual species of the genus Vannella (Amoebozoa, Discosea, Vannellida) with pronounced dorsal ridges and folds.

Amoebae of the order Vannellida (Amoebozoa, Discosea) have a fairly recognizable spatulate, fan-shaped or semi-circular outlines and a wide area of frontal hyaloplasm. They can be easily distinguished from the other groups of lobose amoebae even by light microscopy. The dorsal side of these amoebae is usually smooth and rarely bears ridges or folds, which are never numerous or regular. We have isolated an unusual species of vannellid amoebae, called Vannella primoblina n. sp. from a terrestrial substrate. It has well-developed dorsal relief consisting of regularly appearing folds and ridges. This amoeba superficially resembles members of the genus Thecamoeba. However, molecular analysis showed that this strain belongs to the genus Vannella. This finding indicates that dorsal folds may also be a characteristic of some species of vannellid amoebae and probably are a functional detail of the cell morphology rather than an apomorphy of Thecamoebida lineage. Overall outlines of the cell and the presence of the expanded frontal hyaline area remains the most reliable characters used to differentiate vannellid amoebae from other gymnamoebae lineages.

Range-wide Phylogeography of a Nivicolous Protist Didymium nivicola Meyl. (Myxomycetes, Amoebozoa): Striking Contrasts Between the Northern and the Southern Hemisphere.

Soil protists play a crucial role in terrestrial ecosystems and often show immense taxonomic diversity. However, for many groups, distribution patterns remain largely unknown. We investigated range-wide intraspecific diversity of a specialized airborne protist (Didymium nivicola Meyl.) that occupies a narrow ecological niche associated with long-lasting snow cover. We sampled 122 collections covering all areas where the species was recorded worldwide. We obtained 105 and 41 sequences of small ribosomal subunit rDNA (SSU) and elongation factor 1-alpha (EF1A), respectively. While the species is very diverse in the austral Andes, Southern Hemisphere (SH; 17 SSU ribotypes and 12 EF1A genotypes identified), its populations are genetically uniform across three continents of the Northern Hemisphere (NH; single ribotype, single genotype). Our results indicate the austral Andes as a possible diversification centre for D. nivicola where populations seem to reproduce sexually. Two main parts of the range display highly contrasting genetic patterns, thus biogeographical history and dynamics. Current distribution of D. nivicola in the NH is likely a result of a dispersal event from the SH and subsequent long-distance dispersal (LDD) that might be associated with a shift to asexual mode of reproduction.

Amoeboid protist systematics: A report on the "Systematics of amoeboid protists" symposium at the VIIIth ECOP/ISOP meeting in Rome, 2019.

Amoeboid protists are extremely abundant and diverse in natural systems where they often play outstanding ecological roles. They can be found in almost all major eukaryotic divisions, and genomic approaches are bringing major changes in our perception of their deep evolutionary relationships. At fine taxonomic levels, the generalization of barcoding is revealing a considerable and unsuspected specific diversity that can be appreciated with careful morphometric analyses based on light and electron microscopic observations. We provide examples on the difficulties and advances in amoeboid protists systematics in a selection of groups that were presented at the VIIIth ECOP/ISOP meeting in Rome, 2019. We conclude that, in all studied groups, important taxonomical rearrangements will certainly take place in the next few years, and systematics must be adapted to incorporate these changes. Notably, nomenclature should be flexible enough to integrate many new high level taxa, and a unified policy must be adopted to species description and to the establishment of types.

Thecamoeba foliovenanda n. sp. (Amoebozoa, Discosea, Thecamoebida) - One more case of sibling species among amoebae of the genus Thecamoeba.

Until recently it seemed that amoebae of the genus Thecamoeba can be reliably identified using light-microscopic characters, like the size and shape of the cell, a characteristic pattern of folds and ridges and structure of the nucleus. However, recent molecular studies show the presence of sibling species that can be reliably distinguished based on the gene sequence data only. Here we describe a new terrestrial species, Thecamoeba foliovenanda n. sp. This species is almost identical with Thecamoeba similis in light-microscopy, which has minor differences in the ultrastructure but considerable differences in the SSU rRNA gene sequence. We investigated the light-microscopic data, as well as transmission electron-microscopic images and videorecords on the type strain of T. similis 1583/8 and performed its comparison with the new species. This study further supports the conclusion that species of the genus Thecamoeba nowadays require gene sequencing for reliable identification and species distinction.

Description of a new species of marine amoeba Korotnevella mutabilis n. sp. (Amoebozoa, Dactylopodida).

Korotnevella (Amoebozoa, Dactylopodida) is a genus of naked lobose amoebae with a dactylopodial morphotype. The cell membrane of these amoebae is covered with a monolayer of scales. The structure and size of scales are considered as species-specific features. Here, we describe a new marine species, Korotnevella mutabilis n. sp., isolated from the bottom sediment sample of Nivå Bay (Baltic Sea, The Sound) and studied with light and electron microscopy as well as with molecular phylogenetic analysis. This species has a number of morphological similarities with Korotnevella monacantholepis, such as size of the cell, L/B ratio, the nucleus structure and the type of a biotope from which both species were isolated. At the same time, Korotnevella mutabilis n. sp. differs from K. monacantholepis in the structure of basket-shaped scales: Korotnevella mutabilis n. sp. has an enclosed hammock-shaped latticework basket and up to two spines while K. monacantholepis has an opened two-row latticework basket and never has two spines. According to molecular phylogenetic analyses based on the sequences of the mitochondrial COI gene, Korotnevella mutabilis n. sp. is a distinct species, highly divergent from other Korotnevella species.

Molecular investigation of Phryganella acropodia Hertwig et Lesser, 1874 (Arcellinida, Amoebozoa).

Phryganella acropodia Hertwig and Lesser, 1874, is one of the most common and abundant testate amoeba species. It represents the type species of the genus Phryganella Penard, 1902, which in turn is the type genus for the suborder Phryganellina (Arcellinida) Bovee, 1985, but despite its taxonomic importance it was not yet analyzed with molecular methods. We established two cultures of putative Phryganella acropodia, designed Phryganellina-specific primers, amplified SSU rDNA data and subjected these sequences to phylogenetic analyses. Morphological and genetic differences were found between both strains. With SSU rDNA phylogenetic analyses we confirm that Phryganella acropodia branches with Phryganella paradoxa Penard, 1902 and Cryptodifflugia Penard, 1890 in the Phryganellina. We thus give further evidence that pseudopodia morphology in the Arcellinida is a character of high taxonomic value, as suggested by Bovee and Jung when erecting the suborder Phryganellina. Moreover, we provide evidence for cryptic diversity and for the first time confirm the existence of a naked life stage in Arcellinida by molecular means.

Cunea russae n. sp. (Amoebozoa, Dactylopodida), another cryptic species of Cunea Kudryavtsev and Pawlowski, 2015, inhabits a continental brackish-water biotope.

The genus Cunea Kudryavtsev and Pawlowski, 2015 (Amoebozoa, Dactylopodida) was initially described from the oceanic benthos: C. profundata, from over 5 km depth in the Atlantic Ocean, and C. thuwala from the Red Sea benthos at ca. 60 m depth. Both species are identical to each other in morphology (including cell coat ultrastructure), but differ significantly in the gene sequence data, including barcoding loci of small subunit ribosomal RNA and cytochrome oxidase subunit 1 gene, as well as actin. This paper describes the third species of Cunea, C. russae n. sp. isolated from a brackish water habitat without a direct connection to the ocean, a small spring of brackish water (19‰) emerging from a 246 m deep hole in the earth. This species is morphologically identical to the previous two amoebae, but differs from them significantly in the gene sequence data and ecological preferences. In particular, this species has the broadest salinity tolerance range, being able to reproduce well already at 2.5‰. It is also capable of resisting cold temperatures, like C. profundata. The data obtained suggest that the genus Cunea may comprise a significant taxonomic diversity represented by morphologically identical, but quickly diverging species with significant ecological plasticity.

Bog Microtopography and the Climatic Sensitivity of Testate Amoeba Communities: Implications for Transfer Function-Based Paleo-Water Table Reconstructions.

Although the use of sub-fossil testate amoebae as a proxy for raised bog hydrology in Holocene paleoecological studies is well-established, some detailed aspects of species-environment relationships remain under-researched. One such issue is the effect of bog surface microtopography on the climatic sensitivity of testate amoeba communities. Although it has been suggested that some microforms-especially hummocks-may be less sensitive to climatic forcing than others, this has rarely been objectively tested. To investigate this, subfossil testate amoebae assemblages have been examined in a series of shallow cores collected along a hummock-lawn-hollow transect from a bog in central Ireland and the resulting reconstructed water table records, dated using 210Pb, have been compared with instrumental weather data. Testate amoebae communities in the hollow microform were found to be significantly less diverse than those in the hummock and lawn, and both the hummock and lawn showed statistically significant correlations with instrumental temperature and precipitation data. Therefore, whilst the suggestion that paleoecological investigations should target intermediate bog microforms remains sound, the notion that hummock-based testate amoebae hydrological data are climatically-insensitive is challenged.

Molecular Phylogeny of Polychaos annulatum (Amoebozoa, Tubulinea, Euamoebida) Shows that Genus Polychaos Belongs to the Family Hartmannellidae.

We have obtained a sequence of the 18S rRNA gene of the species Polychaos annulatum (Penard 1902) Smirnov et Goodkov 1998 using the isolation of a single nucleus from an amoeba cell. Attempts to amplify the 18S rRNA gene from the DNA of this species by conventional PCR were not successful, so we applied the whole genome amplification of the nuclear DNA followed by NGS sequencing. The 18S rRNA gene was found among the resulting contigs. The analysis unexpectedly shows that P. annulatum robustly groups within the family Hartmannellidae, but not Amoebidae. This finding warrants revision of the basic morphological criteria used to classify Euamoebida families and show that "proteus-type" amoebae may belong to other families rather than Amoebidae. This makes taxonomic assignments of such species more complex and the borders between Euamoebida families more nuanced. It is getting evident that molecular data are necessary to clarify the position of species even in this most "classical" order of naked lobose amoebae.

Parasitic Infections of Wild Boars (Sus scrofa) in Iran: A Literature Review.

BACKGROUND: Swine species are an important source of meat production worldwide, except in Islamic countries where pig breeding and pork consumption are forbidden. Hence, they are often neglected in these regions. A considerable number of wild boars (Sus scrofa) inhabit Iranian territories, particularly in dense forests of north, west and southwest of the country, but our knowledge regarding their parasites is very limited. OBJECTIVE: The lack of a comprehensive record in this connection encouraged us to review the whole works of literature in the country. METHODS: The current review presents all the information about the parasitic diseases of wild boar in Iran extracted from articles available in both Persian and English databases until June 2017. RESULTS: So far, 8 genera of protozoa (Toxoplasma, Balanthidium, Tritrichomonas, Blastocystis, Entamoeba, Iodamoeba, Chilomastix and Sarcocystis) and 20 helminth species, including four cestode species, two trematode species, thirteen nematode species as well as a single species of Acanthocephala have been described in Iranian wild boars. CONCLUSION: This review sheds light on the veterinary and public health aspects of the parasitic diseases of wild boars in the country and alerts authorities for future preventive measures.

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus.

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister-like glycostyles with hexagonal base and dome-shaped apex; besides, cell surface bears hair-like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.

Stygamoeba cauta n. sp. (Amoebozoa, Discosea) - a new brackish-water species from Nivå Bay (Baltic Sea, The Sound).

Several evolutionary lineages of Amoebozoa are characterized by unusual morphological and ultrastructural features that impede resolving of their position in the phylogenetic tree. Among them is the genus Stygamoeba, not yet reliably placed on the phylogenetic tree even by a phylogenomic analysis. Only two species of Stygamoeba are known at present, and molecular data exists on one species only. Here, we present a description of the mesohaline species Stygamoeba cauta n. sp. isolated from the bottom sediments of Nivå Bay (Baltic Sea, The Sound). This stick-like, flattened amoeba morphologically resembles the previously described species Stygamoeba regulataSmirnov, 1996. However, the molecular analysis based on the 18S rRNA gene sequences and differences in cell behavior and pattern of locomotion provide strong support for establishing a new species.

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

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