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.

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.

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.

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.

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.

Paramoeba aparasomata n. sp., a symbiont-free species, and its relative Paramoeba karteshi n. sp. (Amoebozoa, Dactylopodida).

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.

Testate Amoebae in the 407-Million-Year-Old Rhynie Chert.

The Lower Devonian Rhynie chert is justly famous for the clear glimpse it offers of early terrestrial ecosystems [1]. Seven species of stem- and crown-group vascular plants have been described from Rhynie, many preserved in growth position [2], as well as 14 species of invertebrate animals, all arthropods [3] save for a single nematode population [4]. While these shed welcome light on early tracheophytes and land animals, modern terrestrial ecosystems additionally contain a diversity of microscopic organisms that are key to ecosystem function, including fungi, protists, and bacteria. Fungi ranging from mycorrhizae to saprophytes are well preserved in Rhynie rocks ([5] and references therein), and oomycetes are also present [5]. Both green algae (charophytes) and cyanobacteria have also been documented locally [6, 7, 8]. To date, however, phagotrophic protists have not been observed in Rhynie cherts, even though such organisms contribute importantly to carbon, nitrogen, and silica cycling in modern terrestrial communities [9]. Here, we report a population of organic tests described as Palaeoleptochlamys hassii gen. nov., sp. nov. from a pond along the Rhynie alluvial plain, which we interpret as arcellinid amoebozoans. These fossils expand the ecological dimensions of the Rhynie biota and support the hypothesis that arcellinids transitioned from marine through freshwater environments to colonize soil ecosystems in synchrony with early vascular plants.

Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) - An example of sibling species within the genus Thecamoeba.

A new species Thecamoeba cosmophorea n. sp. (Amoebozoa, Discosea, Thecamoebida) was isolated from leaf litter collected in the surroundings of Saint-Petersburg (Russia). This species resembles T. quadrilineata in light-microscopic morphology, but has certain morphological differences and significantly differs in 18S rRNA gene sequence. We performed a direct comparison of this newly isolated species with the Thecamoeba strain isolated from leaf litter in East Siberia (Russia) and identified as T. quadrilineata both at the morphological and the molecular level. There is no type strain of T. quadrilineata, and the type material on this species is represented with the stained slide by F.C. Page that he designated as neotype in 1977. The sequence of the 18S rRNA gene of this species deposited in the GenBank belongs to the isolate identified as T. quadrilineata by Rolf Michel in 1998 and deposited as CCAP 1583/10 strain. Hence we cannot be entirely sure that morphological and molecular data on T. quadrilineata belong to the same amoebae species. The use of molecular data for reliable species differentiation is getting obligate even within the amoebae genus Thecamoeba, which until recently was believed to be among few genera of naked lobose amoebae allowing morphological identification of species.

Phylogenomics of Thecamoebida (Discosea, Amoebozoa) with the Description of Stratorugosa tubuloviscum gen. nov. sp. nov., a Freshwater Amoeba with a Perinuclear MTOC.

Thecamoebida Smirnov and Cavalier-Smith, 2011 (Discosea, Amoebozoa) has been molecularly understudied. The group until recently consisted of three genera containing species that live in terrestrial or aquatic environments. Here, we describe a fourth genus, Stratorugosa tubuloviscum gen. nov. sp. nov., which was isolated from a freshwater Amoeba proteus Ward's Science culture. Although this species most closely morphologically resembles a large, rugose Thecamoeba, S. tubuloviscum gen. nov. sp. nov. can be differentiated from Thecamoeba spp. by the following: 1) the presence of definitive finger-like (lobate-like) subpseudopodia extending at both the anterior and lateral parts of the cell during locomotion; 2) a peculiar locomotive mechanism with two sections, frontal and back, of the cells moving in a pulling and piggyback movement, respectively; 3) the presence of fibrillar cytoplasmic microtubules (MTs) organized by a prominent, perinuclear microtubule-organizing center (MTOC). A phylogenomic analysis of 511 genes assembled from transcriptomic data showed that this new genus was highly supported as sister to Stenamoeba. Despite the variance in gross morphology, Stenamoeba and S. tubuloviscum gen nov. sp. nov. both have MTOCs unlike two Thecamoeba spp., which display dot-like cytoplasmic MTs and lack an MTOC.

Morphology, biology and phylogeny of Phalansterium arcticum sp. n. (Amoebozoa, Variosea), isolated from ancient Arctic permafrost.

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.

Multiple Roots of Fruiting Body Formation in Amoebozoa.

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.

Two new species of Ripella (Amoebozoa, Vannellida) and unusual intragenomic variability in the SSU rRNA gene of this genus.

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.

Description of Neoparamoeba longipodia n. sp. and a new strain of Neoparamoeba aestuarina (Page, 1970) (Amoebozoa, Dactylopodida) from deep-sea habitats.

Two strains of lobose amoebae have been isolated from the deep-sea bottom sediments of the Sea of Japan (3.6km deep) and Western Atlantic Ocean (5.1km deep). Amoebae of both strains have a dactylopodial mophotype, intracellular kinetoplastid symbiont (Perkinsela-like organism) and have no microscales on the cell surface. The morphology and molecular data of the Sea of Japan strain allow us to unambiguously identify it as Neoparamoeba aestuarina (Page, 1970). At the same time, the Atlantic strain is described as a new species Neoparamoeba longipodia as it differs from other species of the genus Neoparamoeba in morphology and gene sequence data. The data presented expand the range of known habitats for the genus Neoparamoeba and permit further analysis of the phylogenetic relationships within this clade with the expanded set of molecular data.

Morphometric and genetic analysis of Arcella intermedia and Arcella intermedia laevis (Amoebozoa, Arcellinida) illuminate phenotypic plasticity in microbial eukaryotes.

Testate amoebae are eukaryotic microorganisms characterized by the presence of an external shell (test). The shell morphology is used as a diagnostic character, but discordance between morphological and molecular data has been demonstrated in groups of arcellinids (Amoebozoa), one of the principal groups of testate amoebae. Morphology of the test is supposed to differentiate genera and species and it is applied in ecological, monitoring and paleontological studies. However, if phenotype does not reflect genotype, conclusions in these types of studies become severely impaired. The objective of this work is to evaluate the morphometrical and morphological variation of the closely related and morphologically similar taxa Arcella intermedia laevis Tsyganov and Mazei, 2006 and Arcella intermedia (Deflandre 1928) Tsyganov and Mazei, 2006 in nature and in cultured individuals and see how these are correlated with molecular data. Our results demonstrate that phenotypic plasticity in Arcella intermedia make morphological distinctions impossible in both taxa. Arcella intermedia and Arcella intermedia laevis are molecularly identical for SSU rDNA and a mitochondrial molecular marker (NAD9/7). We conclude that morphological techniques alone cannot identify phenotypic plasticity from natural populations. More work is clearly needed to better understand the morphological, morphometric and molecular variability in these organisms.

Genetic structure of a morphological species within the amoeba genus Korotnevella (Amoebozoa: Discosea), revealed by the analysis of two genes.

Amoebae of the genus Korotnevella are covered with scales, the structure of which is believed to be species-specific and allows distinguishing species reliably at the morphological level. We studied members of this genus in order to assess the genetic structure of the local populations of amoebae. For the present study we isolated nine freshwater strains of Korotnevella, belonging to three species, from two locations in North-Western Russia. In order to obtain data on the population structure of these amoebae, we identified all isolates based on the light-microscopic morphology and scale structure and investigated both inter-strain and intra-strain polymorphism of Cox I and 18S rRNA genes. Results show that both genes provide congruent patterns of population structure. The Cox I gene appears to be more reliable DNA barcode while the 18S rRNA gene shows an interesting pattern of polymorphism, which may represent phylotypes of amoebae. Local population of amoebae in every studied species consists of a number of genetic lineages (phylotypes), some shared between the populations while others are unique to a local habitat.

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.

Pseudoparamoeba microlepis n. sp., Korotnevella fousta n. sp. (Amoebozoa, Dactylopodida), with notes on the evolution of scales among dactylopodid amoebae.

The present paper describes two new species of lobose amoebae belonging to the family Paramoebidae within the order Dactylopodida: Pseudoparamoeba microlepis n. sp. and Korotnevella fousta n. sp. Among them, P. microlepis formally fits the diagnosis of the genus Korotnevella, because it has scales and lacks a parasome. At the same time its 18S rDNA gene sequence robustly groups with Pseudoparamoeba pagei and never branches among those of Korotnevella spp. Korotnevella fousta in 18S rDNA tree groups among other species of the genus Korotnevella. It has uniform scales with an elliptical basal plate and spine arising from its central part and surrounded by an "inverted skirt" structure. These findings show that the current differentiation between the genera Korotnevella and Pseudoparamoeba based on the presence of scales is not entirely valid. At the moment only molecular data can reliably differentiate these two genera.

Uncovering Cryptic Diversity in Two Amoebozoan Species Using Complete Mitochondrial Genome Sequences.

The Amoebozoa are a major eukaryotic lineage that encompasses a wide range of amoeboid organisms. The group is understudied from a systematic perspective: molecular tools have only been applied in the last 15 yr. Hence, there is an undersampling of both genes and taxa in the group especially compared to plants, animals, and fungi. Here, we present the complete mitochondrial genomes of two ubiquitous and abundant morpho-species (Acanthamoeba castellanii and Vermamoeba vermiformis). Both have mitochondrial genomes of close relatives previously available, enabling insights into recent divergences at a genomic scale, while simultaneously offering comparisons with divergence estimates obtained from traditionally used single genes, SSU rDNA and cox1. The newly sequenced mt genomes are significantly divergent from their previously sequenced conspecifics (A. castellannii 16.4% divergence at nucleotide level and 10.4% amino acid; V. vermiformis 21.6% and 13.1%, respectively), while divergence at the small subunit ribosomal DNA is below 1% within both species. Morphological analyses determined that these lineages are indistinguishable from their previously sequenced counterparts. Phylogenetic reconstructions using 26 mt genes also indicate a level of divergence that is comparable to divergence among species, while reconstructions using the small subunit ribosomal DNA (SSU rDNA) do not. In addition, we demonstrate that between closely related taxa, there are high levels of synteny, which can be explored for primer design to obtain larger fragments than the traditional barcoding genes. We conclude that, although most systematic work has relied on SSU, this gene alone can severely underestimate diversity. Thus, we suggest that the mt genome emerges as an alternative for unraveling the lower level phylogenetic relationships of Amoebozoa.

Cyst-forming amoebae of the genus Korotnevella (Amoebozoa: Dactylopodida), with description of two new species.

The genus KorotnevellaGoodkov, 1988 (Amoebozoa: Dactylopodida) comprises amoebae with a complex cell coat that include a layer of scales covering the entire cell surface. Until now no Korotnevella species were known to form cysts. Here we describe two new species of genus Korotnevella and provide a light- and electron-microscopic report of encystment in three species (K. stella (Schaeffer, 1926) Goodkov, 1988, K. limbata n. sp. and K. heteracantha n. sp.) of this genus. Korotnevella limbata has two peculiar wide rims along the upper margin of basket scales forming two short spines at their junctions. The basket scales of K. heteracantha are very similar to those of K. bulla, but have a different spine length and a closed latticework basket with polygonal elements. All three studied species have similar cyst wall consisting of three layers. The outer layer contains scales of trophic stage; the middle one includes so called "cyst scales" with meshwork walls; and the inner one is composed of fibrillar material. Cyst structure of this sort appears to be unique for lobose amoebae and probably represents an autapomorphy for genus Korotnevella.