Three New Freshwater Cochliopodium Species (Himatismenida, Amoebozoa) from the Southeastern United States.

Cochliopodium is a lens-shaped genus of Amoebozoa characterized by a flexible layer of microscopic dorsal scales. Recent taxonomic and molecular studies reported cryptic diversity in this group and suggested that the often-used scale morphology is not a reliable character for species delineation in the genus. Here, we described three freshwater Cochliopodium spp. from the southeastern United States based on morphological, immunocytochemistry (ICC), and molecular data. A maximum-likelihood phylogenetic analysis and pairwise comparison of COI sequences of Cochliopodium species showed that each of these monoclonal cultures were genetically distinct from each other and any described species with molecular data. Two of the new isolates, "crystal UK-YT2" (Cochliopodium crystalli n. sp.) and "crystal-like UK-YT3" (C. jaguari n. sp.), formed a clade with C. larifeili, which all share a prominent microtubule organizing center (MTOC) and have cubical-shaped crystals. The "Marrs Spring UK-YT4" isolate, C. marrii n. sp., was 100% identical to "Cochliopodium sp. SG-2014 KJ569724." These sequences formed a clade with C. actinophorum and C. arabianum. While the new isolates can be separated morphologically, most of the taxonomic features used in the group show plasticity; therefore, Cochliopodium species can only be reliably identified with the help of molecular data.

Longamoebia is not monophyletic: Phylogenomic and cytoskeleton analyses provide novel and well-resolved relationships of amoebozoan subclades.

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.

Mycamoeba gemmipara nov. gen., nov. sp., the First Cultured Member of the Environmental Dermamoebidae Clade LKM74 and its Unusual Life Cycle.

Since the first environmental DNA surveys, entire groups of sequences called "environmental clades" did not have any cultured representative. LKM74 is an amoebozoan clade affiliated to Dermamoebidae, whose presence is pervasively reported in soil and freshwater. We obtained an isolate from soil that we assigned to LKM74 by molecular phylogeny, close related to freshwater clones. We described Mycamoeba gemmipara based on observations made with light- and transmission electron microscopy. It is an extremely small amoeba with typical lingulate shape. Unlike other Dermamoebidae, it lacked ornamentation on its cell membrane, and condensed chromatin formed characteristic patterns in the nucleus. M. gemmipara displayed a unique life cycle: trophozoites formed walled coccoid stages which grew through successive buddings and developed into branched structures holding cysts. These structures, measuring hundreds of micrometres, are built as the exclusive product of osmotrophic feeding. To demonstrate that M. gemmipara is a genuine soil inhabitant, we screened its presence in an environmental soil DNA diversity survey performed on an experimental setup where pig cadavers were left to decompose in soils to follow changes in eukaryotic communities. Mycamoeba gemmipara was present in all samples, although related reads were uncommon underneath the cadaver.

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.

A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD).

Neoparamoeba perurans is the aetiological agent of amoebic gill disease (AGD) in salmonids, however multiple other amoeba species colonise the gills and their role in AGD is unknown. Taxonomic assessments of these accompanying amoebae on AGD-affected salmon have previously been based on gross morphology alone. The aim of the present study was to document the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon using a combination of morphological and sequence-based taxonomic methods. Amoebae were characterised morphologically via light microscopy and transmission electron microscopy, and by phylogenetic analyses based on the 18S rRNA gene and cytochrome oxidase subunit I (COI) gene. In addition to N. perurans, 11 other amoebozoans were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. In some cases, such as Paramoeba eilhardi, this is the first time this species has been isolated from the gills of teleost fish. Furthermore, sequencing of both the 18S rRNA and COI gene revealed significant genetic variation within genera. We highlight that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established.

Two new species of the genus Stenamoeba (Discosea, Longamoebia): cytoplasmic MTOC is present in one more amoebae lineage.

Two new species of the recently described genus Stenamoeba, named S. berchidia and S. sardiniensis were isolated from a single soil sample on Sardinia, Italy. Both share morphological features characteristic to Stenamoeba and form in phylogenetic analyses together with other Stenamoeba spp. a highly supported clade within the family Thecamoebidae. The ultrastructural investigation of Stenamoeba sardiniensis revealed the presence of cytoplasmic microtubule-organizing centers (MTOCs), located close to one of several dictyosomes found inside the cell. This is the first report of cytoplasmic MTOCs among Thecamoebidae. The presence of MTOCs is now shown in five of nine orders comprising the class Discosea and potentially could be a phylogenetic marker in this group. We re-isolated Stenamoeba limacina from German soils. This strain shows a similar morphology and an almost complete SSU rDNA sequence identity with the type strain of S. limacina originating from gills of fishes, collected in Czech Republic.