It's time to consider the Arcellinida shell as a weapon.

The shells of testate amoebae are morphologically diverse and persistent in the environment. Accordingly, the examination of the morphology and composition of shells became a standard tool in ecological, palaeoecological, and evolutionary studies. However, so far the function of the shell remains poorly understood and, although based on limited evidence, the shell was considered as a defense mechanism. Based on recent evidence, we propose that the shell of arcellinid testate amoebae is a crucial component facilitating the amoebae's attack of large prey. Accordingly, the shell is not purely protective, but must be considered also as a weapon. This change in perspective opens up numerous new avenues in protistology and will lead to a substantial change in ecological, palaeoecological, and evolutionary research.

Expansion of the cytochrome C oxidase subunit I database and description of four new lobose testate amoebae species (Amoebozoa; Arcellinida).

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.

Novel contributions to the molecular and morphological diversity of freshwater monothalamid foraminifera: Description of six new species.

Non-marine monothalamous foraminifera are common in freshwater and soil habitats. They comprise a poorly-known group lacking sufficient information about diversity, morphology, reproduction, distribution and ecology. Based on an integrative morphological and molecular approach we describe a new family, a new genus and six new species of freshwater monothalamids from different localities in the Netherlands and France. We establish Astroperula as a new genus of organic walled freshwater foraminifera that contains two species, Astroperula dumacki and Astroperula parvipila. Furthermore, two new agglutinated freshwater monothalamids are described and illustrated, Limnogromia leanneae and Lacogromia pawlowskii, one new organic walled, Velamentofex dujardini, and a new naked monothalamid, Haplomyxa retiforma. Additional information is provided about a special form of cell division in Claparedellus lachmannii and L. leanneae, and feeding behavior in cultures of V. dujardini. Morphological observations are added for an unidentified Limnogromia sampled from the Netherlands and two types of Lacogromia sampled from an alpine region in the French Pyrenees and a karst sinkhole in Bosnia and Herzegovina. The present study provides additional insight into the ecology, diversity and occurrence of freshwater monothalamids and emphasizes the fact that a combination of morphological and molecular methods is necessary to clearly distinguish species in this group.

Transfer of the thecate amoebae Lecythium spinosum and Pamphagus armatus to Rhizaspis (Thecofilosea, Cercozoa, Rhizaria).

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. The captivating spine-bearing taxa in Thecofilosea were not yet molecularly characterized due to being very rare. There are only two known spine-bearing species, Pamphagus armatus and Lecythium spinosum, which were synonymized by Penard in 1902. Due to a morphological difference of those taxa, we discuss here that we disagree with this taxonomical act. We further isolated single cells of Pamphagus armatus directly from their habitat and successfully sequenced their SSU rDNA, which we subjected to phylogenetic analyses. We show that Pamphagus armatus branches within the Rhizaspididae (Tectofilosida, Thecofilosea). Accordingly, we transfer Pamphagus armatus and the assumingly closely related species Lecythium spinosum to Rhizaspis.

Broad sampling of monothalamids (Rhizaria, Foraminifera) gives further insight into diversity of non-marine Foraminifera.

Non-marine foraminifera are among the least known groups of protists and only a handful of species have been described since the 19th century. We collected one naked and five morphologically almost identical organic-walled monothalamid species from freshwater and terrestrial environments from Germany and Austria. One of the species was identified as Lieberkuehnia wageneriClaparède and Lachmann, 1859. As its original description is ambiguous and its type specimen has been lost, a neotype is proposed. We describe four new organic-walled monothalamous foraminifera and a novel Reticulomyxa species both morphologically and genetically. Analyses of molecular data of the different isolates revealed that they are distributed across six different clades. Two new genera, Claparedellus gen. nov. and Velamentofex gen. nov., and five new monothalamous families, Lacogromiidae fam. nov., Limnogromiidae fam. nov., Lieberkuehniidae fam. nov., Edaphoallogromiidae fam. nov. and Velamentofexidae fam. nov., are established.

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.

Shell Colour in Cercozoa; a Simple Trait to Distinguish Thecofilosea from Imbricatea?

Rigid and persistent shells of microeukaryotes are widely used as bioindicators in ecological and paleontological studies. Drawing conclusions on ecological or evolutionary patterns depends strongly on the right taxonomic assignment of the observed species, however confusion is common. Especially in filose shelled amoebae it is often unclear whether species belong to the Imbricatea or Thecofilosea when only morphological data are collected. Molecular surveys shed light on their evolutionary relationship; based on these we propose a hypothesis how to differentiate doubtful species even light microscopically.

SSU rDNA Phylogeny Indicates the Scale-lacking Trivalvulariida ord. nov. as a Sister Group to the Euglyphida (Cercozoa, Rhizaria).

The testate amoeba Leptogromia operculata was described by Valkanov in 1970 from marine waters. We re-discovered this species in brackish water along the North Sea in the Netherlands. Based on detailed comparison of morphology and SSU rDNA phylogeny we conclude that this species represents a sister clade to the Euglyphida (Imbricatea, Cercozoa). We further describe a similar species Trivalvularis immunda gen. nov., sp. nov. from freshwater in France and the Netherlands on basis of morphological data. Trivalvularis and Leptogromia share a unique oral apparatus with three valves that can close the aperture of the shell. Due to this unique morphological character and the phylogenetic analysis of L. operculata we place both species in a new family Trivalvulariidae in the new order Trivalvulariida.

The Dancing Star: Reinvestigation of Artodiscus saltans (Variosea, Amoebozoa) Penard 1890.

Artodiscus saltans, first described by Penard (1890), has a unique morphology. Without genetic data it could not yet been reliably placed into a wider taxonomical context. We present morphological data for A. saltans from different aquatic habitats of four European countries. We subjected three cells of one strain from Germany to molecular analyses and, interestingly, obtained six different rDNA sequences. Phylogenetic analyses of these SSU rDNA sequences revealed that A. saltans branches close to the amoebozoan Multicilia marina (Variosea, Amoebozoa).

Rediscovery of the Testate Amoeba Genus Penardeugenia (Thaumatomonadida, Imbricatea).

The genus Penardeugenia DEFLANDRE 1958 accommodates a single species which was described as Pamphagus bathybioticus PENARD 1904. Although this species seems to be well characterized in its description, it has never been recorded again. Despite its possession of silica scales, it was assigned to the scale-lacking Chlamydophryidae (Thecofilosea, Cercozoa). We here present morphological data of three isolates. One of them was successfully cultured and enabled the amplification of its SSU rDNA sequence. Molecular analyses revealed the evolutionary origin of Penardeugenia in the scale-bearing flagellate class Thaumatomonadida, which is backed up by morphological similarities of their scales. We consider Penardeugenia to represent a closely related genus to Thaumatomastix, which apparently lost its ability to form flagella. We describe two new species from Germany, P. huelsmannii and P. hausmannii.

Taxonomic revision of freshwater foraminifera with the description of two new agglutinated species and genera.

Most foraminifera inhabit marine habitats, but some species of monothalamids have been described from freshwater environments, mainly from Swiss water bodies over 100 years ago. Recent environmental DNA surveys revealed the presence of four major phylogenetic clades of freshwater foraminifera. However, until now only one of them (clade 2) has been associated to a morphologically described taxon-the family Reticulomyxidae. Here, we present morphological and molecular data for the genera representing the three remaining clades. We describe two new agglutinated freshwater genera from China and the Netherlands, Lacogromia and Limnogromia, which represent clades 3 and 4, respectively. We also report the first ribosomal DNA sequences of the genus Lieberkuehnia, which place this genus within clade 1. Our study provides the first morphotaxonomic documentation of molecular clades of freshwater foraminifera, showing that the environmental DNA sequences correspond to the agglutinated monothalamous species, morphologically similar to those described 100 years ago.

NAD9/NAD7 (mitochondrial nicotinamide adenine dinucleotide dehydrogenase gene)-A new "Holy Grail" phylogenetic and DNA-barcoding marker for Arcellinida (Amoebozoa)?

Molecular phylogeny is an indispensable tool for assessing evolutionary relationships among protists. The most commonly used marker is the small subunit ribosomal RNA gene, a conserved gene present in many copies in the nuclear genomes. However, this marker is not variable enough at a fine-level taxonomic scale, and intra-genomic polymorphism has already been reported. Finding a marker that could be useful at both deep and fine taxonomic resolution levels seemed like a utopic dream. We designed Amoebozoa-specific primers to amplify a region including partial sequences of two subunits of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase gene (NAD9/NAD7). We applied them to arcellinids belonging to distantly related genera (Arcella, Difflugia, Netzelia and Hyalosphenia) and to Arcellinid-rich environmental samples to obtain additional Amoebozoa sequences. Tree topology was congruent with previous phylogenies, all nodes being highly supported, suggesting that this marker is well-suited for deep phylogenies in Arcellinida and perhaps Amoebozoa. Furthermore, it enabled discrimination of close-related taxa. This short genetic marker (ca. 250bp) can therefore be used at different taxonomic levels, due to a fast-varying intergenic region presenting either a small intergenic sequence or an overlap, depending on the species.