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.

Deconstructing Difflugia: The tangled evolution of lobose testate amoebae shells (Amoebozoa: Arcellinida) illustrates the importance of convergent evolution in protist phylogeny.

Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.

Superficially described and ignored for 92 years, rediscovered and emended: Apodera angatakere (Amoebozoa: Arcellinida: Hyalospheniformes) is a new flagship testate amoeba taxon from Aotearoa (New Zealand).

Eukaryotic microbial diversity is known to be extensive but remains largely undescribed and uncharted. While much of this unknown diversity is composed of inconspicuous flagellates and parasites, larger and morphologically distinct protists are regularly discovered, most notably from poorly studied regions. Here we report a new flagship species of hyalospheniid (Amoebozoa; Arcellinida; Hyalospheniformes) testate amoeba from New Zealand and an unusual story of overlooked description under a preoccupied name and subsequent oversight for nearly one century. Through a process involving The Maori Language Commission, we named the species Apodera angatakere, meaning "a shell with a keel." This species resembles Apodera vas but differs by the presence of a distinctive hollow keel. Cytochrome Oxidase Subunit 1 (COI) sequence data show that this species forms a distinct clade nested within genus Apodera. This conspicuous species is so far known only from New Zealand and is restricted to peatlands. It is one of the few examples of endemic microorganisms from this biodiversity hotspot and biogeographer's paradise. As over 90% of New Zealand's peatlands have been lost since European colonization and much of the remaining surfaces are threatened, Apodera angatakere could be a flagship species not only for microbial biogeography but also for island biodiversity conservation.

The testate amoebae of New Zealand: A checklist, identification key and assessment of biogeographic patterns.

New Zealand (NZ) is a well-known hotspot of biodiversity and endemism for macroscopic organisms, but its microbial diversity is comparatively poorly documented. We assembled all records on NZ testate amoebae published since the early 20th century and present a comprehensive taxonomic checklist for NZ. Testate amoebae are reported from six major habitat types across both the North and South Islands of NZ, but the sampling effort is ecologically and geographically biased in favour of wetlands and the South Island. As a result, 93% of all 128 morphotypes recorded in NZ occur in wetlands, 28% are restricted to the South Island, and diversity is greater at higher latitudes. Around 50% of morphotypes have a broad latitudinal distribution across the NZ mainland, whereas 15% have narrow latitudinal ranges. Future research should aim to broaden the geographical and ecological ranges. We predict that our list of NZ testate amoebae will expand substantially with future work, and that the latitudinal diversity gradient will be inverted. We also introduce an interactive, fully illustrated, online Lucid key for the rapid identification of NZ testate amoebae. As many morphospecies are cosmopolitan, this key provides a useful tool for testate amoebae identification in other parts of the world.

Protist taxonomic and functional diversity in soil, freshwater and marine ecosystems.

Protists dominate eukaryotic diversity and play key functional roles in all ecosystems, particularly by catalyzing carbon and nutrient cycling. To date, however, a comparative analysis of their taxonomic and functional diversity that compares the major ecosystems on Earth (soil, freshwater and marine systems) is missing. Here, we present a comparison of protist diversity based on standardized high throughput 18S rRNA gene sequencing of soil, freshwater and marine environmental DNA. Soil and freshwater protist communities were more similar to each other than to marine protist communities, with virtually no overlap of Operational Taxonomic Units (OTUs) between terrestrial and marine habitats. Soil protists showed higher γ diversity than aquatic samples. Differences in taxonomic composition of the communities led to changes in a functional diversity among ecosystems, as expressed in relative abundance of consumers, phototrophs and parasites. Phototrophs (eukaryotic algae) dominated freshwater systems (49% of the sequences) and consumers soil and marine ecosystems (59% and 48%, respectively). The individual functional groups were composed of ecosystem- specific taxonomic groups. Parasites were equally common in all ecosystems, yet, terrestrial systems hosted more OTUs assigned to parasites of macro-organisms while aquatic systems contained mostly microbial parasitoids. Together, we show biogeographic patterns of protist diversity across major ecosystems on Earth, preparing the way for more focused studies that will help understanding the multiple roles of protists in the biosphere.

Description of Phaeobola aeris gen. nov., sp. nov (Rhizaria, Cercozoa, Euglyphida) Sheds Light on Euglyphida's Dark Matter.

The majority of Euglyphida species are characterised by shells with imbricated silica scales. Environmental surveys indicate a large unexplored diversity and recent efforts hinted at a certain diversity of yet undescribed, inconspicuous, scale-lacking Euglyphida. Here we describe Phaeobola aeris gen. nov., sp. nov. that shows a variety of morphological characters typical for the Euglyphida but lacks silica scales-instead, this species bears an agglutinated test. Neither its morphology nor phylogenetic placement allows its assignment to any currently described family. We erected the yet monospecific genus Phaeobola gen. nov., which with yet available data remain Euglyphida incertae sedis.

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.

Dispersal limitations and historical factors determine the biogeography of specialized terrestrial protists.

Recent studies show that soil eukaryotic diversity is immense and dominated by micro-organisms. However, it is unclear to what extent the processes that shape the distribution of diversity in plants and animals also apply to micro-organisms. Major diversification events in multicellular organisms have often been attributed to long-term climatic and geological processes, but the impact of such processes on protist diversity has received much less attention as their distribution has often been believed to be largely cosmopolitan. Here, we quantified phylogeographical patterns in Hyalosphenia papilio, a large testate amoeba restricted to Holarctic Sphagnum-dominated peatlands, to test if the current distribution of its genetic diversity can be explained by historical factors or by the current distribution of suitable habitats. Phylogenetic diversity was higher in Western North America, corresponding to the inferred geographical origin of the H. papilio complex, and was lower in Eurasia despite extensive suitable habitats. These results suggest that patterns of phylogenetic diversity and distribution can be explained by the history of Holarctic Sphagnum peatland range expansions and contractions in response to Quaternary glaciations that promoted cladogenetic range evolution, rather than the contemporary distribution of suitable habitats. Species distributions were positively correlated with climatic niche breadth, suggesting that climatic tolerance is key to dispersal ability in H. papilio. This implies that, at least for large and specialized terrestrial micro-organisms, propagule dispersal is slow enough that historical processes may contribute to their diversification and phylogeographical patterns and may partly explain their very high overall diversity.

Assessing the ecological value of small testate amoebae (<45 µm) in New Zealand peatlands.

Methodological advances are essential for robust ecological research. Quantitative reconstructions of environmental conditions using testate amoebae rely on sound taxonomy. While the taxonomy of large species is relatively well resolved, this is not the case for most small taxa (typically <45 µm long). In New Zealand, peatlands contain a diversity of both cosmopolitan and characteristic large southern endemic taxa, but also have a high abundance of small taxa. The latter are often lumped into morphotypes reducing their value as ecological indicators. In this study, we demonstrate how (a) lumping small taxa versus splitting them into unique types, and (b) including or excluding them from community analysis influenced their ecological inference. We assessed testate amoeba composition in six peat bogs from New Zealand, three that were moderately-to-highly impacted, and three that were non-impacted. Environmental variables were measured at each sampling site and the surface testate amoeba community patterns and community-environment relationships compared. We found a clear division between impacted and non-impacted sites. Several distinct small taxa were more strongly related to water-table depth and conductivity, while the larger taxa were more correlated to pH. These results show that improved taxonomic resolution of small taxa can provide more informed environmental assessment.

En garde! Redefinition of Nebela militaris (Arcellinida, Hyalospheniidae) and erection of Alabasta gen. nov.

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.

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.