Amoebozoan testate amoebae illuminate the diversity of heterotrophs and the complexity of ecosystems throughout geological time.

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

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.

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.

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.

Phylogenetic reconstruction and evolution of the Rab GTPase gene family in Amoebozoa.

Rab GTPase is a paralog-rich gene family that controls the maintenance of the eukaryotic cell compartmentalization system. Diverse eukaryotes have varying numbers of Rab paralogs. Currently, little is known about the evolutionary pattern of Rab GTPase in most major eukaryotic 'supergroups'. Here, we present a comprehensive phylogenetic reconstruction of the Rab GTPase gene family in the eukaryotic 'supergroup' Amoebozoa, a diverse lineage represented by unicellular and multicellular organisms. We demonstrate that Amoebozoa conserved 20 of the 23 ancestral Rab GTPases predicted to be present in the last eukaryotic common ancestor and massively expanded several 'novel' in-paralogs. Due to these 'novel' in-paralogs, the Rab family composition dramatically varies between the members of Amoebozoa; as a consequence, 'supergroup'-based studies may significantly change our current understanding of the evolution and diversity of this gene family. The high diversity of the Rab GTPase gene family in Amoebozoa makes this 'supergroup' a key lineage to study and advance our knowledge of the evolution of Rab in Eukaryotes.

The Sexual Ancestor of all Eukaryotes: A Defense of the "Meiosis Toolkit": A Rigorous Survey Supports the Obligate Link between Meiosis Machinery and Sexual Recombination.

The distribution pattern of the meiotic machinery in known eukaryotes is most parsimoniously explained by the hypothesis that all eukaryotes are ancestrally sexual. However, this assumption is questioned by preliminary results, in culture conditions. These suggested that Acanthamoeba, an organism considered to be largely asexual, constitutively expresses meiosis genes nevertheless-at least in the lab. This apparent disconnect between the "meiosis toolkit" and sexual processes in Acanthamoeba led to the conclusion that the eukaryotic ancestor is asexual. In this review, the "meiosis toolkit" is rigorously defended, drawing on numerous research articles. Additionally, the claim of constitutive meiotic gene expression is probed in Acanthamoeba via the same transcriptomics data. The results show that the expression of the meiotic machinery is not constitutive in Acanthamoeba as claimed before. Furthermore, it is argued that this would have no implications for understanding the nature of the eukaryotic ancestor, regardless of the result.

De novo Sequencing, Assembly, and Annotation of the Transcriptome for the Free-Living Testate Amoeba Arcella intermedia.

Arcella, a diverse understudied genus of testate amoebae is a member of Tubulinea in Amoebozoa group. Transcriptomes are a powerful tool for characterization of these organisms as they are an efficient way of characterizing the protein-coding potential of the genome. In this work, we employed both single-cell and clonal populations transcriptomics to create a reference transcriptome for Arcella. We compared our results with annotations of Dictyostelium discoideum, a model Amoebozoan. We assembled a pool of 38 Arcella intermedia transcriptomes, which after filtering are composed of a total of 14,712 translated proteins. There are GO categories enriched in Arcella including mainly intracellular signal transduction pathways; we also used KEGG to annotate 11,546 contigs, which also have similar distribution to Dictyostelium. A large portion of data is still impossible to assign to a gene family, probably due to a combination of lineage-specific genes, incomplete sequences in the transcriptome and rapidly evolved genes. Some absences in pathways could also be related to low expression of these genes. We provide a reference database for Arcella, and we highlight the emergence of the need for further gene discovery in Arcella.

Phylogenomics and Morphological Reconstruction of Arcellinida Testate Amoebae Highlight Diversity of Microbial Eukaryotes in the Neoproterozoic.

Life was microbial for the majority of Earth's history, but as very few microbial lineages leave a fossil record, the Precambrian evolution of life remains shrouded in mystery. Shelled (testate) amoebae stand out as an exception with rich documented diversity in the Neoproterozoic as vase-shaped microfossils (VSMs). While there is general consensus that most of these can be attributed to the Arcellinida lineage in Amoebozoa, it is still unclear whether they can be used as key fossils for interpretation of early eukaryotic evolution. Here, we present a well-resolved phylogenomic reconstruction based on 250 genes, obtained using single-cell transcriptomic techniques from a representative selection of 19 Arcellinid testate amoeba taxa. The robust phylogenetic framework enables deeper interpretations of evolution in this lineage and demanded an updated classification of the group. Additionally, we performed reconstruction of ancestral morphologies, yielding hypothetical ancestors remarkably similar to existing Neoproterozoic VSMs. We demonstrate that major lineages of testate amoebae were already diversified before the Sturtian glaciation (720 mya), supporting the hypothesis that massive eukaryotic diversification took place in the early Neoproterozoic and congruent with the interpretation that VSM are arcellinid testate amoebae.

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.