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.

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea).

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies.

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.

A comparative study indicates vertical inheritance and horizontal gene transfer of arsenic resistance-related genes in eukaryotes.

Arsenic is a ubiquitous element in the environment, a source of constant evolutionary pressure on organisms. The arsenic resistance machinery is thoroughly described for bacteria. Highly resistant lineages are also common in eukaryotes, but evolutionary knowledge is much more limited. While the origin of the resistance machinery in eukaryotes is loosely attributed to horizontal gene transfer (HGT) from bacteria, only a handful of eukaryotes were deeply studied. Here we investigate the origin and evolution of the core genes in arsenic resistance in eukaryotes using a broad phylogenetic framework. We hypothesize that, as arsenic pressure is constant throughout Earth's history, resistance mechanisms are probably ancestral to eukaryotes. We identified homologs for each of the arsenic resistance genes in eukaryotes and traced their possible origin using phylogenetic reconstruction. We reveal that: i. an important component of the arsenic-resistant machinery originated before the last eukaryotic common ancestor; ii. later events of gene duplication and HGT generated new homologs that, in many cases, replaced ancestral ones. Even though HGT has an important contribution to the expansion of arsenic metabolism in eukaryotes, we propose the hypothesis of ancestral origin and differential retention of arsenic resistance mechanisms in the group. Key-words: Environmental adaptation; resistance to toxic metalloids; detoxification; comparative genomics; functional phylogenomics.

Exploring the protist microbiome: The diversity of bacterial communities associated with Arcella spp. (Tubulina: Amoebozoa).

Research on protist-bacteria interactions is increasingly relevant as these associations are now known to play important roles in ecosystem and human health. Free-living amoebae are abundant in all environments and are frequent hosts for bacterial endosymbionts including pathogenic bacteria. However, to date, only a small fraction of these symbionts have been identified, while the structure and composition of the total symbiotic bacterial communities still remains largely unknown. Here, we use the testate amoeba Arcella spp. as model organisms to investigate the specificity and diversity of Arcella-associated microbial communities. High-throughputamplicon sequencing from the V4 region of the 16S rRNA gene revealed high diversity in the bacterial communities associated with the wild Arcella spp. To investigate the specificity of the associated bacterial community with greater precision, we investigated the bacterial communities of two lab-cultured Arcella species, A. hemispherica and A. intermedia, grown in two different media types. Our results suggest that Arcella-bacteria associations are species-specific, and that the associated bacterial community of lab-cultured Arcella spp. remains distinct from that of the surrounding media. Further, each host Arcella species could be distinguished based on its bacterial composition. Our findings provide insight into the understanding of eukaryotic-bacterial symbiosis.

Population and molecular responses to warming in Netzelia tuberspinifera - An endemic and sensitive protist from East Asia.

With the effects of global warming becoming ever more obvious, biodiversity conservation is facing severe challenges. Currently, a deeper understanding the mechanisms of the effects of warming on sensitive species has become an important topic in aquatic biodiversity and ecological management. Our study first overcame the "challenge" for a sensitive indicator species (Netzelia tuberspinifera, an endemic testate amoeba species in East Asia) of culturing under laboratory conditions, and then explored its molecular response mechanisms to warming using transcriptomic analysis. Our data indicate that temperature mainly drove the geographical and seasonal variation of N. tuberspinifera populations. Transcriptomic results indicate that when the temperature is <25 °C, rising temperature triggers the biosynthesis of ribosomes; while the temperature is >25 °C, it triggers molecular processes related with cell division, test formation and general biomass increase. However, once the temperature exceeds 40 °C, N. tuberspinifera is unable to survive. Following from these results, the distribution of N. tuberspinifera might expand towards higher altitude or latitude regions under global warming. For the first time, our study showed direct evidence for sensitive protozoa species that presents a very narrow adaptation mechanism to local climate. Our work provides fundamental data for regional biodiversity conservation and scientific reference in subtropical and tropical waterbodies.

The integrin-mediated adhesive complex in the ancestor of animals, fungi, and amoebae.

Integrins are transmembrane receptors that activate signal transduction pathways upon extracellular matrix binding. The integrin-mediated adhesive complex (IMAC) mediates various cell physiological processes. Although the IMAC was thought to be specific to animals, in the past ten years these complexes were discovered in other lineages of Obazoa, the group containing animals, fungi, and several microbial eukaryotes. Very recently, many genomes and transcriptomes from Amoebozoa (the eukaryotic supergroup sister to Obazoa), other obazoans, orphan protist lineages, and the eukaryotes' closest prokaryotic relatives, have become available. To increase the resolution of where and when IMAC proteins exist and have emerged, we surveyed these newly available genomes and transcriptomes for the presence of IMAC proteins. Our results highlight that many of these proteins appear to have evolved earlier in eukaryote evolution than previously thought and that co-option of this apparently ancient protein complex was key to the emergence of animal-type multicellularity. The role of the IMACs in amoebozoans is unknown, but they play critical adhesive roles in at least some unicellular organisms.

Complex Evolution of the Mismatch Repair System in Eukaryotes is Illuminated by Novel Archaeal Genomes.

Repairing DNA damage is one of the most important functions of the 'housekeeping' proteins, as DNA molecules are constantly subject to different kinds of damage. An important mechanism of DNA repair is the mismatch repair system (MMR). In eukaryotes, it is more complex than it is in bacteria or Archaea due to an inflated number of paralogues produced as a result of an extensive process of gene duplication and further specialization upon the evolution of the first eukaryotes, including an important part of the meiotic machinery. Recently, the discovery and sequencing of Asgard Archaea allowed us to revisit the MMR system evolution with the addition of new data from a group that is closely related to the eukaryotic ancestor. This new analysis provided evidence for a complex evolutionary history of eukaryotic MMR: an archaeal origin for the nuclear MMR system in eukaryotes, with subsequent acquisitions of other MMR systems from organelles.

Comparative Characterization of Mitogenomes From Five Orders of Cestodes (Eucestoda: Tapeworms).

The recognized potential of using mitogenomics in phylogenetics and the more accessible use of high-throughput sequencing (HTS) offer an opportunity to investigate groups of neglected organisms. Here, we leveraged HTS to execute the most comprehensive documentation of mitogenomes for cestodes based on the number of terminals sequenced. We adopted modern approaches to obtain the complete mitogenome sequences of 86 specimens representing five orders of cestodes (three reported for the first time: Phyllobothriidea, "Tetraphyllidea" and Trypanorhyncha). These complete mitogenomes represent an increase of 41% of the mitogenomes available for cestodes (61-147) and an addition of 33% in the representativeness of the cestode orders. The complete mitochondrial genomes are conserved, circular, encoded in the same strand, and transcribed in the same direction, following the pattern observed previously for tapeworms. Their length varies from 13,369 to 13,795 bp, containing 36 genes in total. Except for the Trypanorhyncha specimen, the gene order of the other four cestode orders sequenced here suggests that it could be a synapomorphy for the acetabulate group (with a reversion for taenids). Our results also suggest that no single gene can tell all the evolutionary history contained in the mitogenome. Therefore, cestodes phylogenies based on a single mitochondrial marker may fail to capture their evolutionary history. We predict that such phylogenies would be improved if conducted under a total evidence framework. The characterization of the new mitochondrial genomes is the first step to provide a valuable resource for future studies on the evolutionary relationships of these groups of parasites.

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.

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.

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.

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.

Genome skimming is a low-cost and robust strategy to assemble complete mitochondrial genomes from ethanol preserved specimens in biodiversity studies.

Global loss of biodiversity is an ongoing process that concerns both local and global authorities. Studies of biodiversity mainly involve traditional methods using morphological characters and molecular protocols. However, conventional methods are a time consuming and resource demanding task. The development of high-throughput sequencing (HTS) techniques has reshaped the way we explore biodiversity and opened a path to new questions and novel empirical approaches. With the emergence of HTS, sequencing the complete mitochondrial genome became more accessible, and the number of genome sequences published has increased exponentially during the last decades. Despite the current state of knowledge about the potential of mitogenomics in phylogenetics, this is still a relatively under-explored area for a multitude of taxonomic groups, especially for those without commercial relevance, non-models organisms and with preserved DNA. Here we take the first step to assemble and annotate the genomes from HTS data using a new protocol of genome skimming which will offer an opportunity to extend the field of mitogenomics to under-studied organisms. We extracted genomic DNA from specimens preserved in ethanol. We used Nextera XT DNA to prepare indexed paired-end libraries since it is a powerful tool for working with diverse samples, requiring a low amount of input DNA. We sequenced the samples in two different Illumina platform (MiSeq or NextSeq 550). We trimmed raw reads, filtered and had their quality tested accordingly. We performed the assembly using a baiting and iterative mapping strategy, and the annotated the putative mitochondrion through a semi-automatic procedure. We applied the contiguity index to access the completeness of each new mitogenome. Our results reveal the efficiency of the proposed method to recover the whole mitogenomes of preserved DNA from non-model organisms even if there are gene rearrangement in the specimens. Our findings suggest the potential of combining the adequate platform and library to the genome skimming as an innovative approach, which opens a new range of possibilities of its use to obtain molecular data from organisms with different levels of preservation.

All Eukaryotes Are Sexual, unless Proven Otherwise: Many So-Called Asexuals Present Meiotic Machinery and Might Be Able to Have Sex.

Here a wide distribution of meiotic machinery is shown, indicating the occurrence of sexual processes in all major eukaryotic groups, without exceptions, including the putative "asexuals." Meiotic machinery has evolved from archaeal DNA repair machinery by means of ancestral gene duplications. Sex is very conserved and widespread in eukaryotes, even though its evolutionary importance is still a matter of debate. The main processes in sex are plasmogamy, followed by karyogamy and meiosis. Meiosis is fundamentally a chromosomal process, which implies recombination and ploidy reduction. Several eukaryotic lineages are proposed to be asexual because their sexual processes are never observed, but presumed asexuality correlates with lack of study. The authors stress the complete lack of meiotic proteins in nucleomorphs and their almost complete loss in the fungus Malassezia. Inversely, complete sets of meiotic proteins are present in fungal groups Glomeromycotina, Trichophyton, and Cryptococcus. Endosymbiont Perkinsela and endoparasitic Microsporidia also present meiotic proteins.

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.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Reinvestigation of Phryganella paradoxa (Arcellinida, Amoebozoa) Penard 1902.

A major drawback in testate amoeba research is a general lack of scientific studies combining molecular approaches and classical laboratory experiments. We isolated five yet uncultured testate amoebae of the genus Phryganella Penard, 1902 from three different rivers and one pond in Germany. Based on established cultures we show their morphology, which we studied by light and electron microscopy, and present their unique feeding mode on abundant and common pennate diatoms like Nitzschia spp. and Synedra spp., whose frustules are bent and frequently, but not always, broken during the feeding process. We further obtained the first SSU rDNA sequences of strains of the family Phryganellidae, all of which contain introns. We used the sequences to confirm the taxonomic placement of the Phryganellidae in the Arcellinida (Amoebozoa), branching as a sister group to the Cryptodifflugiidae.

Comparative Genomics Supports Sex and Meiosis in Diverse Amoebozoa.

Sex and reproduction are often treated as a single phenomenon in animals and plants, as in these organisms reproduction implies mixis and meiosis. In contrast, sex and reproduction are independent biological phenomena that may or may not be linked in the majority of other eukaryotes. Current evidence supports a eukaryotic ancestor bearing a mating type system and meiosis, which is a process exclusive to eukaryotes. Even though sex is ancestral, the literature regarding life cycles of amoeboid lineages depicts them as asexual organisms. Why would loss of sex be common in amoebae, if it is rarely lost, if ever, in plants and animals, as well as in fungi? One way to approach the question of meiosis in the "asexuals" is to evaluate the patterns of occurrence of genes for the proteins involved in syngamy and meiosis. We have applied a comparative genomic approach to study the occurrence of the machinery for plasmogamy, karyogamy, and meiosis in Amoebozoa, a major amoeboid supergroup. Our results support a putative occurrence of syngamy and meiotic processes in all major amoebozoan lineages. We conclude that most amoebozoans may perform mixis, recombination, and ploidy reduction through canonical meiotic processes. The present evidence indicates the possibility of sexual cycles in many lineages traditionally held as asexual.

Topological assessment of metabolic networks reveals evolutionary information.

Evolutionary information was inferred from the topology of metabolic networks corresponding to 17 plant species belonging to major plant lineages Chlorophytes, Bryophytes, Lycophytes and Angiosperms. The plant metabolic networks were built using the substrate-product network modeling based on the metabolic reactions available on the PlantCyc database (version 9.5), from which their local topological properties such as degree, in-degree, out-degree, clustering coefficient, hub-score, authority-score, local efficiency, betweenness and eigencentrality were measured. The topological measurements corresponding to each metabolite within the networks were considered as a set of metabolic characters to compound a feature vector representing each plant. Our results revealed that some local topological characters are able to discern among plant kinships, since similar phylogenies were found when comparing dendrograms obtained by topological metrics to the one obtained by DNA sequences of chloroplast genes. Furthermore, we also found that even a smaller number of metabolic characters is able to separate among major clades with high bootstrap support (BS > 95), while for some suborders a bigger content has been required.