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.

GTRpmix: A Linked General Time-Reversible Model for Profile Mixture Models.

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common matrix of exchangeabilities and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile. Currently, empirically estimated exchangeability matrices (e.g. the LG matrix) are widely used for phylogenetic inference under profile mixture models. However, these were estimated using a single profile and are unlikely optimal for profile mixture models. Here, we describe the GTRpmix model that allows maximum likelihood estimation of a common exchangeability matrix under any profile mixture model. We show that exchangeability matrices estimated under profile mixture models differ from the LG matrix, dramatically improving model fit and topological estimation accuracy for empirical test cases. Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic-supermatrices to be used for phylogenetic analyses. One, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archaeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices, combined with profile mixture models, fit data better and have improved topology estimation relative to the LG matrix combined with the same mixture models. Starting with version 2.3.1, IQ-TREE2 allows users to estimate linked exchangeabilities (i.e. amino acid exchange rates) under profile mixture models.

Poor Diagnostic Reproducibility in the Identification of Nonconventional Dysplasia in Colitis Impacts the Application of Histologic Stratification Tools.

Due to their increased cancer risk, patients with longstanding inflammatory bowel disease are offered endoscopic surveillance with concomitant histopathologic assessments, aimed at identifying dysplasia as a precursor lesion of colitis-associated colorectal cancer. However, this strategy is beset with difficulties and limitations. Recently, a novel classification criterion for colitis-associated low-grade dysplasia has been proposed, and an association between nonconventional dysplasia and progression was reported, suggesting the possibility of histology-based stratification of patients with colitis-associated lesions. Here, a cohort of colitis-associated lesions was assessed by a panel of 6 experienced pathologists to test the applicability of the published classification criteria and try and validate the association between nonconventional dysplasia and progression. While confirming the presence of different morphologic patterns of colitis-associated dysplasia, the study demonstrated difficulties concerning diagnostic reproducibility between pathologists and was unable to validate the association of nonconventional dysplasia with cancer progression. Our study highlights the overall difficulty of using histologic assessment of precursor lesions for cancer risk prediction in inflammatory bowel disease patients and suggests the need for a different diagnostic strategy that can objectively identify high-risk phenotypes.

PhyloFisher: A phylogenomic package for resolving eukaryotic relationships.

Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. However, no software currently exists that includes tools for dataset construction and subsequent analysis with diverse validation strategies to assess robustness. Furthermore, there are no publicly available high-quality curated databases designed to assess deep (>100 million years) relationships in the tree of eukaryotes. To address these issues, we developed an easy-to-use software package, PhyloFisher (https://github.com/TheBrownLab/PhyloFisher), written in Python 3. PhyloFisher includes a manually curated database of 240 protein-coding genes from 304 eukaryotic taxa covering known eukaryotic diversity, a novel tool for ortholog selection, and utilities that will perform diverse analyses required by state-of-the-art phylogenomic investigations. Through phylogenetic reconstructions of the tree of eukaryotes and of the Saccharomycetaceae clade of budding yeasts, we demonstrate the utility of the PhyloFisher workflow and the provided starting database to address phylogenetic questions across a large range of evolutionary time points for diverse groups of organisms. We also demonstrate that undetected paralogy can remain in phylogenomic "single-copy orthogroup" datasets constructed using widely accepted methods such as all vs. all BLAST searches followed by Markov Cluster Algorithm (MCL) clustering and application of automated tree pruning algorithms. Finally, we show how the PhyloFisher workflow helps detect inadvertent paralog inclusions, allowing the user to make more informed decisions regarding orthology assignments, leading to a more accurate final dataset.

Isolation of Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea) from the feces of Ambystoma annulatum (Ringed Salamander) in Northwest Arkansas.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

Living in the cracks: Two novel genera of Variosea (Amoebozoa) discovered on an urban sidewalk.

Biological soil crusts represent a rich habitat for diverse and complex eukaryotic microbial communities. A unique but extremely common habitat is the urban sidewalk and its cracks that collect detritus. While these habitats are ubiquitous across the globe, little to no work has been conducted to characterize protists found there. Amoeboid protists are major predators of bacteria and other microbial eukaryotes in these microhabitats and therefore play a substantial ecological role. From sidewalk crack soil crusts, we have isolated three naked amoebae with finely tapered subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a holistic approach including light, electron, and fluorescence microscopy as well as phylogenetics using the ribosomal small subunit rRNA gene and phylogenomics using 230 nuclear genes, we find that these amoeboid organisms fail to match any previously described eukaryote genus. However, we determined the amoebae belong to the amoebozoan lineage Variosea based on phylogenetics. The molecular analyses place our isolates in two novel genera forming a grade at the base of the variosean group Protosteliida. These three novel varioseans among two novel genera and species are herein named "Kanabo kenzan" and "Parakanabo toge."

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis.

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

Phylogenetic placement of the protosteloid amoeba Microglomus paxillus identifies another case of sporocarpic fruiting in Discosea (Amoebozoa).

Protosteloid amoebae are a paraphyletic assemblage of amoeboid protists found exclusively in the eukaryotic assemblage Amoebozoa. These amoebae can facultatively form a dispersal structure known as a fruiting body, or more specifically, a sporocarp, from a single amoeboid cell. Sporocarps consist of one to a few spores atop a noncellular stalk. Protosteloid amoebae are known in two out of three well-established major assemblages of Amoebozoa. Amoebae with a protosteloid life cycle are known in the major Amoebozoa lineages Discosea and Evosea but not in Tubulinea. To date, only one genus, which is monotypic, lacks sequence data and, therefore, remains phylogenetically homeless. To further clarify the evolutionary milieu of sporocarpic fruiting we used single-cell transcriptomics to obtain data from individual sporocarps of isolates of the protosteloid amoeba Microglomus paxillus. Our phylogenomic analyses using 229 protein coding markers suggest that M. paxillus is a member of the Discosea lineage of Amoebozoa most closely related to Mycamoeba gemmipara. Due to the hypervariable nature of the SSU rRNA sequence we were unable to further resolve the phylogenetic position of M. paxillus in taxon rich datasets using only this marker. Regardless, our results widen the known distribution of sporocarpy in Discosea and stimulate the debate between a single or multiple origins of sporocarpic fruiting in Amoebozoa.

Novel Doublesex Duplication Associated with Sexually Dimorphic Development of Dogface Butterfly Wings.

Sexually dimorphic development is responsible for some of the most remarkable phenotypic variation found in nature. Alternative splicing of the transcription factor gene doublesex (dsx) is a highly conserved developmental switch controlling the expression of sex-specific pathways. Here, we leverage sex-specific differences in butterfly wing color pattern to characterize the genetic basis of sexually dimorphic development. We use RNA-seq, immunolocalization, and motif binding site analysis to test specific predictions about the role of dsx in the development of structurally based ultraviolet (UV) wing patterns in Zerene cesonia (Southern Dogface). Unexpectedly, we discover a novel duplication of dsx that shows a sex-specific burst of expression associated with the sexually dimorphic UV coloration. The derived copy consists of a single exon that encodes a DNA binding but no protein-binding domain and has experienced rapid amino-acid divergence. We propose the novel dsx paralog may suppress UV scale differentiation in females, which is supported by an excess of Dsx-binding sites at cytoskeletal and chitin-related genes with sex-biased expression. These findings illustrate the molecular flexibility of the dsx gene in mediating the differentiation of secondary sexual characteristics.

Environmental Breviatea harbour mutualistic Arcobacter epibionts.

Breviatea form a lineage of free living, unicellular protists, distantly related to animals and fungi. This lineage emerged almost one billion years ago, when the oceanic oxygen content was low, and extant Breviatea have evolved or retained an anaerobic lifestyle. Here we report the cultivation of Lenisia limosa, gen. et sp. nov., a newly discovered breviate colonized by relatives of animal-associated Arcobacter. Physiological experiments show that the association of L. limosa with Arcobacter is driven by the transfer of hydrogen and is mutualistic, providing benefits to both partners. With whole-genome sequencing and differential proteomics, we show that an experimentally observed fitness gain of L. limosa could be explained by the activity of a so far unknown type of NAD(P)H-accepting hydrogenase, which is expressed in the presence, but not in the absence, of Arcobacter. Differential proteomics further reveal that the presence of Lenisia stimulates expression of known 'virulence' factors by Arcobacter. These proteins typically enable colonization of animal cells during infection, but may in the present case act for mutual benefit. Finally, re-investigation of two currently available transcriptomic data sets of other Breviatea reveals the presence and activity of related hydrogen-consuming Arcobacter, indicating that mutualistic interaction between these two groups of microbes might be pervasive. Our results support the notion that molecular mechanisms involved in virulence can also support mutualism, as shown here for Arcobacter and Breviatea.

Ancient Adaptive Lateral Gene Transfers in the Symbiotic Opalina-Blastocystis Stramenopile Lineage.

Lateral gene transfer is a very common process in bacterial and archaeal evolution, playing an important role in the adaptation to new environments. In eukaryotes, its role and frequency remain highly debated, although recent research supports that gene transfer from bacteria to diverse eukaryotes may be much more common than previously appreciated. However, most of this research focused on animals and the true phylogenetic and functional impact of bacterial genes in less-studied microbial eukaryotic groups remains largely unknown. Here, we have analyzed transcriptome data from the deep-branching stramenopile Opalinidae, common members of frog gut microbiomes, and distantly related to the well-known genus Blastocystis. Phylogenetic analyses suggest the early acquisition of several bacterial genes in a common ancestor of both lineages. Those lateral gene transfers most likely facilitated the adaptation of the free-living ancestor of the Opalinidae-Blastocystis symbiotic group to new niches in the oxygen-depleted animal gut environment.

EukRef: Phylogenetic curation of ribosomal RNA to enhance understanding of eukaryotic diversity and distribution.

Environmental sequencing has greatly expanded our knowledge of micro-eukaryotic diversity and ecology by revealing previously unknown lineages and their distribution. However, the value of these data is critically dependent on the quality of the reference databases used to assign an identity to environmental sequences. Existing databases contain errors and struggle to keep pace with rapidly changing eukaryotic taxonomy, the influx of novel diversity, and computational challenges related to assembling the high-quality alignments and trees needed for accurate characterization of lineage diversity. EukRef (eukref.org) is an ongoing community-driven initiative that addresses these challenges by bringing together taxonomists with expertise spanning the eukaryotic tree of life and microbial ecologists, who use environmental sequence data to develop reliable reference databases across the diversity of microbial eukaryotes. EukRef organizes and facilitates rigorous mining and annotation of sequence data by providing protocols, guidelines, and tools. The EukRef pipeline and tools allow users interested in a particular group of microbial eukaryotes to retrieve all sequences belonging to that group from International Nucleotide Sequence Database Collaboration (INSDC) (GenBank, the European Nucleotide Archive [ENA], or the DNA DataBank of Japan [DDBJ]), to place those sequences in a phylogenetic tree, and to curate taxonomic and environmental information for the group. We provide guidelines to facilitate the process and to standardize taxonomic annotations. The final outputs of this process are (1) a reference tree and alignment, (2) a reference sequence database, including taxonomic and environmental information, and (3) a list of putative chimeras and other artifactual sequences. These products will be useful for the broad community as they become publicly available (at eukref.org) and are shared with existing reference databases.

An efficient single-cell transcriptomics workflow for microbial eukaryotes benchmarked on Giardia intestinalis cells.

BACKGROUND: Most diversity in the eukaryotic tree of life is represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, some protists remain difficult to maintain in cultures, which further influences what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists is often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed. RESULTS: In this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we reduced the reaction volume and purified the amplified cDNA with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and reducing the volumes by half led to significantly fewer genes detected. We also added a 5' biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency. CONCLUSIONS: The presented single-cell RNA sequencing workflow represents an efficient method to explore the diversity and cell biology of individual protist cells.

Integrated analysis of multiple receptor tyrosine kinases identifies Axl as a therapeutic target and mediator of resistance to sorafenib in hepatocellular carcinoma.

BACKGROUND: Aberrant activation of Axl is implicated in the progression of hepatocellular carcinoma (HCC). We explored the biologic significance and preclinical efficacy of Axl inhibition as a therapeutic strategy in sorafenib-naive and resistant HCC. METHODS: We evaluated Axl expression in sorafenib-naive and resistant (SR) clones of epithelial (HuH7) and mesenchymal origin (SKHep-1) using antibody arrays and confirmed tissue expression. We tested the effect of Axl inhibition with RNA-interference and pharmacologically with R428 on a number of phenotypic assays. RESULTS: Axl mRNA overexpression in cell lines (n = 28) and RNA-seq tissue datasets (n = 373) correlated with epithelial-to-mesenchymal transition (EMT). Axl was overexpressed in HCC compared to cirrhosis and normal liver. We confirmed sorafenib resistance to be associated with EMT and enhanced motility in both HuH7-SR and SKHep-1-SR cells documenting a 4-fold increase in Axl phosphorylation as an adaptive feature of chronic sorafenib treatment in SKHep-1-SR cells. Axl inhibition reduced motility and enhanced sensitivity to sorafenib in SKHep-1SR cells. In patients treated with sorafenib (n = 40), circulating Axl levels correlated with shorter survival. CONCLUSIONS: Suppression of Axl-dependent signalling influences the transformed phenotype in HCC cells and contributes to adaptive resistance to sorafenib, providing a pre-clinical rationale for the development of Axl inhibitors as a measure to overcome sorafenib resistance.

Description of Armaparvus languidus n. gen. n. sp. Confirms Ultrastructural Unity of Cutosea (Amoebozoa, Evosea).

The American Type Culture Collection (ATCC) PRA-29 isolate has a publicly available transcriptome, which has led to its inclusion in recent phylogenomic analyses. The ATCC PRA-29 isolate was originally identified and deposited as "Pessonella sp." This taxon branches robustly within the recently discovered clade Cutosea, very distantly related to the clade in which the genus Pessonella is believed to branch based on morphological data. Using detailed light and electron microscopy, we studied the morphology and ultrastructure of ATCC PRA-29 as well as other cutosean amoebae to better elucidate the morphological affinity of ATCC PRA-29 to other amoebozoans. Here, we show that ATCC PRA-29 was misidentified by the original depositor as Pessonella and name it Armaparvus languidus n. gen. n. sp. We show that a cell coat of microscales separated from the cell membrane is a unique trait found in all known cutosean amoebae. As Cutosea represents a clade at the deepest bifurcation in the amoebozoan group Evosea and because this clade is currently taxon-poor, but likely represents a major understudied group it will be important to isolate and describe more cutosean amoebae in the future.

A Non-Flagellated Member of the Myxogastria and Expansion of the Echinosteliida.

Myxogastria (also called Myxomycetes or plasmodial slime-moulds) are mostly known through their usually conspicuous fruiting bodies. Another unifying trait is the presence of a facultative flagellate stage along with the obligate amoeboid stage. Here we show with two-gene phylogenies (SSU rRNA and EF-1alpha genes) that the incertae sedis, non-flagellate Echinosteliopsis oligospora belongs to the dark-spore clade (Fuscisporidia) of the Myxogastria. In addition, we confirm that Echinostelium bisporum, firstly described as a protostelid, belongs to the Echinosteliida, which are divided into three major clades and are paraphyletic to the remaining Fuscisporidia.

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.

Between a Pod and a Hard Test: The Deep Evolution of Amoebae.

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

Coprophilic amoebae and flagellates, including Guttulinopsis, Rosculus and Helkesimastix, characterise a divergent and diverse rhizarian radiation and contribute to a large diversity of faecal-associated protists.

A wide diversity of organisms utilize faecal habitats as a rich nutrient source or a mechanism to traverse through animal hosts. We sequenced the 18S rRNA genes of the coprophilic, fruiting body-forming amoeba Guttulinopsis vulgaris and its non-fruiting relatives Rosculus 'ithacus' CCAP 1571/3, R. terrestris n. sp. and R. elongata n. sp. and demonstrate that they are related to the coprophilic flagellate Helkesimastix in a strongly supported, but highly divergent 18S sister clade. PCR primers specific to both clades were used to generate 18S amplicons from a range of environmental and faecal DNA samples. Phylogenetic analysis of the cloned sequences demonstrated a high diversity of uncharacterised sequence types within this clade, likely representing previously described members of the genera Guttulinopsis, Rosculus and Helkesimastix, as well as so-far unobserved organisms. Further, an Illumina MiSeq sequenced set of 18S V4-region amplicons generated from faecal DNAs using universal eukaryote primers showed that core-cercozoan assemblages in faecal samples are as diverse as those found in more conventionally examined habitats. These results reveal many novel lineages, some of which appear to occur preferentially in faecal material, in particular cercomonads and glissomonads. More broadly, we show that faecal habitats are likely untapped reservoirs of microbial eukaryotic diversity.