Create, Analyze, and Visualize Phylogenomic Datasets Using PhyloFisher.

PhyloFisher is a software package written primarily in Python3 that can be used for the creation, analysis, and visualization of phylogenomic datasets that consist of protein sequences from eukaryotic organisms. Unlike many existing phylogenomic pipelines, PhyloFisher comes with a manually curated database of 240 protein-coding genes, a subset of a previous phylogenetic dataset sampled from 304 eukaryotic taxa. The software package can also utilize a user-created database of eukaryotic proteins, which may be more appropriate for shallow evolutionary questions. PhyloFisher is also equipped with a set of utilities to aid in running routine analyses, such as the prediction of alternative genetic codes, removal of genes and/or taxa based on occupancy/completeness of the dataset, testing for amino acid compositional heterogeneity among sequences, removal of heterotachious and/or fast-evolving sites, removal of fast-evolving taxa, supermatrix creation from randomly resampled genes, and supermatrix creation from nucleotide sequences. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Constructing a phylogenomic dataset Basic Protocol 2: Performing phylogenomic analyses Support Protocol 1: Installing PhyloFisher Support Protocol 2: Creating a custom phylogenomic database.

Encyclopedia of Family A DNA Polymerases Localized in Organelles: Evolutionary Contribution of Bacteria Including the Proto-Mitochondrion.

DNA polymerases synthesize DNA from deoxyribonucleotides in a semiconservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are 2 genome-containing organelles, mitochondria, and plastids, which were derived from an alphaproteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNA polymerases that localize and work in them to maintain their genomes. The evolution of organellar DNA polymerases has yet to be fully understood because of 2 unsettled issues. First, the diversity of organellar DNA polymerases has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNA polymerases that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNA polymerases known to date show no phylogenetic affinity to those of the extant alphaproteobacteria or cyanobacteria. In this study, we identified from diverse eukaryotes 134 family A DNA polymerase sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNA polymerases were further examined experimentally. The results presented here suggest that the diversity of organellar DNA polymerases has been shaped by multiple transfers of the PolI gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed 2 mitochondrial DNA polymerases, POP, and a candidate of the direct descendant of the proto-mitochondrial DNA polymerase I, rdxPolA, identified in this study.

Genomics of Preaxostyla Flagellates Illuminates the Path Towards the Loss of Mitochondria.

The notion that mitochondria cannot be lost was shattered with the report of an oxymonad Monocercomonoides exilis, the first eukaryote arguably without any mitochondrion. Yet, questions remain about whether this extends beyond the single species and how this transition took place. The Oxymonadida is a group of gut endobionts taxonomically housed in the Preaxostyla which also contains free-living flagellates of the genera Trimastix and Paratrimastix. The latter two taxa harbour conspicuous mitochondrion-related organelles (MROs). Here we report high-quality genome and transcriptome assemblies of two Preaxostyla representatives, the free-living Paratrimastix pyriformis and the oxymonad Blattamonas nauphoetae. We performed thorough comparisons among all available genomic and transcriptomic data of Preaxostyla to further decipher the evolutionary changes towards amitochondriality, endobiosis, and unstacked Golgi. Our results provide insights into the metabolic and endomembrane evolution, but most strikingly the data confirm the complete loss of mitochondria for all three oxymonad species investigated (M. exilis, B. nauphoetae, and Streblomastix strix), suggesting the amitochondriate status is common to a large part if not the whole group of Oxymonadida. This observation moves this unique loss to 100 MYA when oxymonad lineage diversified.

Hydrocytium expands the phylogenetic, morphological, and genomic diversity of the poorly known green algal order Chaetopeltidales.

PREMISE: Chaetopeltidales is a small, understudied order of the green algal class Chlorophyceae, that is slowly expanding with the occasional discoveries of novel algae. Here we demonstrate that hitherto unrecognized chaetopeltidaleans also exist among previously described but neglected and misclassified species. METHODS: Strain SAG 40.91 of Characium acuminatum, shown by previous preliminary evidence to have affinities with the orders Oedogoniales, Chaetophorales, and Chaetopeltidales (together constituting the OCC clade), was investigated with light and electron microscopy to characterize its morphology and ultrastructure. Sequence assemblies of the organellar and nuclear genomes were obtained and utilized in bioinformatic and phylogenetic analyses to address the phylogenetic position of the alga and its salient genomic features. RESULTS: The characterization of strain SAG 40.91 and a critical literature review led us to reinstate the forgotten genus Hydrocytium A.Braun 1855, with SAG 40.91 representing its type species, Hydrocytium acuminatum. Independent molecular markers converged on placing H. acuminatum as a deeply diverged lineage of the order Chaetopeltidales, formalized as the new family Hydrocytiaceae. Both chloroplast and mitochondrial genomes shared characteristics with other members of Chaetopeltidales and were bloated by repetitive sequences. Notably, the mitochondrial cox2a gene was transferred into the nuclear genome in the H. acuminatum lineage, independently of the same event in Volvocales. The nuclear genome data from H. acuminatum and from another chaetopeltidalean that was reported by others revealed endogenized viral sequences corresponding to novel members of the phylum Nucleocytoviricota. CONCLUSIONS: The resurrected genus Hydrocytium expands the known diversity of chaetopeltidalean algae and provides the first glimpse into their virosphere.

A Novel Group of Dynamin-Related Proteins Shared by Eukaryotes and Giant Viruses Is Able to Remodel Mitochondria From Within the Matrix.

The diverse GTPases of the dynamin superfamily play various roles in the cell, as exemplified by the dynamin-related proteins (DRPs) Mgm1 and Opa1, which remodel the mitochondrial inner membrane in fungi and metazoans, respectively. Via an exhaustive search of genomic and metagenomic databases, we found previously unknown DRP types occurring in diverse eukaryotes and giant viruses (phylum Nucleocytoviricota). One novel DRP clade, termed MidX, combined hitherto uncharacterized proteins from giant viruses and six distantly related eukaryote taxa (Stramenopiles, Telonemia, Picozoa, Amoebozoa, Apusomonadida, and Choanoflagellata). MidX stood out because it was not only predicted to be mitochondria-targeted but also to assume a tertiary structure not observed in other DRPs before. To understand how MidX affects mitochondria, we exogenously expressed MidX from Hyperionvirus in the kinetoplastid Trypanosoma brucei, which lacks Mgm1 or Opa1 orthologs. MidX massively affected mitochondrial morphology from inside the matrix, where it closely associates with the inner membrane. This unprecedented mode of action contrasts to those of Mgm1 and Opa1, which mediate inner membrane remodeling in the intermembrane space. We speculate that MidX was acquired in Nucleocytoviricota evolution by horizontal gene transfer from eukaryotes and is used by giant viruses to remodel host mitochondria during infection. MidX's unique structure may be an adaptation for reshaping mitochondria from the inside. Finally, Mgm1 forms a sister group to MidX and not Opa1 in our phylogenetic analysis, throwing into question the long-presumed homology of these DRPs with similar roles in sister lineages.

A single cryptomonad cell harbors a complex community of organelles, bacteria, a phage, and selfish elements.

Symbiosis between prokaryotes and microbial eukaryotes (protists) has broadly impacted both evolution and ecology. Endosymbiosis led to mitochondria and plastids, the latter spreading across the tree of eukaryotes by subsequent rounds of endosymbiosis. Present-day endosymbionts in protists remain both common and diverse, although what function they serve is often unknown. Here, we describe a highly complex community of endosymbionts and a bacteriophage (phage) within a single cryptomonad cell. Cryptomonads are a model for organelle evolution because their secondary plastid retains a relict endosymbiont nucleus, but only one previously unidentified Cryptomonas strain (SAG 25.80) is known to harbor bacterial endosymbionts. We carried out electron microscopy and FISH imaging as well as genomic sequencing on Cryptomonas SAG 25.80, which revealed a stable, complex community even after over 50 years in continuous cultivation. We identified the host strain as Cryptomonas gyropyrenoidosa, and sequenced genomes from its mitochondria, plastid, and nucleomorph (and partially its nucleus), as well as two symbionts, Megaira polyxenophila and Grellia numerosa, and one phage (MAnkyphage) infecting M. polyxenophila. Comparing closely related endosymbionts from other hosts revealed similar metabolic and genomic features, with the exception of abundant transposons and genome plasticity in M. polyxenophila from Cryptomonas. We found an abundance of eukaryote-interacting genes as well as many toxin-antitoxin systems, including in the MAnkyphage genome that also encodes several eukaryotic-like proteins. Overall, the Cryptomonas cell is an endosymbiotic conglomeration with seven distinct evolving genomes that all show evidence of inter-lineage conflict but nevertheless remain stable, even after more than 4,000 generations in culture.

Redefining Chlorobotryaceae as one of the principal and most diverse lineages of eustigmatophyte algae.

Eustigmatophyceae is one of the ∼17 classes of the vast algal phylum Ochrophyta. Over the last decade, the eustigmatophytes emerged as an expansive group that has grown from the initially recognized handful of species to well over 200 genetically distinct entities (potential species). Yet the majority of eustigs, remain represented by unidentified strains, or even only metabarcode sequences obtained from environmental samples. Moreover, the formal classification of the group has not yet been harmonized with the recently uncovered diversity and phylogenetic relationships within the class. Here we make a major step towards resolving this issue by addressing the diversity, phylogeny and classification of one of the most prominent eustigmatophyte clades previously informally called the "Eustigmataceae group". We obtained 18S rDNA and rbcL gene sequences from four new strains from the "Eustigmataceae group", and from several additional eustig strains, and performed the most comprehensive phylogenetic analyses of Eustigmatophyceae to date. Our results of these analyses confirm the monophyly of the "Eustigmataceae group" and define its major subclades. We also sequenced plastid genomes of five "Eustigmataceae group" strains to not only improve our understanding of the plastid gene content evolution in eustigs, but also to obtain a robustly resolved eustigmatophyte phylogeny. With this new genomic data, we have solidified the view of the "Eustigmataceae group" as a well-defined family level clade. Crucially, we also have firmly established the genus Chlorobotrys as a member of the "Eustigmataceae group". This new molecular evidence, together with a critical analysis of the literature going back to the 19th century, provided the basis to radically redefine the historical concept of the family Chlorobotryaceae as the formal taxonomic rubric corresponding to the "Eustigmataceae group". With this change, the family names Eustigmataceae and Characiopsidaceae are reduced to synonymy with the Chlorobotryaceae, with the latter having taxonomic priority. We additionally studied in detail the morphology and ultrastructure of two Chlorobotryaceae members, which we describe as Neustupella aerophytica gen. et sp. nov. and Lietzensia polymorpha gen. et sp. nov. Finally, our analyses of partial genomic data from several Chlorobotryaceae representatives identified genes for hallmark flagellar proteins in all of these strains. The presence of the flagellar proteins strongly suggests that zoosporogenesis is a common trait of the family and also occurs in the members never observed to produce flagellated stages. Altogether, our work paints a rich picture of one of the most diverse principal lineages of eustigmatophyte algae.

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates.

Fornicata, a lineage of a broader and ancient anaerobic eukaryotic clade Metamonada, contains diverse taxa that are ideally suited for evolutionary studies addressing various fundamental biological questions, such as the evolutionary trajectory of mitochondrion-related organelles (MROs), the transition between free-living and endobiotic lifestyles, and the derivation of alternative genetic codes. To this end, we conducted detailed microscopic and transcriptome analyses in a poorly documented strain of an anaerobic free-living marine flagellate, PCS, in the so-called CL3 fornicate lineage. Fortuitously, we discovered that the original culture contained two morphologically similar and closely related CL3 representatives, which doubles the taxon representation within this lineage. We obtained a monoeukaryotic culture of one of them and formally describe it as a new member of the family Caviomonadidae, Euthynema mutabile gen. et sp. nov. In contrast to previously studied caviomonads, the endobiotic Caviomonas mobilis and Iotanema spirale, E. mutabile possesses an ultrastructurally discernible MRO. We sequenced and assembled the transcriptome of E. mutabile, and by sequence subtraction, obtained transcriptome data from the other CL3 clade representative present in the original PCS culture, denoted PCS-ghost. Transcriptome analyses showed that the reassignment of only one of the UAR stop codons to encode Gln previously reported from I. spirale does not extend to its free-living relatives and is likely due to a unique amino acid substitution in I. spirale's eRF1 protein domain responsible for termination codon recognition. The backbone fornicate phylogeny was robustly resolved in a phylogenomic analysis, with the CL3 clade amongst the earliest branching lineages. Metabolic and MRO functional reconstructions of CL3 clade members revealed that all three, including I. spirale, encode homologs of key components of the mitochondrial protein import apparatus and the ISC pathway, indicating the presence of a MRO in all of them. In silico evidence indicates that the organelles of E. mutabile and PCS-ghost host ATP and H2 production, unlike the cryptic MRO of I. spirale. These data suggest that the CL3 clade has experienced a hydrogenosome-to-mitosome transition independent from that previously documented for the lineage leading to Giardia.

A new lineage of non-photosynthetic green algae with extreme organellar genomes.

BACKGROUND: The plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them. RESULTS: We discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms, we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes. CONCLUSIONS: With its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution.

When you Like Other Algae: Adglutina synurophila gen. et sp. nov. (Moewusinia, Chlorophyceae), a Clingy Green Microalga Associated with Synura Colonies.

Unicellular green biflagellates of the order Volvocales (Chlorophyceae, Chlorophyta) are common inhabitants of various types of habitats, and can also form peculiar interspecific relationships. Most of their morphological diversity has historically been assigned to the two prominent genera Chlamydomonas and Chloromonas. Ongoing reclassification of these algae, aided by molecular phylogenetics, has resulted in numerous newly proposed genera, but there are certainly brand-new taxa awaiting recognition. In this study, based on morphological and ultrastructural observations together with sequence data of the nuclear 18S and ITS2 rDNA and the plastid rbcL gene, we describe Adglutina synurophila gen. et sp. nov., a volvocalean biflagellate isolated from colonies of the golden-brown alga Synura petersenii (Chrysophyceae). Phylogenetic analyses placed Adglutina in the phylogroup Moewusinia as a sister lineage to the acidophilic "Chlamydomonas" species. It is characterised by having oval to broadly ellipsoidal cells with a low keel-shaped papilla and a cup-shaped chloroplast lacking a pyrenoid, but possessing a lateral eyespot of a variable position. The unique set of features, together with its Synura-loving nature, anchor Adglutina as a well distinguishable phylogenetic lineage within the Moewusinia. The novel alga has a widespread distribution; it has been found in three European countries to date.

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E.

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogues in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in mouse embryonic fibroblasts (MEFs) results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 knockout (KO) in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other intraflagellar transport (IFT) proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi.

Biodegradable Nanohybrid Materials as Candidates for Self-Sanitizing Filters Aimed at Protection from SARS-CoV-2 in Public Areas.

The COVID-19 pandemic has raised the problem of efficient, low-cost materials enabling the effective protection of people from viruses transmitted through the air or via surfaces. Nanofibers can be a great candidate for efficient air filtration due to their structure, although they cannot protect from viruses. In this work, we prepared a wide range of nanofibrous biodegradable samples containing Ag (up to 0.6 at.%) and Cu (up to 20.4 at.%) exhibiting various wettability. By adjusting the magnetron current (0.3 A) and implanter voltage (5 kV), the deposition of TiO2 and Ag+ implantation into PCL/PEO nanofibers was optimized in order to achieve implantation of Ag+ without damaging the nanofibrous structure of the PCL/PEO. The optimal conditions to implant silver were achieved for the PCL-Ti0.3-Ag-5kV sample. The coating of PCL nanofibers by a Cu layer was successfully realized by magnetron sputtering. The antiviral activity evaluated by widely used methodology involving the cultivation of VeroE6 cells was the highest for PCL-Cu and PCL-COOH, where the VeroE6 viability was 73.1 and 68.1%, respectively, which is significantly higher compared to SARS-CoV-2 samples without self-sanitizing (42.8%). Interestingly, the samples with implanted silver and TiO2 exhibited no antiviral effect. This difference between Cu and Ag containing nanofibers might be related to the different concentrations of ions released from the samples: 80 µg/L/day for Cu2+ versus 15 µg/L/day for Ag+. The high antiviral activity of PCL-Cu opens up an exciting opportunity to prepare low-cost self-sanitizing surfaces for anti-SARS-CoV-2 protection and can be essential for air filtration application and facemasks. The rough cost estimation for the production of a biodegradable nanohybrid PCL-Cu facemask revealed ~$0.28/piece, and the business case for the production of these facemasks would be highly positive, with an Internal Rate of Return of 34%.

Electrospun Biodegradable Nanofibers Coated Homogenously by Cu Magnetron Sputtering Exhibit Fast Ion Release. Computational and Experimental Study.

Copper-coated nanofibrous materials are desirable for catalysis, electrochemistry, sensing, and biomedical use. The preparation of copper or copper-coated nanofibers can be pretty challenging, requiring many chemical steps that we eliminated in our robust approach, where for the first time, Cu was deposited by magnetron sputtering onto temperature-sensitive polymer nanofibers. For the first time, the large-scale modeling of PCL films irradiation by molecular dynamics simulation was performed and allowed to predict the ions penetration depth and tune the deposition conditions. The Cu-coated polycaprolactone (PCL) nanofibers were thoroughly characterized and tested as antibacterial agents for various Gram-positive and Gram-negative bacteria. Fast release of Cu2+ ions (concentration up to 3.4 µg/mL) led to significant suppression of E. coli and S. aureus colonies but was insufficient against S. typhimurium and Ps. aeruginosa. The effect of Cu layer oxidation upon contact with liquid media was investigated by X-ray photoelectron spectroscopy revealing that, after two hours, 55% of Cu atoms are in form of CuO or Cu(OH)2. The Cu-coated nanofibers will be great candidates for wound dressings thanks to an interesting synergistic effect: on the one hand, the rapid release of copper ions kills bacteria, while on the other hand, it stimulates the regeneration with the activation of immune cells. Indeed, copper ions are necessary for the bacteriostatic action of cells of the immune system. The reactive CO2/C2H4 plasma polymers deposited onto PCL-Cu nanofibers can be applied to grafting of viable proteins, peptides, or drugs, and it further explores the versatility of developed nanofibers for biomedical applications use.

Monodopsis and Vischeria Genomes Shed New Light on the Biology of Eustigmatophyte Algae.

Members of eustigmatophyte algae, especially Nannochloropsis and Microchloropsis, have been tapped for biofuel production owing to their exceptionally high lipid content. Although extensive genomic, transcriptomic, and synthetic biology toolkits have been made available for Nannochloropsis and Microchloropsis, very little is known about other eustigmatophytes. Here we present three near-chromosomal and gapless genome assemblies of Monodopsis strains C73 and C141 (60 Mb) and Vischeria strain C74 (106 Mb), which are the sister groups to Nannochloropsis and Microchloropsis in the order Eustigmatales. These genomes contain unusually high percentages of simple repeats, ranging from 12% to 21% of the total assembly size. Unlike Nannochloropsis and Microchloropsis, long interspersed nuclear element repeats are abundant in Monodopsis and Vischeria and might constitute the centromeric regions. We found that both mevalonate and nonmevalonate pathways for terpenoid biosynthesis are present in Monodopsis and Vischeria, which is different from Nannochloropsis and Microchloropsis that have only the latter. Our analysis further revealed extensive spliced leader trans-splicing in Monodopsis and Vischeria at 36-61% of genes. Altogether, the high-quality genomes of Monodopsis and Vischeria not only serve as the much-needed outgroups to advance Nannochloropsis and Microchloropsis research, but also shed new light on the biology and evolution of eustigmatophyte algae.

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.

A Eukaryote-Wide Perspective on the Diversity and Evolution of the ARF GTPase Protein Family.

The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a data set of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor, nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13, and Arl16 is presented for the first time. Moreover, Arl17, Arl18, and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our data set revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells.

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BACKGROUND: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. RESULTS: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. CONCLUSIONS: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Protist diversity: Novel groups enrich the algal tree of life.

In recent years, the diversity of eukaryotic microbes has been greatly expanded by recognising or discovering new major branches of the algal tree of life. A new study defines the phylogenetic home for an elusive marine planktonic lineage previously known only by plastidial rRNA genes, placing it in a new class of the phylum Haptophyta.

Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system.

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

Expansion and transformation of the minor spliceosomal system in the slime mold Physarum polycephalum.

Spliceosomal introns interrupt nuclear genes and are removed from RNA transcripts ("spliced") by machinery called spliceosomes. Although the vast majority of spliceosomal introns are removed by the so-called major (or "U2") spliceosome, diverse eukaryotes also contain a rare second form, the minor ("U12") spliceosome, and associated ("U12-type") introns.1-3 In all characterized species, U12-type introns are distinguished by several features, including being rare in the genome (∼0.5% of all introns),4-6 containing extended evolutionarily conserved splicing motifs,4,5,7,8 being generally ancient,9,10 and being inefficiently spliced.11-13 Here, we report a remarkable exception in the slime mold Physarum polycephalum. The P. polycephalum genome contains >20,000 U12-type introns-25 times more than any other species-enriched in a diversity of non-canonical splice boundaries as well as transformed splicing signals that appear to have co-evolved with the spliceosome due to massive gain of efficiently spliced U12-type introns. These results reveal an unappreciated dynamism of minor spliceosomal introns and spliceosomal introns in general.