The large phylogenetic distance separating eukaryotic genes and their archaeal orthologs has prevented identification of the position of the eukaryotic root in phylogenomic studies. Recently, an innovative approach has been proposed to circumvent this issue: the use as phylogenetic markers of proteins that have been transferred from bacterial donor sources to eukaryotes, after their emergence from Archaea. Using this approach, two recent independent studies have built phylogenomic datasets based on bacterial sequences, leading to different predictions of the eukaryotic root. Taking advantage of additional genome sequences from the jakobid Andalucia godoyi and the two known malawimonad species (Malawimonas jakobiformis and Malawimonas californiana), we reanalyzed these two phylogenomic datasets. We show that both datasets pinpoint the same phylogenetic position of the eukaryotic root that is between "Unikonta" and "Bikonta," with malawimonad and collodictyonid lineages on the Unikonta side of the root. Our results firmly indicate that (i) the supergroup Excavata is not monophyletic and (ii) the last common ancestor of eukaryotes was a biflagellate organism. Based on our results, we propose to rename the two major eukaryotic groups Unikonta and Bikonta as Opimoda and Diphoda, respectively. eukaryote phylogeny | phylogenomics | Opimoda | Diphoda | LECA

Algae with secondary plastids of a red algal origin, such as ochrophytes (photosynthetic stramenopiles), are diverse and ecologically important, yet their evolutionary history remains controversial. We sequenced plastid genomes of two ochrophytes, Ochromonas sp. CCMP1393 (Chrysophyceae) and Trachydiscus minutus (Eustigmatophyceae). A shared split of the clpC gene as well as phylogenomic analyses of concatenated protein sequences demonstrated that chrysophytes and eustigmatophytes form a clade, the Limnista, exhibiting an unexpectedly elevated rate of plastid gene evolution. Our analyses also indicate that the root of the ochrophyte phylogeny falls between the recently redefined Khakista and Phaeista assemblages. Taking advantage of the expanded sampling of plastid genome sequences, we revisited the phylogenetic position of the plastid of Vitrella brassicaformis, a member of Alveolata with the least derived plastid genome known for the whole group. The results varied depending on the dataset and phylogenetic method employed, but suggested that the Vitrella plastids emerged from a deep ochrophyte lineage rather than being derived vertically from a hypothetical plastid-bearing common ancestor of alveolates and stramenopiles. Thus, we hypothesize that the plastid in Vitrella, and potentially in other alveolates, may have been acquired by an endosymbiosis of an early ochrophyte.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%–61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than β-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.

Background: Comparative analyses have indicated that the mitochondrion of the last eukaryotic common ancestor likely possessed all the key core structures and functions that are widely conserved throughout the domain Eucarya. To date, such studies have largely focused on animals, fungi, and land plants (primarily multicellular eukaryotes); relatively few mitochondrial proteomes from protists (primarily unicellular eukaryotic microbes) have been examined. To gauge the full extent of mitochondrial structural and functional complexity and to identify potential evolutionary trends in mitochondrial proteomes, more comprehensive explorations of phylogenetically diverse mitochondrial proteomes are required. In this regard, a key group is the jakobids, a clade of protists belonging to the eukaryotic supergroup Discoba, distinguished by having the most gene-rich and most bacteria-like mitochondrial genomes discovered to date. Results: In this study, we assembled the draft nuclear genome sequence for the jakobid Andalucia godoyi and used a comprehensive in silico approach to infer the nucleus-encoded portion of the mitochondrial proteome of this protist, identifying 864 candidate mitochondrial proteins. The A. godoyi mitochondrial proteome has a complexity that parallels that of other eukaryotes, while exhibiting an unusually large number of ancestral features that have been lost particularly in opisthokont (animal and fungal) mitochondria. Notably, we find no evidence that the A. godoyi nuclear genome has or had a gene encoding a single-subunit, T3/T7 bacteriophage-like RNA polymerase, which functions as the mitochondrial transcriptase in all eukaryotes except the jakobids.

Euglenophytes are a familiar algal group with green alga-derived secondary plastids, but the knowledge of euglenophyte plastid function and evolution is still highly incomplete. With this in mind we sequenced and analysed the transcriptome of the non-photosynthetic species Euglena longa. The transcriptomic data confirmed the absence of genes for the photosynthetic machinery, but provided candidate plastid-localised proteins bearing N-terminal bipartite topogenic signals (BTSs) of the characteristic euglenophyte type. Further comparative analyses including transcriptome assemblies available for photosynthetic euglenophytes enabled us to unveil salient aspects of the basic euglenophyte plastid infrastructure, such as plastidial targeting of several proteins as C-terminal translational fusions with other BTS-bearing proteins or replacement of the conventional eubacteria-derived plastidial ribosomal protein L24 by homologs of archaeo-eukaryotic origin. Strikingly, no homologs of any key component of the TOC/TIC system and the plastid division apparatus are discernible in euglenophytes, and the machinery for intraplastidial protein targeting has been simplified by the loss of the cpSRP/cpFtsY system and the SEC2 translocon. Lastly, euglenophytes proved to encode a plastid-targeted homolog of the termination factor Rho horizontally acquired from a Lambdaproteobacteria-related donor. Our study thus further documents a substantial remodelling of the euglenophyte plastid compared to its green algal progenitor.

Rickettsiales are obligate intracellular bacteria originally found in metazoans, but more recently recognized as widespread endosymbionts of various protists. One genus was detected also in several green algae, but reports on rickettsialean endosymbionts in other algal groups are lacking. Here we show that several distantly related eustigmatophytes (coccoid algae belonging to Ochrophyta, Stramenopiles) are infected by Candidatus Phycorickettsia gen. nov., a new member of the family Rickettsiaceae. The genome sequence of Ca. Phycorickettsia trachydisci sp. nov., an endosymbiont of Trachydiscus minutus CCALA 838, revealed genomic features (size, GC content, number of genes) typical for other Rickettsiales, but some unusual aspects of the gene content were noted. Specifically, Phycorickettsia lacks genes for several components of the respiration chain, haem biosynthesis pathway, or c-di-GMP-based signalling. On the other hand, it uniquely harbours a six-gene operon of enigmatic function that we recently reported from plastid genomes of two distantly related eustigmatophytes and from various non-rickettsialean bacteria. Strikingly, the eustigmatophyte operon is closely related to the one from Phycorickettsia, suggesting a gene transfer event between the endosymbiont and host lineages in early eustigmatophyte evolution. We hypothesize an important role of the operon in the physiology of Phycorickettsia infection and a long-term eustigmatophyte-Phycorickettsia coexistence.

Eustigmatophyceae (Ochrophyta, Stramenopiles) is a small algal group with species of the genus Nannochloropsis being its best studied representatives. Nuclear and organellar genomes have been recently sequenced for several Nannochloropsis spp., but phylogenetically wider genomic studies are missing for eustigmatophytes. We sequenced mitochondrial genomes (mitogenomes) of three species representing most major eustigmatophyte lineages, Monodopsis sp. MarTras21, Vischeria sp. CAUP Q 202 and Trachydiscus minutus, and carried out their comparative analysis in the context of available data from Nannochloropsis and other stramenopiles, revealing a number of noticeable findings. First, mitogenomes of most eustigmatophytes are highly collinear and similar in the gene content, but extensive rearrangements and loss of three otherwise ubiquitous genes happened in the Vischeria lineage; this correlates with an accelerated evolution of mitochondrial gene sequences in this lineage. Second, eustigmatophytes appear to be the only ochrophyte group with the Atp1 protein encoded by the mitogenome. Third, eustigmatophyte mitogenomes uniquely share a truncated nad11 gene encoding only the C-terminal part of the Nad11 protein, while the N-terminal part is encoded by a separate gene in the nuclear genome. Fourth, UGA as a termination codon and the cognate release factor mRF2 were lost from mitochondria independently by the Nannochloropsis and T. minutus lineages. Finally, the rps3 gene in the mitogenome of Vischeria sp. is interrupted by the UAG codon, but the genome includes a gene for an unusual tRNA with an extended anticodon loop that we speculate may serve as a suppressor tRNA to properly decode the rps3 gene.

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 dataset 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 (LECA), 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 dataset 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.