Vladimír Klimeš

Bacterial proteins pinpoint a single eukaryotic root

Significance The root of eukaryote phylogeny formally represents the last eukaryotic common ancestor (LECA), but its position has remained controversial. Using new genome sequences, we revised and expanded two datasets of eukaryotic proteins of bacterial origin, which previously yielded conflicting views on the eukaryotic root. Analyses using state-of-the-art phylogenomic methodology revealed that both expanded datasets now support the same root position. Our results justify a new nomenclature for the two main eukaryotic groups and provide a robust phylogenetic framework to investigate the early evolution of the eukaryotic cell. 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.

Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?

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.

Rho GTPases: deciphering the evolutionary history of a complex protein family.

Rho GTPases constitute a significant subgroup of the eukaryotic Ras superfamily of small GTPases implicated in the regulation of diverse cellular processes, such as the dynamics of the actin cytoskeleton, establishment, and maintenance of cell polarity and membrane trafficking. Whereas a few eukaryotes lack Rho genes, a majority of species typically bear multiple Rho paralogs, raising a question about the origin of the family and the paths of its diversification in individual eukaryotic lineages. In this chapter, we ruminate on several aspects of the evolutionary history of the Rho family and methodological challenges of its reconstruction. First, we provide an updated survey of Rho GTPases in diverse eukaryotic branches, demonstrating almost ubiquitous occurrence of Rho genes across the eukaryotic phylogeny most consistent with the presence of at least one Rho gene already in the last eukaryotic common ancestor. Second, we discuss the obstacles in reconstructing the history of gene duplications giving rise to the extant diversity of Rho paralogs in different species, and point to numerous limitations posed by the current phylogenetic methodology. Third, as a case study demonstrating various issues of data collection, phylogenetic analyses and interpretations of trees, we present an analysis of the Rho family in the fungal kingdom, revealing the existence of at least four separate paralogs (Cdc42, Rac, Rho1, and Rho4) in early fungi and subsequent potentially independent expansions of the family in different fungal subgroups. We conclude with the warning that the currently dominating perception of the Rho phylogeny is biased by the metazoan (and especially vertebrate) perspective, and a new, more global view is to be worked out when a better genome sampling and more adequate methods of phylogenetic inference are employed.

A paneukaryotic genomic analysis of the small GTPase RABL2 underscores the significance of recurrent gene loss in eukaryote evolution.

BackgroundThe cilium (flagellum) is a complex cellular structure inherited from the last eukaryotic common ancestor (LECA). A large number of ciliary proteins have been characterized in a few model organisms, but their evolutionary history often remains unexplored. One such protein is the small GTPase RABL2, recently implicated in the assembly of the sperm tail in mammals.ResultsUsing the wealth of currently available genome and transcriptome sequences, including data from our on-going sequencing projects, we systematically analyzed the phylogenetic distribution and evolutionary history of RABL2 orthologs. Our dense taxonomic sampling revealed the presence of RABL2 genes in nearly all major eukaryotic lineages, including small “obscure” taxa such as breviates, ancyromonads, malawimonads, jakobids, picozoans, or palpitomonads. The phyletic pattern of RABL2 genes indicates that it was present already in the LECA. However, some organisms lack RABL2 as a result of secondary loss and our present sampling predicts well over 30 such independent events during the eukaryote evolution. The distribution of RABL2 genes correlates with the presence/absence of cilia: not a single well-established cilium-lacking species has retained a RABL2 ortholog. However, several ciliated taxa, most notably nematodes, some arthropods and platyhelminths, diplomonads, and ciliated subgroups of apicomplexans and embryophytes, lack RABL2 as well, suggesting some simplification in their cilium-associated functions. On the other hand, several algae currently unknown to form cilia, e.g., the “prasinophytes” of the genus Prasinoderma or the ochrophytes Pelagococcus subviridis and Pinguiococcus pyrenoidosus, turned out to encode not only RABL2, but also homologs of some hallmark ciliary proteins, suggesting the existence of a cryptic flagellated stage in their life cycles. We additionally obtained insights into the evolution of the RABL2 gene architecture, which seems to have ancestrally consisted of eight exons subsequently modified not only by lineage-specific intron loss and gain, but also by recurrent loss of the terminal exon encoding a poorly conserved C-terminal extension.ConclusionsOur comparative analysis supports the notion that RABL2 is an ancestral component of the eukaryotic cilium and underscores the still underappreciated magnitude of recurrent gene loss, or reductive evolution in general, in the history of eukaryotic genomes and cells.ReviewersThis article was reviewed by Berend Snel and James O. McInerney.

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis

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.

A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons.

Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3′-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3′-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left.

A Comparative Analysis of Mitochondrial Genomes in Eustigmatophyte Algae

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.

Nuclear genetic codes with a different meaning of the UAG and the UAA codon

BackgroundDepartures from the standard genetic code in eukaryotic nuclear genomes are known for only a handful of lineages and only a few genetic code variants seem to exist outside the ciliates, the most creative group in this regard. Most frequent code modifications entail reassignment of the UAG and UAA codons, with evidence for at least 13 independent cases of a coordinated change in the meaning of both codons. However, no change affecting each of the two codons separately has been documented, suggesting the existence of underlying evolutionary or mechanistic constraints.ResultsHere, we present the discovery of two new variants of the nuclear genetic code, in which UAG is translated as an amino acid while UAA is kept as a termination codon (along with UGA). The first variant occurs in an organism noticed in a (meta)transcriptome from the heteropteran Lygus hesperus and demonstrated to be a novel insect-dwelling member of Rhizaria (specifically Sainouroidea). This first documented case of a rhizarian with a non-canonical genetic code employs UAG to encode leucine and represents an unprecedented change among nuclear codon reassignments. The second code variant was found in the recently described anaerobic flagellate Iotanema spirale (Metamonada: Fornicata). Analyses of transcriptomic data revealed that I. spirale uses UAG to encode glutamine, similarly to the most common variant of a non-canonical code known from several unrelated eukaryotic groups, including hexamitin diplomonads (also a lineage of fornicates). However, in these organisms, UAA also encodes glutamine, whereas it is the primary termination codon in I. spirale. Along with phylogenetic evidence for distant relationship of I. spirale and hexamitins, this indicates two independent genetic code changes in fornicates.ConclusionsOur study documents, for the first time, that evolutionary changes of the meaning of UAG and UAA codons in nuclear genomes can be decoupled and that the interpretation of the two codons by the cytoplasmic translation apparatus is mechanistically separable. The latter conclusion has interesting implications for possibilities of genetic code engineering in eukaryotes. We also present a newly developed generally applicable phylogeny-informed method for inferring the meaning of reassigned codons.

A gene transfer event suggests a long-term partnership between eustigmatophyte algae and a novel lineage of endosymbiotic bacteria

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.

Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses

Background: Euglenophytes are an interesting algal group that emerged within the ancestrally plastid-lacking Euglenozoa phylum by acquiring a plastid from a green algal donor. However, the knowledge of euglenophyte plastid biology and evolution is highly incomplete, partly because euglenophytes have so far been little studied on a genome- and transcriptome-wide scale. Transcriptome data from only a single species, Euglena gracilis, have been exploited to functional insights, but aspects of the plastid biology have been largely neglected. Results: To expand the resources for studying euglenophyte biology and to improve our knowledge of the euglenophyte plastid function and evolution, 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 in this species, but provided a number of candidate plastid-localized proteins bearing the same type of N-terminal bipartite topogenic signals (BTSs) as known from the photosynthetic species E. gracilis. Further comparative analyses using transcriptome assemblies available for E. gracilis and two additional photosynthetic euglenophytes of the genus Eutreptiella enabled us to unveil several salient aspects of the basic plastid infrastructure in euglenophytes. First, a number of plastidial proteins seem to reach the organelle as C-terminal translational fusions with other BTS-bearing proteins. Second, the conventional eubacteria-derived plastidial ribosomal protein L24 is missing and seems to have been replaced by very different homologs of the archaeo-eukaryotic origin. Third, no homologs of any key component of the TOC/TIC system (translocon of the outer/inner chloroplast membrane) 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, suggesting an unprecedented modification of the transcription mechanism in their plastid. Conclusions: Our study suggests that the euglenophyte plastid has been substantially remodelled comparted to its green algal progenitor by both loss of original and acquisition of novel molecular components, making it a particularly interesting subject for further studies.