Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and,with the exception of endosymbiotic gene transfers,few ancient transfer events persist

While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence–absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages.     

Functional replacement of a primary metabolic pathway via multiple independent eukaryote‐to‐eukaryote gene transfers and selective retention

Although lateral gene transfer (LGT) is now recognized as a major force in the evolution of prokaryotes, the contribution of LGT to the evolution and diversification of eukaryotes is less understood. Notably, transfers of complete pathways are believed to be less likely between eukaryotes, because the successful transfer of a pathway requires the physical clustering of functionally related genes. Here, we report that in one of the closest unicellular relatives of animals, the choanoflagellate, Monosiga, three genes whose products work together in the glutamate synthase cycle are of algal origin. The concerted retention of these three independently acquired genes is best explained as the consequence of a series of adaptive replacement events. More generally, this study argues that (i) eukaryote‐to‐eukaryote transfers of entire metabolic pathways are possible, (ii) adaptive functional replacements of primary pathways can occur, and (iii) functional replacements involving eukaryotic genes are likely to have also contributed to the evolution of eukaryotes. Lastly, these data underscore the potential contribution of algal genes to the evolution of nonphotosynthetic lineages.     

The rate and pattern of cladogenesis in microbes

Theories of macroevolution rarely have been extended to include microbes; however, because microbes represent the most ancient and diverse assemblage of organismal diversity, such oversight limits our understanding of evolutionary history. Our analysis of phylogenetic trees for microbes suggests that macroevolution may differ between prokaryotes and both micro- and macroeukaryotes (mainly plants and animals). Phylogenetic trees inferred for prokaryotes and some microbial eukaryotes conformed to expectations assuming a constant rate of cladogenesis over time and among lineages: nevertheless, microbial eukaryote trees exhibited more variation in rates of cladogenesis than prokaryote trees. We hypothesize that the contrast of macroevolutionary dynamics between prokaryotes and many eukaryotes is due, at least in part, to differences in the prevalence of lateral gene transfer (LGT) between the two groups. Inheritance is predominantly, if not wholly, vertical within eukaryotes, a feature that allows for the emergence and maintenance of heritable variation among lineages. By contrast, frequent LGT in prokaryotes may ameliorate heritable variation in rate of cladogenesis resulting from the emergence of key innovations; thus, the inferred difference in macroevolution might reflect exclusivity of key innovations in eukaryotes and their promiscuous nature in prokaryotes.     

Detection of Lateral Gene Transfer Events in the Prokaryotic tRNA Synthetases by the Ratios of Evolutionary Distances Method

The availability of large numbers of genomic sequences has demonstrated the importance of lateral gene transfer (LGT) in prokaryotic evolution. However, considerable uncertainty remains concerning the frequency of LGT compared to other evolutionary processes. To examine LGTs in ancient lineages of prokaryotes a method was developed that utilizes the ratios of evolutionary distances (RED) to distinguish between alternative evolutionary histories. The advantages of this approach are that the variability inherent in comparing protein sequences is transparent, the direction of LGT and the relative rates of evolution are readily identified, and it is possible to detect other types of evolutionary events. This method was standardized using 35 genes encoding ribosomal proteins that were believed to share a vertical evolution. Using RED-T, an original computer program designed to implement the RED method, the evolution of the genes encoding the 20 aminoacyl-tRNA synthetases was examined. Although LGTs were common in the evolution of the aminoacyl-tRNA synthetases, they were not sufficient to obscure the organismal phylogeny. Moreover, much of the apparent complexity of the gene tree was consistent with the formation of the paralogs in the ancestors to the modern lineages followed by more recent loss of one paralog or the other.     

The Vein Patterning 1 (VEP1) gene family laterally spread through an ecological network

Lateral gene transfer (LGT) is a major evolutionary mechanism in prokaryotes. Knowledge about LGT--particularly, multicellular--eukaryotes has only recently started to accumulate. A widespread assumption sees the gene as the unit of LGT, largely because little is yet known about how LGT chances are affected by structural/functional features at the subgenic level. Here we trace the evolutionary trajectory of VEin Patterning 1, a novel gene family known to be essential for plant development and defense. At the subgenic level VEP1 encodes a dinucleotide-binding Rossmann-fold domain, in common with members of the short-chain dehydrogenase/reductase (SDR) protein family. We found: i) VEP1 likely originated in an aerobic, mesophilic and chemoorganotrophic α-proteobacterium, and was laterally propagated through nets of ecological interactions, including multiple LGTs between phylogenetically distant green plant/fungi-associated bacteria, and five independent LGTs to eukaryotes. Of these latest five transfers, three are ancient LGTs, implicating an ancestral fungus, the last common ancestor of land plants and an ancestral trebouxiophyte green alga, and two are recent LGTs to modern embryophytes. ii) VEP1's rampant LGT behavior was enabled by the robustness and broad utility of the dinucleotide-binding Rossmann-fold, which provided a platform for the evolution of two unprecedented departures from the canonical SDR catalytic triad. iii) The fate of VEP1 in eukaryotes has been different in different lineages, being ubiquitous and highly conserved in land plants, whereas fungi underwent multiple losses. And iv) VEP1-harboring bacteria include non-phytopathogenic and phytopathogenic symbionts which are non-randomly distributed with respect to the type of harbored VEP1 gene. Our findings suggest that VEP1 may have been instrumental for the evolutionary transition of green plants to land, and point to a LGT-mediated 'Trojan Horse' mechanism for the evolution of bacterial pathogenesis against plants. VEP1 may serve as tool for revealing microbial interactions in plant/fungi-associated environments.     

Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affecting eukaryotes

BACKGROUND: Lateral gene transfer (LGT) is an important evolutionary mechanism among prokaryotes. The situation in eukaryotes is less clear; the human genome sequence failed to give strong support for any recent transfers from prokaryotes to vertebrates, yet a number of LGTs from prokaryotes to protists (unicellular eukaryotes) have been documented. Here, we perform a systematic analysis to investigate the impact of LGT on the evolution of diplomonads, a group of anaerobic protists.RESULTS: Phylogenetic analyses of 15 genes present in the genome of the Atlantic Salmon parasite Spironucleus barkhanus and/or the intestinal parasite Giardia lamblia show that most of these genes originated via LGT. Half of the genes are putatively involved in processes related to an anaerobic lifestyle, and this finding suggests that a common ancestor, which most probably was aerobic, of Spironucleus and Giardia adapted to an anaerobic environment in part by acquiring genes via LGT from prokaryotes. The sources of the transferred diplomonad genes are found among all three domains of life, including other eukaryotes. Many of the phylogenetic reconstructions show eukaryotes emerging in several distinct regions of the tree, strongly suggesting that LGT not only involved diplomonads, but also involved other eukaryotic groups.CONCLUSIONS: Our study shows that LGT is a significant evolutionary mechanism among diplomonads in particular and protists in general. These findings provide insights into the evolution of biochemical pathways in early eukaryote evolution and have important implications for studies of eukaryotic genome evolution and organismal relationships. Furthermore, "fusion" hypotheses for the origin of eukaryotes need to be rigorously reexamined in the light of these results.     

Evolutionary origins of genomic repertoires in bacteria

Explaining the diversity of gene repertoires has been a major problem in modern evolutionary biology. In eukaryotes, this diversity is believed to result mainly from gene duplication and loss, but in prokaryotes, lateral gene transfer (LGT) can also contribute substantially to genome contents. To determine the histories of gene inventories, we conducted an exhaustive analysis of gene phylogenies for all gene families in a widely sampled group, the γ-Proteobacteria. We show that, although these bacterial genomes display striking differences in gene repertoires, most gene families having representatives in several species have congruent histories. Other than the few vast multigene families, gene duplication has contributed relatively little to the contents of these genomes; instead, LGT, over time, provides most of the diversity in genomic repertoires. Most such acquired genes are lost, but the majority of those that persist in genomes are transmitted strictly vertically. Although our analyses are limited to the γ-Proteobacteria, these results resolve a long-standing paradox—i.e., the ability to make robust phylogenetic inferences in light of substantial LGT.     

Characterization of an Ancient Lepidopteran Lateral Gene Transfer

Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.     

The molecular evolution of catalatic hydroperoxidases: evidence for multiple lateral transfer of genes between prokaryota and from bacteria into eukaryota

The past decade has produced an increasing number of reports on horizontal gene transfer between prokaryotic organisms. Only recently, with the flood of available whole genome sequence data and a renewed intensity of the debate about the universal tree of life, a very few reports on lateral gene transfer (LGT) from prokaryotes into the Eukaryota have been published. We have investigated and report here on the molecular evolution of the gene families that encode catalatic hydroperoxidases. We have found that this process included not only frequent horizontal gene transfer among prokaryotes but also several lateral gene transfer events between bacteria and fungi and between bacteria and the protistan ancestor of the alga/plant lineage.     

Transfer of energy pathway genes in microbial enhanced biological phosphorus removal communities

Background

Lateral gene transfer (LGT) is an important evolutionary process in microbial evolution. In sewage treatment plants, LGT of antibiotic resistance and xenobiotic degradation-related proteins has been suggested, but the role of LGT outside these processes is unknown. Microbial communities involved in Enhanced Biological Phosphorus Removal (EBPR) have been used to treat wastewater in the last 50 years and may provide insights into adaptation to an engineered environment. We introduce two different types of analysis to identify LGT in EBPR sewage communities, based on identifying assembled sequences with more than one strong taxonomic match, and on unusual phylogenetic patterns. We applied these methods to investigate the role of LGT in six energy-related metabolic pathways.

Results

The analyses identified overlapping but non-identical sets of transferred enzymes. All of these were homologous with sequences from known mobile genetic elements, and many were also in close proximity to transposases and integrases in the EBPR data set. The taxonomic method had higher sensitivity than the phylogenetic method, identifying more potential LGTs. Both analyses identified the putative transfer of five enzymes within an Australian community, two in a Danish community, and none in a US-derived culture.

Conclusions

Our methods were able to identify sequences with unusual phylogenetic or compositional properties as candidate LGT events. The association of these candidates with known mobile elements supports the hypothesis of transfer. The results of our analysis strongly suggest that LGT has influenced the development of functionally important energy-related pathways in EBPR systems, but transfers may be unique to each community due to different operating conditions or taxonomic composition.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1752-5) contains supplementary material, which is available to authorized users.     

Replacement of the Arginine Biosynthesis Operon in Xanthomonadales by Lateral Gene Transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the γ-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.     

Eukaryotic origin of a metabolic pathway in virus by horizontal gene transfer

Horizontal gene transfer, the movement of genetic materials across the normal mating barriers between organisms occurs frequently and contributes significantly to the evolution of both eukaryotic and prokaryotic genomes. However, few concurrent transfers of functionally related genes implemented in a pathway from eukaryotes to prokaryotes are observed. Here, we did phylogenetic analyses to support that the genes, i.e. dihydrofolate reductase, glycine hydroxymethyltransferase, and thymidylate synthase involved in thymidylate metabolism, in Hz-1 virus were obtained from insect genome recently by independent horizontal gene transfers. In addition, five other related genes in nucleotide metabolism show evidences of horizontal gene transfers. These genes demonstrate similar expression pattern, and they may have formatted a functionally related pathway (e.g. thymidylate synthesis, and DNA replication) in Hz-1 virus. In conclusion, we provide an example of horizontal gene transfer of functionally related genes in a pathway to prokaryote from eukaryote.     

Quartet mapping and the extent of lateral transfer in bacterial genomes

Several recent analyses have used quartet-based methods to assess the congruence among phylogenies derived for large sets of genes from prokaryotic genomes. The principal conclusion from these studies is that lateral gene transfer (LGT) has blurred prokaryotic phylogenies to such a degree that the darwinian scheme of treelike evolution might be abandoned in favor of a net or web. Here, we focus on one of these methods, quartet mapping, and show that its application can lead to overestimation of the extent of inferred LGT in prokaryotes, particularly when applied to distantly related taxa.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

ANALYSIS OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) GENES REVEALS THE COMPLEX EVOLUTIONARY HISTORY OF A MICROBIAL EUKARYOTE1

Microbial eukaryotes may extinguish much of their nuclear phylogenetic history due to endosymbiotic/horizontal gene transfer (E/HGT). We studied E/HGT in 32,110 contigs of expressed sequence tags (ESTs) from the dinoflagellate Alexandrium tamarense (Dinophyceae) using a conservative phylogenomic approach. The vast majority of predicted proteins (86.4%) in this alga are novel or dinoflagellate‐specific. We searched for putative homologs of these predicted proteins against a taxonomically broadly sampled protein database that includes all currently available data from algae and protists, and reconstructed a phylogeny from each of the putative homologous protein sets. Of the 2,523 resulting phylogenies, 14%–17% are potentially impacted by E/HGT involving both prokaryote and eukaryote lineages, with 2%–4% showing clear evidence of reticulate evolution. The complex evolutionary histories of the remaining proteins, many of which may also have been affected by E/HGT, cannot be interpreted using our approach with currently available gene data. We present empirical evidence of reticulate genome evolution that combined with inadequate or highly complex phylogenetic signal in many proteins may impede genome‐wide approaches to infer the tree of microbial eukaryotes.     

Lateral gene transfer of O1 serogroup encoding genes of Vibrio cholerae

In Gram-negative bacteria, the O-antigen-encoding genes may be transferred between lineages, although mechanisms are not fully understood. To assess possible lateral gene transfer (LGT), 21 Argentinean Vibrio cholerae O-group 1 (O1) isolates were examined using multilocus sequence typing (MLST) to determine the genetic relatedness of housekeeping genes and genes from the O1 gene cluster. MSLT analysis revealed that 4.4% of the nucleotides in the seven housekeeping loci were variable, with six distinct genetic lineages identified among O1 isolates. In contrast, MLST analysis of the eight loci from the O1 serogroup region revealed that 0.24% of the 4943 nucleotides were variable. A putative breakpoint was identified in the JUMPstart sequence. Nine conserved nucleotides differed by a single nucleotide from a DNA uptake signal sequence (USS) also found in Pastuerellaceae . Our data indicate that genes in the O1 biogenesis region are closely related even in distinct genetic lineages, indicative of LGT, with a putative DNA USS identified at the defined boundary for the DNA exchange.     

Evolution of the cutinase gene family: evidence for lateral gene transfer of a candidate Phytophthora virulence factor

Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases -- well documented virulence factors in fungi -- between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens.     

Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes

Background

Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH), to evaluate and compare the patterns and rates of lateral gene transfer (LGT) in prokaryotes and eukaryotes.

Results

We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists).

Conclusion

LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

     

Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae

Background

Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome.

Results

Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F).

Conclusions

This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1097) contains supplementary material, which is available to authorized users.     

Horizontal gene transfer as a possible evolutionary predecessor of sexual reproduction

Computer modeling shows that high rates of lateral gene transfer (LGT), combined with homologous recombination, enhance selection efficiency, thus making it possible for organisms to acquire larger genomes without irreversible growth of mutation load. In prokaryotes, the optimal (high) rate of LGT cannot evolve because of the “suicidal effect” of modifiers, i.e., alleles that enhance LGT are systematically replaced by alleles that hinder LGT and never vice versa. Therefore, the latter alleles spread despite the fact that they reduce fitness of organisms and populations. This “conflict of interests” can be resolved via evolution of whole-genome reciprocal recombination, which effectively removes the basis for propagation of “selfish” modifiers. Eukaryotic sexual reproduction (amphimixis) probably appeared as the final result of evolution of mechanisms of LGT driven by selection for high interindividual recombination rate. Such selection could have been facilitated at the early stages of eukaryogenesis due to an increase in genome size and high mutation rate caused by higher O₂ concentration and invasion of group II introns.