Phylogenomic Testing of Root Hypotheses.

The determination of the last common ancestor (LCA) of a group of species plays a vital role in evolutionary theory. Traditionally, an LCA is inferred by the rooting of a fully resolved species tree. From a theoretical perspective, however, inference of the LCA amounts to the reconstruction of just one branch-the root branch-of the true species tree and should therefore be a much easier task than the full resolution of the species tree. Discarding the reliance on a hypothesized species tree and its rooting leads us to reevaluate what phylogenetic signal is directly relevant to LCA inference and to recast the task as that of sampling the total evidence from all gene families at the genomic scope. Here, we reformulate LCA and root inference in the framework of statistical hypothesis testing and outline an analytical procedure to formally test competing a priori LCA hypotheses and to infer confidence sets for the earliest speciation events in the history of a group of species. Applying our methods to two demonstrative data sets, we show that our inference of the opisthokonta LCA is well in agreement with the common knowledge. Inference of the proteobacteria LCA shows that it is most closely related to modern Epsilonproteobacteria, raising the possibility that it may have been characterized by a chemolithoautotrophic and anaerobic life style. Our inference is based on data comprising between 43% (opisthokonta) and 86% (proteobacteria) of all gene families. Approaching LCA inference within a statistical framework renders the phylogenomic inference powerful and robust.

The Ancestral Mitotic State: Closed Orthomitosis With Intranuclear Spindles in the Syncytial Last Eukaryotic Common Ancestor.

All eukaryotes have linear chromosomes that are distributed to daughter nuclei during mitotic division, but the ancestral state of nuclear division in the last eukaryotic common ancestor (LECA) is so far unresolved. To address this issue, we have employed ancestral state reconstructions for mitotic states that can be found across the eukaryotic tree concerning the intactness of the nuclear envelope during mitosis (open or closed), the position of spindles (intranuclear or extranuclear), and the symmetry of spindles being either axial (orthomitosis) or bilateral (pleuromitosis). The data indicate that the LECA possessed closed orthomitosis with intranuclear spindles. Our reconstruction is compatible with recent findings indicating a syncytial state of the LECA, because it decouples three main processes: chromosome division, chromosome partitioning, and cell division (cytokinesis). The possession of closed mitosis using intranuclear spindles adds to the number of cellular traits that can now be attributed to LECA, providing insights into the lifestyle of this otherwise elusive biological entity at the origin of eukaryotic cells. Closed mitosis in a syncytial eukaryotic common ancestor would buffer mutations arising at the origin of mitotic division by allowing nuclei with viable chromosome sets to complement defective nuclei via mRNA in the cytosol.

Realistic Gene Transfer to Gene Duplication Ratios Identify Different Roots in the Bacterial Phylogeny Using a Tree Reconciliation Method.

The rooting of phylogenetic trees permits important inferences about ancestral states and the polarity of evolutionary events. Recently, methods that reconcile discordance between gene-trees and species-trees-tree reconciliation methods-are becoming increasingly popular for rooting species trees. Rooting via reconciliation requires values for a particular parameter, the gene transfer to gene duplication ratio (T:D), which in current practice is estimated on the fly from discordances observed in the trees. To date, the accuracy of T:D estimates obtained by reconciliation analyses has not been compared to T:D estimates obtained by independent means, hence the effect of T:D upon inferences of species tree roots is altogether unexplored. Here we investigated the issue in detail by performing tree reconciliations of more than 10,000 gene trees under a variety of T:D ratios for two phylogenetic cases: a bacterial (prokaryotic) tree with 265 species and a fungal-metazoan (eukaryotic) tree with 31 species. We show that the T:D ratios automatically estimated by a current tree reconciliation method, ALE, generate virtually identical T:D ratios across bacterial genes and fungal-metazoan genes. The T:D ratios estimated by ALE differ 10- to 100-fold from robust, ALE-independent estimates from real data. More important is our finding that the root inferences using ALE in both datasets are strongly dependent upon T:D. Using more realistic T:D ratios, the number of roots inferred by ALE consistently increases and, in some cases, clearly incorrect roots are inferred. Furthermore, our analyses reveal that gene duplications have a far greater impact on ALE's preferences for phylogenetic root placement than gene transfers or gene losses do. Overall, we show that obtaining reliable species tree roots with ALE is only possible when gene duplications are abundant in the data and the number of falsely inferred gene duplications is low. Finding a sufficient sample of true gene duplications for rooting species trees critically depends on the T:D ratios used in the analyses. T:D ratios, while being important parameters of genome evolution in their own right, affect the root inferences with tree reconciliations to an unanticipated degree.

Ancestral State Reconstructions Trace Mitochondria But Not Phagocytosis to the Last Eukaryotic Common Ancestor.

Two main theories have been put forward to explain the origin of mitochondria in eukaryotes: phagotrophic engulfment (undigested food) and microbial symbiosis (physiological interactions). The two theories generate mutually exclusive predictions about the order in which mitochondria and phagocytosis arose. To discriminate the alternatives, we have employed ancestral state reconstructions (ASR) for phagocytosis as a trait, phagotrophy as a feeding habit, the presence of mitochondria, the presence of plastids, and the multinucleated organization across major eukaryotic lineages. To mitigate the bias introduced by assuming a particular eukaryotic phylogeny, we reconstructed the appearance of these traits across 1789 different rooted gene trees, each having species from opisthokonts, mycetozoa, hacrobia, excavate, archeplastida, and Stramenopiles, Alveolates and Rhizaria. The trees reflect conflicting relationships and different positions of the root. We employed a novel phylogenomic test that summarizes ASR across trees which reconstructs a last eukaryotic common ancestor that possessed mitochondria, was multinucleated, lacked plastids, and was non-phagotrophic as well as non-phagocytic. This indicates that both phagocytosis and phagotrophy arose subsequent to the origin of mitochondria, consistent with findings from comparative physiology. Furthermore, our ASRs uncovered multiple origins of phagocytosis and of phagotrophy across eukaryotes, indicating that, like wings in animals, these traits are useful but neither ancestral nor homologous across groups. The data indicate that mitochondria preceded the origin of phagocytosis, such that phagocytosis cannot have been the mechanism by which mitochondria were acquired.

Gene Duplications Are At Least 50 Times Less Frequent than Gene Transfers in Prokaryotic Genomes.

The contribution of gene duplications to the evolution of eukaryotic genomes is well studied. By contrast, studies of gene duplications in prokaryotes are scarce and generally limited to a handful of genes or careful analysis of a few prokaryotic lineages. Systematic broad-scale studies of prokaryotic genomes that sample available data are lacking, leaving gaps in our understanding of the contribution of gene duplications as a source of genetic novelty in the prokaryotic world. Here, we report conservative and robust estimates for the frequency of recent gene duplications within prokaryotic genomes relative to recent lateral gene transfer (LGT), as mechanisms to generate multiple copies of related sequences in the same genome. We obtain our estimates by focusing on evolutionarily recent events among 5,655 prokaryotic genomes, thereby avoiding vagaries of deep phylogenetic inference and confounding effects of ancient events and differential loss. We find that recent, genome-specific gene duplications are at least 50 times less frequent and probably 100 times less frequent than recent, genome-specific, gene acquisitions via LGT. The frequency of gene duplications varies across lineages and functional categories. The findings improve our understanding of genome evolution in prokaryotes and have far-reaching implications for evolutionary models that entail LGT to gene duplications ratio as a parameter.

Evidence for a Syncytial Origin of Eukaryotes from Ancestral State Reconstruction.

Modern accounts of eukaryogenesis entail an endosymbiotic encounter between an archaeal host and a proteobacterial endosymbiont, with subsequent evolution giving rise to a unicell possessing a single nucleus and mitochondria. The mononucleate state of the last eukaryotic common ancestor (LECA) is seldom, if ever, questioned, even though cells harboring multiple (syncytia, coenocytes, and polykaryons) are surprisingly common across eukaryotic supergroups. Here, we present a survey of multinucleated forms. Ancestral character state reconstruction for representatives of 106 eukaryotic taxa using 16 different possible roots and supergroup sister relationships, indicate that LECA, in addition to being mitochondriate, sexual, and meiotic, was multinucleate. LECA exhibited closed mitosis, which is the rule for modern syncytial forms, shedding light on the mechanics of its chromosome segregation. A simple mathematical model shows that within LECA's multinucleate cytosol, relationships among mitochondria and nuclei were neither one-to-one, nor one-to-many, but many-to-many, placing mitonuclear interactions and cytonuclear compatibility at the evolutionary base of eukaryotic cell origin. Within a syncytium, individual nuclei and individual mitochondria function as the initial lower-level evolutionary units of selection, as opposed to individual cells, during eukaryogenesis. Nuclei within a syncytium rescue each other's lethal mutations, thereby postponing selection for viable nuclei and cytonuclear compatibility to the generation of spores, buffering transitional bottlenecks at eukaryogenesis. The prokaryote-to-eukaryote transition is traditionally thought to have left no intermediates, yet if eukaryogenesis proceeded via a syncytial common ancestor, intermediate forms have persisted to the present throughout the eukaryotic tree as syncytia but have so far gone unrecognized.

The metabolic network of the last bacterial common ancestor.

Bacteria are the most abundant cells on Earth. They are generally regarded as ancient, but due to striking diversity in their metabolic capacities and widespread lateral gene transfer, the physiology of the first bacteria is unknown. From 1089 reference genomes of bacterial anaerobes, we identified 146 protein families that trace to the last bacterial common ancestor, LBCA, and form the conserved predicted core of its metabolic network, which requires only nine genes to encompass all universal metabolites. Our results indicate that LBCA performed gluconeogenesis towards cell wall synthesis, and had numerous RNA modifications and multifunctional enzymes that permitted life with low gene content. In accordance with recent findings for LUCA and LACA, analyses of thousands of individual gene trees indicate that LBCA was rod-shaped and the first lineage to diverge from the ancestral bacterial stem was most similar to modern Clostridia, followed by other autotrophs that harbor the acetyl-CoA pathway.

Gene Duplications Trace Mitochondria to the Onset of Eukaryote Complexity.

The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here, we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial-derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal-derived and eukaryote-specific genes. The surplus of bacterial-derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage-specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggests that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin.

Anomalous Phylogenetic Behavior of Ribosomal Proteins in Metagenome-Assembled Asgard Archaea.

Metagenomic studies permit the exploration of microbial diversity in a defined habitat, and binning procedures enable phylogenomic analyses, taxon description, and even phenotypic characterizations in the absence of morphological evidence. Such lineages include asgard archaea, which were initially reported to represent archaea with eukaryotic cell complexity, although the first images of such an archaeon show simple cells with prokaryotic characteristics. However, these metagenome-assembled genomes (MAGs) might suffer from data quality problems not encountered in sequences from cultured organisms due to two common analytical procedures of bioinformatics: assembly of metagenomic sequences and binning of assembled sequences on the basis of innate sequence properties and abundance across samples. Consequently, genomic sequences of distantly related taxa, or domains, can in principle be assigned to the same MAG and result in chimeric sequences. The impacts of low-quality or chimeric MAGs on phylogenomic and metabolic prediction remain unknown. Debates that asgard archaeal data are contaminated with eukaryotic sequences are overshadowed by the lack of evidence indicating that individual asgard MAGs stem from the same chromosome. Here, we show that universal proteins including ribosomal proteins of asgard archaeal MAGs fail to meet the basic phylogenetic criterion fulfilled by genome sequences of cultured archaea investigated to date: These proteins do not share common evolutionary histories to the same extent as pure culture genomes do, pointing to a chimeric nature of asgard archaeal MAGs. Our analysis suggests that some asgard archaeal MAGs represent unnatural constructs, genome-like patchworks of genes resulting from assembly and/or the binning process.

A spectrum of verticality across genes.

Lateral gene transfer (LGT) has impacted prokaryotic genome evolution, yet the extent to which LGT compromises vertical evolution across individual genes and individual phyla is unknown, as are the factors that govern LGT frequency across genes. Estimating LGT frequency from tree comparisons is problematic when thousands of genomes are compared, because LGT becomes difficult to distinguish from phylogenetic artefacts. Here we report quantitative estimates for verticality across all genes and genomes, leveraging a well-known property of phylogenetic inference: phylogeny works best at the tips of trees. From terminal (tip) phylum level relationships, we calculate the verticality for 19,050,992 genes from 101,422 clusters in 5,655 prokaryotic genomes and rank them by their verticality. Among functional classes, translation, followed by nucleotide and cofactor biosynthesis, and DNA replication and repair are the most vertical. The most vertically evolving lineages are those rich in ecological specialists such as Acidithiobacilli, Chlamydiae, Chlorobi and Methanococcales. Lineages most affected by LGT are the α-, ß-, γ-, and δ- classes of Proteobacteria and the Firmicutes. The 2,587 eukaryotic clusters in our sample having prokaryotic homologues fail to reject eukaryotic monophyly using the likelihood ratio test. The low verticality of α-proteobacterial and cyanobacterial genomes requires only three partners-an archaeal host, a mitochondrial symbiont, and a plastid ancestor-each with mosaic chromosomes, to directly account for the prokaryotic origin of eukaryotic genes. In terms of phylogeny, the 100 most vertically evolving prokaryotic genes are neither representative nor predictive for the remaining 97% of an average genome. In search of factors that govern LGT frequency, we find a simple but natural principle: Verticality correlates strongly with gene distribution density, LGT being least likely for intruding genes that must replace a preexisting homologue in recipient chromosomes. LGT is most likely for novel genetic material, intruding genes that encounter no competing copy.

The Future of Origin of Life Research: Bridging Decades-Old Divisions.

Research on the origin of life is highly heterogeneous. After a peculiar historical development, it still includes strongly opposed views which potentially hinder progress. In the 1st Interdisciplinary Origin of Life Meeting, early-career researchers gathered to explore the commonalities between theories and approaches, critical divergence points, and expectations for the future. We find that even though classical approaches and theories-e.g. bottom-up and top-down, RNA world vs. metabolism-first-have been prevalent in origin of life research, they are ceasing to be mutually exclusive and they can and should feed integrating approaches. Here we focus on pressing questions and recent developments that bridge the classical disciplines and approaches, and highlight expectations for future endeavours in origin of life research.

New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases.

Pyruvate kinases (PKs) synthesize ATP as the final step of glycolysis in the three domains of life. PKs from most bacteria and eukarya are allosteric enzymes that are activated by sugar phosphates; for example, the feed-forward regulator fructose-1,6-bisphosphate, or AMP as a sensor of energy charge. Archaea utilize unusual glycolytic pathways, but the allosteric properties of PKs from these species are largely unknown. Here, we present an analysis of 24 PKs from most archaeal clades with respect to allosteric properties, together with phylogenetic analyses constructed using a novel mode of rooting protein trees. We find that PKs from many Thermoproteales, an order of crenarchaeota, are allosterically activated by 3-phosphoglycerate (3PG). We also identify five conserved amino acids that form the binding pocket for 3PG. 3PG is generated via an irreversible reaction in the modified glycolytic pathway of these archaea and therefore functions as a feed-forward regulator. We also show that PKs from hyperthermophilic Methanococcales, an order of euryarchaeota, are activated by AMP. Phylogenetic analyses indicate that 3PG-activated PKs form an evolutionary lineage that is distinct from that of sugar-phosphate activated PKs, and that sugar phosphate-activated PKs originated as AMP-regulated PKs in hyperthermophilic Methanococcales. Since the phospho group of sugar phosphates and 3PG overlap in the allosteric site, our data indicate that the allostery in PKs first started from a progenitor phosphate-binding site that evolved in two spatially distinct directions: one direction generated the canonical site that responds to sugar phosphates and the other gave rise to the 3PG site present in Thermoproteales. Overall, our data suggest an intimate connection between the allosteric properties and evolution of PKs.