Uncovering the Genomic Origins of Life.

The Origin of Life Domain (OLD) is the period during which life on Earth began. Here, we derive and use a new phylogenetic algorithm to analyze Protein Families in order to reconstruct the chronological steps by which the OLD evolved. During this period, life began with the appearance of the fundamental components of life such as RNAs, DNAs, amino acids, and membranes. Chronologically, the Origin of Life preceded the Last Universal Common Ancestor, which then subsequently engendered modern life on Earth. Our phylogenetic algorithm allows us to explicitly answer previously unknown origin of life questions. Specifically, we explain and illustrate our computational methods by reconstructing the rings describing the evolution of the RNA and DNA worlds. We phylogenetically reconstruct how the RNA and DNA worlds evolved, infer the origins and chronological order of appearance of the first genetic codes, test whether the Ribosomal RNA world preceded the Membrane world, and interpret these new findings with respect to the experimental and theoretical origin of life studies by others.

Rings Reconcile Genotypic and Phenotypic Evolution within the Proteobacteria.

Although prokaryotes are usually classified using molecular phylogenies instead of phenotypes after the advent of gene sequencing, neither of these methods is satisfactory because the phenotypes cannot explain the molecular trees and the trees do not fit the phenotypes. This scientific crisis still exists and the profound disconnection between these two pillars of evolutionary biology--genotypes and phenotypes--grows larger. We use rings and a genomic form of goods thinking to resolve this conundrum (McInerney JO, Cummins C, Haggerty L. 2011. Goods thinking vs. tree thinking. Mobile Genet Elements. 1:304-308; Nelson-Sathi S, et al. 2015. Origins of major archaeal clades correspond to gene acquisitions from bacteria. Nature 517:77-80). The Proteobacteria is the most speciose prokaryotic phylum known. It is an ideal phylogenetic model for reconstructing Earth's evolutionary history. It contains diverse free living, pathogenic, photosynthetic, sulfur metabolizing, and symbiotic species. Due to its large number of species (Whitman WB, Coleman DC, Wiebe WJ. 1998. Prokaryotes: the unseen majority. Proc Nat Acad Sci U S A. 95:6578-6583) it was initially expected to provide strong phylogenetic support for a proteobacterial tree of life. But despite its many species, sequence-based tree analyses are unable to resolve its topology. Here we develop new rooted ring analyses and study proteobacterial evolution. Using protein family data and new genome-based outgroup rooting procedures, we reconstruct the complex evolutionary history of the proteobacterial rings (combinations of tree-like divergences and endosymbiotic-like convergences). We identify and map the origins of major gene flows within the rooted proteobacterial rings (P < 3.6 × 10(-6)) and find that the evolution of the "Alpha-," "Beta-," and "Gammaproteobacteria" is represented by a unique set of rings. Using new techniques presented here we also root these rings using outgroups. We also map the independent flows of genes involved in DNA-, RNA-, ATP-, and membrane- related processes within the Proteobacteria and thereby demonstrate that these large gene flows are consistent with endosymbioses (P < 3.6 × 10(-9)). Our analyses illustrate what it means to find that a gene is present, or absent, within a gene flow, and thereby clarify the origin of the apparent conflicts between genotypes and phenotypes. Here we identify the gene flows that introduced photosynthesis into the Alpha-, Beta-, and Gammaproteobacteria from the common ancestor of the Actinobacteria and the Firmicutes. Our results also explain why rooted rings, unlike trees, are consistent with the observed genotypic and phenotypic relationships observed among the various proteobacterial classes. We find that ring phylogenies can explain the genotypes and the phenotypes of biological processes within large and complex groups like the Proteobacteria.

Eukaryotic origins.

The origin of the eukaryotes is a fundamental scientific question that for over 30 years has generated a spirited debate between the competing Archaea (or three domains) tree and the eocyte tree. As eukaryotes ourselves, humans have a personal interest in our origins. Eukaryotes contain their defining organelle, the nucleus, after which they are named. They have a complex evolutionary history, over time acquiring multiple organelles, including mitochondria, chloroplasts, smooth and rough endoplasmic reticula, and other organelles all of which may hint at their origins. It is the evolutionary history of the nucleus and their other organelles that have intrigued molecular evolutionists, myself included, for the past 30 years and which continues to hold our interest as increasingly compelling evidence favours the eocyte tree. As with any orthodoxy, it takes time to embrace new concepts and techniques.

Comparison of the Mitochondrial Genomes and Steady State Transcriptomes of Two Strains of the Trypanosomatid Parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3' processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.

Dissociation of ribosomes into large and small subunits.

Structural and functional studies of ribosomal subunits require the dissociation of intact ribosomes into individual small and large ribosomal subunits. The dissociation of the prokaryotic 70S ribosomes into the 50S and 30S subunits is achieved by dialysis against a buffer containing a lower Mg(2+) concentration. Eukaryotic 80S ribosomes are dissociated into 60S and 40S subunits by incubation in a buffer containing puromycin and higher KCl and Mg(2+) concentrations.

Purification of 70S ribosomes.

Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.

Purification of polysomes.

Isolated polysomes (also known as translating ribosomes or polyribosomes) are mRNA-ribosome complexes that are frequently used for the in vitro study of the regulation of protein synthesis. Here we describe a protocol for the isolation of prokaryotic and plant polysomes by sucrose gradient sedimentation. The protocol allows for the separation of multiple ribosomes attached to mRNA from run-off ribosome monomers. In addition to collecting the polysome pellet, if the sucrose gradients used in this protocol are fractionated, it is possible to isolate and quantify the polysomes, the ribosome monomers, and the ribosomal subunits.

Isolation of ribosomes and polysomes.

Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

The deep roots of the rings of life.

Reconstructing early evolutionary events like the origins of informational and operational genes, membranes, and photophosphorylation is difficult because early evolutionary events can be masked by subsequent gene flows. Furthermore, as evolution progresses through both Darwinian survival of the fittest (tree-like evolution) and symbiotic/endosymbiotic cooperation (ring-like evolution), trees alone are not adequate to represent Earth's evolutionary history. Here, we reconstruct and root the New Rings of Life and use it as a framework for interpreting early events in the evolution of life. Unlike the three-domain hypothesis, the rings do not fit all life into one of three immutable categories, but rather accommodate new gene flows as novel organisms are discovered. A draft of the Rooted Rings of Life is reconstructed by analyzing the phylogenetic distributions of indels (insertions/deletions) and genes coding for fundamental molecular processes. Their phylogenetic distributions are inconsistent with all trees. Hypergeometric distribution analyses of them strongly localize the root of the rings to a segment of the deepest ring (P < 10(-21) and P < 10(-194)), and whole-genome analyses independently confirm the topology of the rooted rings (P < 7.1 × 10(-6)). The rings identify several large gene flows, including a flow of a thousand genes into the Halobacteria and the Eubacteria, the related photocyte flow, the flow of genes into the last common ancestor of the eocytes and the eukaryotes, and the informational and operational gene flows into the eukaryotes. The rooted rings also chronologically order steps in the evolution of extant taxa, that is, phototrophy evolved from Halobacteria (photophosphorylation) → Heliobacteria (photosynthesis) → Cyanobacteria (oxygenic photosynthesis).

Rooting gene trees without outgroups: EP rooting.

Gene sequences are routinely used to determine the topologies of unrooted phylogenetic trees, but many of the most important questions in evolution require knowing both the topologies and the roots of trees. However, general algorithms for calculating rooted trees from gene and genomic sequences in the absence of gene paralogs are few. Using the principles of evolutionary parsimony (EP) (Lake JA. 1987a. A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony. Mol Biol Evol. 4:167-181) and its extensions (Cavender, J. 1989. Mechanized derivation of linear invariants. Mol Biol Evol. 6:301-316; Nguyen T, Speed TP. 1992. A derivation of all linear invariants for a nonbalanced transversion model. J Mol Evol. 35:60-76), we explicitly enumerate all linear invariants that solely contain rooting information and derive algorithms for rooting gene trees directly from gene and genomic sequences. These new EP linear rooting invariants allow one to determine rooted trees, even in the complete absence of outgroups and gene paralogs. EP rooting invariants are explicitly derived for three taxon trees, and rules for their extension to four or more taxa are provided. The method is demonstrated using 18S ribosomal DNA to illustrate how the new animal phylogeny (Aguinaldo AMA et al. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489-493; Lake JA. 1990. Origin of the metazoa. Proc Natl Acad Sci USA 87:763-766) may be rooted directly from sequences, even when they are short and paralogs are unavailable. These results are consistent with the current root (Philippe H et al. 2011. Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470:255-260).

Evidence for an early prokaryotic endosymbiosis.

Endosymbioses have dramatically altered eukaryotic life, but are thought to have negligibly affected prokaryotic evolution. Here, by analysing the flows of protein families, I present evidence that the double-membrane, gram-negative prokaryotes were formed as the result of a symbiosis between an ancient actinobacterium and an ancient clostridium. The resulting taxon has been extraordinarily successful, and has profoundly altered the evolution of life by providing endosymbionts necessary for the emergence of eukaryotes and by generating Earth's oxygen atmosphere. Their double-membrane architecture and the observed genome flows into them suggest a common evolutionary mechanism for their origin: an endosymbiosis between a clostridium and actinobacterium.

Genome beginnings: rooting the tree of life.

A rooted tree of life provides a framework to answer central questions about the evolution of life. Here we review progress on rooting the tree of life and introduce a new root of life obtained through the analysis of indels, insertions and deletions, found within paralogous gene sets. Through the analysis of indels in eight paralogous gene sets, the root is localized to the branch between the clade consisting of the Actinobacteria and the double-membrane (Gram-negative) prokaryotes and one consisting of the archaebacteria and the firmicutes. This root provides a new perspective on the habitats of early life, including the evolution of methanogenesis, membranes and hyperthermophily, and the speciation of major prokaryotic taxa. Our analyses exclude methanogenesis as a primitive metabolism, in contrast to previous findings. They parsimoniously imply that the ether archaebacterial lipids are not primitive and that the cenancestral prokaryotic population consisted of organisms enclosed by a single, ester-linked lipid membrane, covered by a peptidoglycan layer. These results explain the similarities previously noted by others between the lipid synthesis pathways in eubacteria and archaebacteria. The new root also implies that the last common ancestor was not hyperthermophilic, although moderate thermophily cannot be excluded.

Evidence for a new root of the tree of life.

Directed indels, insertions or deletions within paralogous genes, have the potential to root the tree of life. Here we apply the top-down rooting algorithm to indels found in PyrD (dihydroorotate dehydrogenase), a key enzyme involved in the de novo biosynthesis of pyrimidines, and HisA (P-ribosylformimino-AICAR-P-isomerase), an essential enzyme in the histidine biosynthesis pathway. Through the comparison of each indel with its two paralogous outgroups, we exclude the root of the tree of life from the clade that encompasses the Actinobacteria, the double-membrane prokaryotes, and their last common ancestor. In combination with previous indel rooting studies excluding the root from a clade consisting of the Firmicutes, the Archaea, and their last common ancestor, this provides evidence for a unique eubacterial root for the tree of life located between the actinobacterial-double-membrane clade and the archaeal-firmicute clade. Mapping the phylogenetic distributions of genes involved in peptidoglycan and lipid synthesis onto this rooted tree parsimoniously implies that the cenancestral prokaryotic population consisted of organisms enclosed by a single, ester-linked lipid membrane, covered by a peptidoglycan layer.

Reconstructing evolutionary graphs: 3D parsimony.

The increasing recognition that symbioses have greatly altered evolution through genome fusions is creating a need for algorithms that can reliably detect and reconstruct fusions. Here, we generalize the bootstrappers gambit algorithm (a quartet method) in order to permit it to analyze both bifurcations and fusions under a single mathematical model, and thereby detect past genomic branchings and endosymbioses. This new method, 3-dimensional parsimony, can be applied to aligned sequences, such as gene, indel, or other genomic presence/absence sequences. It also provides a statistical measure of support for each possible graph. The usefulness of this method is demonstrated by applying it to the ring of life.

Evidence excluding the root of the tree of life from the actinobacteria.

The Actinobacteria are found in aquatic and terrestrial habitats throughout the world and are among the most morphologically varied prokaryotes. They manufacture unusual compounds, utilize novel metabolic pathways, and contain unique genes. This diversity may suggest that the root of the tree of life could be within the Actinobacteria, although there is little or no convincing evidence for such a root. Here, using gene insertions and deletions found in the DNA gyrase, GyrA, and in the paralogous DNA topoisomerase, ParC, we present evidence that the root of life is outside the Actinobacteria.

Evidence for a gram-positive, eubacterial root of the tree of life.

Directed indels, insertions, and deletions within paralogous genes, have the potential to root the tree of life. Here we apply a newly developed rooting algorithm, top-down rooting, to indels found in informational and operational gene sets, introduce new computational tools for indel analyses, and present evidence (P < .01) that the root of the tree of life is not present in its traditional location, between the Eubacteria and the Archaebacteria. Using indels contained in the dihydroorotate dehydrogenase/uroporphyrinogen decarboxylase gene pair and in the ribosomal protein S12/beta prime subunit of the RNA polymerase gene pair, we exclude the root from within the clade consisting of the Firmicutes plus the Archaebacteria and their most recent common ancestor. These results, plus previous directed indel studies excluding the root from the eukaryotes, restrict the root to just four possible sites. One potential root is on the branch leading to the double-membrane prokaryotes, another is on the branch leading to the Actinobacteria, another is within the Actinobacteria, and the fourth is on the branch leading to the Firmicutes-Archaea clade. These results imply (1) that the cenancestral population was not hyperthermophilic, but moderate thermophily cannot be excluded for the root on the branch leading to the Firmicutes-Archaea clade, (2) that the cenancestral population was surrounded by ester lipids and a peptidoglycan layer, and (3) that parts of the mevalonate synthesis pathway were present in the population ancestral to the Bacilli and the Archaebacteria, including geranylgeranylglyceryl phosphate synthase, an enzyme thought to be partially responsible for the unique sn-1 stereochemistry of the archaeal glycerol phosphate backbone.