GEITP had posted a brief blog on this topic (of the last universal common ancestor, LUCA) on 17 Jul 2024, reporting on an abstract and a talk given at a meeting in Puerto Vallarta, Mexico in March 2024. In contrast, this full paper [see attached pdf file, Sept 2024 publication] of course gives FAR more details — which GEITP finds to be extremely helpful in understanding all the calculations (and assumptions) by the authors…
To review briefly our last GEITP blog, featuring the “Lipid First” hypothesis, Earth was first formed 4.54 Billion years ago (4,540 million years ago). The earliest life forms so far proven to exist are microscopic organisms (microbes) that left signals of their presence in rocks ~3.7 Billion years ago (~3,700 million years ago) — meaning that Life must have begun within the first 840 million years after formation of the planet. However, the 2024 publication by E.R.R. Moody et al. [see attached pdf file] shows data suggesting the last universal common ancestor (LUCA) likely existed and was highly sophisticated by 4.2 Billion years ago (4,200 million is just 340 million years after Earth was first formed). But what kind of “compartmentalization” or “protection” from the harsh environment of early Earth preceded the LUCA’s nucleic acids and proteins that Moody et al. propose in the attached pdf file? If the “Lipid First” hypothesis is correct, replicating lipid micelles would be ideal candidates for having populated Earth — within the first 340 million years after Earth first formed.
The common ancestry of all extant cellular life is apparently evidenced by: [a] the universal genetic code, [b] machinery for protein synthesis, [c] shared chirality [the property of a molecule that is non-superimposable on its mirror image] of the almost-universal set of 20 amino acids, and [d] use of ATP as a common energy source. The last universal common ancestor (LUCA) is the node on the tree-of-life from which the fundamental prokaryotic domains (Archaea and Bacteria) diverged. As such, our understanding of LUCA impacts our understanding of the early evolution of life on Earth.
Was LUCA a simple or complex organism? What kind of environment did it inhabit and when? Previous estimates of LUCA are in conflict — either due to conceptual disagreement about what LUCA is, or because of different methodological approaches and data. Published analyses differ in their inferences of LUCA’s genome, from conservative estimates of 80 orthologous proteins, up to 1,529 different potential gene families. Interpretations range from little beyond an information-processing and metabolic core, through to a prokaryote-grade organism with much of the gene repertoire of modern Archaea and Bacteria. Here the authors have used molecular clock methodology, horizontal gene-transfer-aware phylogenetic reconciliation, and existing biogeochemical models to address questions about LUCA’s age, gene content, metabolism, and impact on the early Earth system.
Life’s evolutionary timescale has been typically calibrated to the oldest fossil occurrences. However, the veracity of fossil discoveries from the early Archaean period has been contested. Relaxed Bayesian node-calibrated molecular clock approaches provide a better means of integrating the sparse fossil and geochemical record of early life with the information provided by molecular data; however, constraining LUCA’s age is challenging due to limited prokaryote fossil calibrations and the uncertainty in their placement on the phylogeny. Molecular clock estimates of LUCA have relied on conserved universal single-copy marker genes within phylogenies for which LUCA represents the root.
Dating the root of a tree is difficult because errors propagate from the tips to the root of the dated phylogeny, and information is not available to estimate the rate of evolution for the branch incident on the root node. Therefore, authors analyzed genes that duplicated before LUCA with two (or more) copies in LUCA’s genome.
The root in these gene trees represents this duplication preceding LUCA, whereas LUCA is represented by two descendant nodes. Use of these universal paralogs also has the advantage that the same calibrations can be applied at least twice. After duplication, the same species divergences are represented on both sides of the gene tree and thus can be assumed to have the same age. This considerably reduces the uncertainty when genetic distance (branch length) is resolved into absolute time and rate. When a shared node is assigned a fossil calibration, such cross-bracing also serves to double the number of calibrations on the phylogeny — thus improving divergence-time estimates. Authors calibrated their molecular clock analyses using 13 calibrations.
The calibration on the root of the tree of life is of particular importance. Some previous studies have placed a younger maximum constraint on the age of LUCA based on the assumption that Life could not have survived Late Heavy Bombardment (LHB) [~3.7–3.9 billion years ago (Ga)]. However, the LHB hypothesis is extrapolated and
scaled from the Moon’s impact record — the interpretation of which has been questioned in terms of the intensity, duration, and even the accurateness of an LHB episode. Thus, the LHB hypothesis should not be
considered a credible maximum constraint on the age of LUCA. Authors used soft-uniform bounds, with the maximum-age bound based on the time of the Moon-forming impact (4,510 million years ago), which would have effectively sterilized Earth’s precursors, Tellus and Theia [according to the Giant Impact Theory, our Moon was formed when a Mars-sized protoplanet, Theia, collided with the proto-Earth, Tellus — which created today’s Earth]. Authors’ minimum boundary on the age of LUCA is based on low-δ98Mo isotope values (indicative of Mn oxidation, compatible with oxygenic photosynthesis) and, therefore, total-group Oxyphotobacteria in the Mozaan Group, Pongola Supergroup, South Africa, dated minimally to ~2,954 million years ago [this geological time (1,586 million years after Earth first formed) falls within the Archean Eon — during the Neoarchean Era, which ranges from 2,800 MYA to 1,500 MYA, and was a time when Earth’s crust was still forming — and the first forms of life were likely emerging].
Authors’ estimates for the age of LUCA are inferred with a concatenated and a partitioned dataset, both consisting of five pre-LUCA paralogs: (catalytic and non-catalytic subunits from ATP synthases, elongation factors Tu and G, signal recognition protein and signal recognition particle receptor, tyrosyl-tRNA and tryptophanyl-tRNA synthetases, and leucyl- and valyl-tRNA synthetases). Marginal densities (commonly denoted as “effective priors”) fall within calibration densities (i.e., user-specified priors) when topologically adjacent calibrations do not overlap temporally, but may differ when they overlap — to ensure the relative age relationships between ancestor-descendant nodes.
Authors consider the marginal densities a reasonable interpretation of the calibration evidence — given the phylogeny (authors are not attempting to test the hypothesis that the fossil record is an accurate temporal archive of evolutionary history, because it is not). The duplicated LUCA node-age estimates that authors obtained under the autocorrelated rates [geometric Brownian motion (GBM)] and independent-rates log-normal (ILN) relaxed-clock models with the authors’ partitioned dataset (GBM = 4.18–4.33 Ga; ILN, 4.09–4.32 Ga; see Fig. 1 of attached) fall within our composite age estimate for LUCA — ranging from 3.94 BYA to 4.52 BYA, are comparable to three previous studies. Dating analyses based on single genes, or concatenations that excluded each gene in turn, returned compatible timescales (see Extended Data Figs. 1 and 2 and ‘Additional methods’ in Methods; see attached pdf).
To estimate the physiology of LUCA, authors first inferred an updated microbial phylogeny from 57 phylogenetic marker genes on 700 genomes(!!!), comprising 350 Archaea and 350 Bacteria. This tree was in good agreement with recent phylogenies of the archaeal and bacterial domains of life. For example, recently the TACK and Asgard clades of Archaea and Gracilicutes within Bacteria were recovered as monophyletic. Unfortunately, this analysis is equivocal as to the phylogenetic placement of the Patescibacteria (CPR) and DPANN, which are two small-genome lineages that have been difficult to place in trees. Approximately unbiased tests could not distinguish the placement of these clades — neither at the root of their respective domains nor in derived positions, with CPR sister to Chloroflexota (as reported recently) and DPANN sister to Euryarchaeota. To account for this phylogenetic uncertainty, authors performed LUCA reconstructions on two trees: their maximum likelihood (ML) tree, and a tree in which CPR were placed as the sister of Chloroflexota, with DPANN sister to all other Archaea. In both cases, the gene families mapped to LUCA were very similar.
Authors used the probabilistic gene- and species-tree reconciliation algorithm ALE to infer the evolution of gene family trees for each sampled entry in the KEGG Orthology (KO) database on their species tree. [ALE infers the history of gene duplications, transfers and losses based on a comparison between a distribution of bootstrapped gene trees and the reference species tree, allowing authors to estimate the probability that the gene family was present at a node in the tree.] This reconciliation approach has several advantages for drawing inferences about LUCA. Most gene families have experienced gene transfer since the time of LUCA; therefore, explicitly modeling transfers enables authors to include many more gene families in the analysis — than has been possible using previous approaches. Because the analysis is probabilistic, authors can also account for uncertainty in gene family origins and evolutionary history by averaging over different scenarios using the reconciliation model. Using this approach, authors estimated the probability that each KEGG gene family (KO; KEGG Orthology) was present in LUCA and then used the resulting probabilities to construct a hypothetical model of LUCA’s gene content, metabolic potential (Fig. 2) and environmental context (Fig. 3).
Using the KEGG annotation is beneficial because it allows the authors to connect their inferences to curated functional annotations; however, it has the drawback that some widespread gene families that were likely present in LUCA are divided into multiple KO families that individually appear to be restricted to taxonomic groups and are inferred to have arisen later. To account for this limitation, authors also performed an analysis of COG (Clusters of Orthologous Genes) gene families (which correspond to more coarse-grained functional annotations)
By using modern prokaryotic genomes as training data, authors used a predictive model to estimate the genome size and the number of protein families encoded by LUCA based on the relationship between the number of KEGG gene families and the total number of proteins encoded by modern prokaryote genomes. On the basis of the prokaryotic proteins (PPs) for KEGG Orthology (KO) gene families, authors identified a conservative subset of 399 KO gene families that were likely to be present in LUCA (with PPs ≥0.75, and found in both Archaea and Bacteria); these families formed the basis of the authors’ metabolic reconstruction. However, by integrating over the inferred PPs of all KO gene families — including those with low probabilities — authors also estimated LUCA’s genome size. Their predictive model estimates a genome size of 2.75 Mb (2.49–2.99 Mb) encoding 2,657 (2,451–2,855) proteins…!!
A list of 298 universal marker genes was identified by creating a non-redundant list of markers used in previous studies on archaeal and bacterial phylogenies. Although authors can estimate the number of genes in LUCA’s genome, it is more difficult to identify the specific gene families that might have already been present in LUCA — based on the genomes of modern Archaea and Bacteria (it is likely that the modern version of the pathways would be considered incomplete based on LUCA’s gene content through subsequent evolutionary changes). We should therefore expect reconstructions of metabolic pathways to be incomplete — due to this phylogenetic noise and other limitations of the analysis pipeline. For example, when looking at genes and pathways that can uncontroversially be mapped to LUCA, (e.g., the ribosome and aminoacyl-tRNA synthetases for implementing the genetic code), authors found that they have mapped many (but not all) of the key components to LUCA. Authors interpret this to mean that their reconstruction is probably incomplete — but their interpretation of LUCA’s metabolism relies on their inference of pathways, not individual genes.
The inferred gene content of LUCA therefore suggests it was an anaerobe — because authors did not find support for the presence of terminal oxidases. Instead, authors identified almost all genes encoding proteins of the archaeal (and most of the bacterial) versions of the Wood–Ljungdahl pathway (WLP) (prokaryotic proteins PPs > 0.7), indicating that LUCA had the potential for acetogenic growth and/or carbon fixation. LUCA encoded some Ni-,Fe-hydrogenase subunits, which may have enabled growth in hydrogen. [Enzyme complexes involved in methanogenesis such as methyl-coenzyme M reductase and tetrahydromethanopterin S-methyltransferase were inferred to be absent, suggesting that LUCA was unlikely to function as a modern methanogen.]
Authors found strong support for some components of the TCA cycle (including subunits of oxoglutarate/2-oxoacid ferredoxin oxidoreductase, succinate dehydrogenase, and homocitrate synthase) — although some steps are missing. LUCA was probably capable of gluconeogenesis/glycolysis in that they found support for most subunits of enzymes involved in these pathways. Considering the presence of the Wood–Ljungdahl pathway (WLP), this may indicate that LUCA had the ability to grow organoheterotrophically and potentially also autotrophically. Gluconeogenesis would have been important in linking carbon fixation to nucleotide biosynthesis via the pentose phosphate pathway — most enzymes of which seem to be present in LUCA. Authors found no evidence that LUCA was photosynthetic — with low P-values for almost all components of oxygenic and anoxygenic photosystems.
Authors found strong support for the presence of ATP synthase, specifically, the A and B subunit components of the hydrophilic V/A1 subunit, and the Ia and Kc subunits of the transmembrane V/A0 subunit. This finding is consistent with many previous studies that have mapped ATP synthase subunits to LUCA. Authors also found support for the presence of pathways for assimilatory nitrate (ferredoxin-nitrate reductase), ferredoxin-nitrite reductase, and sulfate reduction (sulfate adenylyltransferase, sulfite reductase, and phosphoadenosine phosphosulfate reductase) — probably to fuel amino acid biosynthesis, for which authors had inferred the presence of 37 partially or complete pathways.
Authors also found support for the presence of 19 class 1 CRISPR–Cas effector protein families in the genome of LUCA — including types I and III, cas10, and cas7. The absence of Cas1 and Cas2 may suggest that the LUCA encoded an early Cas system with the means to deliver an RNA-based immune response by cutting (Cas6/Cas3) and binding (CSM/Cas10) RNA, but lacking the full immune-system-site CRISPR. This finding supports the idea that the effector stage of CRISPR–Cas immunity evolved from RNA-sensing for signal transduction, based on the similarities in RNA-binding modules of the proteins. [This conclusion is consistent with the idea that cellular life was already involved in an arms race with viruses at the time of the LUCA.] The results of this study [see attached] indicate that an early Cas system was an ancestral immune system of extant cellular life.
Altogether, these metabolic reconstructions suggest that LUCA was a relatively complex organism, similar to existing Archaea and Bacteria. On the basis of ancient duplications of the Sec and ATP synthase genes before LUCA, along with highly significant P-values for key components of those systems (e.g., membrane-bound ATP synthase subunits, genes involved in peptidoglycan synthesis, and the cytoskeletal actin-like protein, MreB) — it is highly likely that LUCA possessed the core cellular apparatus of modern prokaryotic life. This also might include the basic constituents of a phospholipid membrane, although the authors’ analysis did not conclusively establish its composition. In particular, authors recovered the following enzymes involved in the synthesis of ether and
ester lipids (alkyldihydroxyacetonephosphate synthase, glycerol 3-phosphate, and glycerol 1-phosphate), and components of the mevalonate pathway (mevalonate 5-phosphate dehydratase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, and hydroxymethylglutaryl-CoA synthase).
Compared with previous estimates of LUCA’s gene content — authors found 81 overlapping COG (clusters of orthologous genes) gene families with the consensus dataset of ref. 7; and 69 overlapping KEGG Orthology genes (KOs) with the dataset of ref. 6. Key points of agreement between the present study and previous studies include: the presence of signal recognition particle protein, ffh [homolog of the mammalian Signal Recognition Particle 54 (SRP54) protein] used in the targeting and delivery of proteins for the plasma membrane, a high number of aminoacyl-tRNA synthetases for amino acid synthesis, and glycolysis/gluconeogenesis bacterial enzymes.
Ref. 6 inferred LUCA to be a thermophilic anaerobic autotroph using the Wood–Ljungdahl pathway (WLP) for carbon fixation — based on the presence of a single enzyme [carbon monoxide dehydrogenase (CODH)] — and similarly suggested that LUCA was capable of nitrogen fixation using a nitrogenase.
Authors used a probabilistic approach to reconstruct LUCA (i.e., they estimated the probability with which each gene family was present in LUCA, based on a model of how gene families evolve along an overarching species tree). [This approach differs from analyses of phylogenetic presence–absence profiles, or those that used filtering criteria (such as broadly distributed or highly vertically evolving families) to define a high-confidence subset of modern genes that might have been present in LUCA.] This study reconstruction maps many more genes to LUCA — albeit each with lower probability — than previous analyses and yields an estimate of LUCA’s genome size that is within the range of modern prokaryotes. The result is an incomplete picture of a cellular organism that was prokaryote grade (rather than progenotic) and that, similarly to prokaryotes today, probably existed as part of an ecosystem. As the common ancestor of sampled, extant prokaryotic life, the LUCA is the oldest node on the species tree that can be reconstructed via phylogenomics but, as Fig. 3 illustrates, it was already the product of a highly innovative period in evolutionary history — during which most of the core components of cells were established. Authors cannot reconstruct LUCA’s contemporaries using phylogenomics, but they can propose hypotheses about their physiologies, based on the reconstructed LUCA whose features immediately suggest the potential for interactions with other prokaryotic metabolisms.
The inference that LUCA used the Wood–Ljungdahl pathway (WLP) helps constrain the environment and ecology in which it could have lived. Modern acetogens can grow autotrophically on H2 (and CO2) or heterotrophically on a wide range of alternative electron donors — including alcohols, sugars, and carboxylic acids. This metabolic flexibility is key to their modern ecological success. Acetogenesis — whether autotrophic or heterotrophic — has a low energy yield and low growth efficiency (although use of the reductive acetyl-CoA pathway for both energy production and biosynthesis would decrease the energy cost of biosynthesis); this would be consistent with an energy-limited early Earth biosphere.
If LUCA functioned as an organoheterotrophic acetogen, it was necessarily part of an ecosystem containing autotrophs providing a source of organic compounds (because the abiotic source flux of organic molecules was minimal on the early planet). Alternatively, if LUCA functioned as a chemoautotrophic acetogen, it could (in principle) have lived independently off an abiotic source of H2 (and CO2). However, it is implausible that LUCA would have existed in isolation — because the by-products of its chemoautotrophic metabolism would have created a niche for a consortium of other metabolisms (as found today in modern sediments) (see Fig. 3d). This would include the potential for LUCA itself to grow as an organoheterotroph.
A chemoautotrophic acetogenic LUCA could have occupied two major potential habitats (Fig. 3e): the first is the deep ocean floor, where hydrothermal vents and serpentinization of sea-floor provided a source of H2. Consistent with this, authors found support for the presence of reverse gyrase (prokaryotic protein PP = 0.97, a hallmark enzyme of hyperthermophilic prokaryotes), which would not be expected if early life existed at the ocean surface (although the evolution of reverse gyrase is complex).
The second habitat is the ocean surface where the atmosphere would have provided a source of H2 derived from volcanoes and metamorphism [the process in which rocks are transformed into new rocks with different mineral compositions or textures due to factors like heat, pressure, and chemical reactions with fluids]. Indeed, authors detected the presence of spore photoproduct lyase, which in extant organisms repairs methylene-bridged thymine dimers occurring in spore DNA because of damage induced through ultraviolet (UV) radiation. Previous studies have often favored a deep-ocean environment for LUCA, because early life would have been better protected there from an episode of Late Heavy Bombardment (LHB). However, if the LHB was less intense than initially proposed, or just a sampling artefact — these arguments weaken. Another possibility may be that LUCA inhabited a shallow hydrothermal vent or a hot spring.
Diatomic hydrogen (H2) fluxes in these ecosystems could have been several times higher on the early Earth (with its greater internal heat source) than today. With the present H2 flux and the known scaling of the H2-escape rate to space, an abiotic atmospheric concentration of H2 of ~150 ppmv (parts per million by volume) is predicted. Chemoautotrophic acetogens would have locally drawn down the concentration of H2 (in either surface or deep niche) but their low growth efficiency would ensure H2 (and CO2) remained available. This — and the organic matter and acetate produced — would have created niches for other metabolisms, including methanogenesis (see Fig. 3d).
Based on thermodynamic considerations — methane (CH4) and carbon dioxide (CO2) are expected to be the eventual metabolic end-products of the resulting ecosystem, with a small fraction of the initial hydrogen consumption buried as organic matter; the resulting CH4 flux into the atmosphere would fuel photochemical H2 regeneration and associated productivity in the surface ocean (see Fig. 3e of attached). Existing models suggest that the resulting global H2-recycling system is highly effective, such that the supply flux of H2 to the surface could have exceeded the volcanic input of H2 to the atmosphere by at least an order of magnitude — in turn implying that the productivity of such a biosphere was boosted by a comparable factor. Photochemical recycling to carbon monoxide (CO) would also have supported a surface niche for organisms consuming CO.
In deep-ocean habitats, there could be some localized recycling of electrons (see Fig. 3d of attached), but a quantitative loss of highly insoluble H2 and CH4 to the atmosphere, and minimal return after photochemical conversion of CH4 to H2, would mean global recycling to depth would be minimal (see Fig. 3e). Hence, the surface environment for LUCA could have become dominant — although recycling of the resulting organic matter could be spread through the ocean depths (i.e., ‘deep heterotrophic ecosystem’ in Fig. 3e). The global net primary productivity of an early chemoautotrophic biosphere — including acetogenic LUCA and methanogens — could have been about three orders of magnitude less than today.
The nutrient supply (for example, N) required to support such a biosphere would need to balance that amount lost in the burial flux of organic matter. Earth surface redox balance dictates that hydrogen loss to space and burial of electrons/hydrogen must together balance input of electrons/hydrogen. Lightning would have provided a source of nitrite and nitrate — consistent with LUCA’s inferred pathways of nitrite and (possibly) nitrate reduction.
In conclusion, by treating gene presence probabilistically, the authors’ reconstruction maps many more genes (2,657 genes) to LUCA than previous analyses and results — contained in an estimate of LUCA’s genome size (2.75 Mb) that is within the range of modern prokaryotes. The result is a picture of a cellular organism that was prokaryote grade, rather than progenotic grade, and that probably existed as a component of an ecosystem, using the Wood–Ljungdahl pathway (WLP) for acetogenic growth and carbon fixation. [The Wood-Ljungdahl pathway (WLP), also known as the reductive acetyl-CoA pathway, is a metabolic pathway primarily used by anaerobic bacteria and archaea for carbon fixation and energy conservation. The WLP is a crucial process in acetogenesis, where carbon dioxide (CO2) is converted into acetate; and in methanogenesis, where CO2 is used to produce methane.]
Authors cannot use phylogenetics to reconstruct other members of this early ecosystem, but they felt that they could infer their physiologies based on the metabolic inputs and outputs of LUCA. How evolution proceeded from the origin of life — to early communities at the time of LUCA — remains an open question, but the inferred age of LUCA (~4.2 Ga; 340 million years after Earth first formed), compared with the origin of the Earth and Moon, suggests that the process required a surprisingly short interval of geologic time…!!
The nature of LUCA, its age, and its impact on the Earth system — have been the subject of vigorous debate across many diverse disciplines, often based on disparate data and methods. Age estimates for LUCA have usually been based on the fossil record, varying with every reinterpretation. The nature of LUCA’s metabolism has proven equally contentious — with some attributing all core metabolisms to LUCA, whereas others reconstructing a simpler life form, dependent on geochemistry. Here the authors have inferred that LUCA lived ~4.2 Ga (range of 4,009 million to 4,330 million years ago) through divergence-time analysis of pre-LUCA gene duplicates, calibrated using microbial fossils and isotope records under a new cross-bracing implementation.
Phylogenetic reconciliation suggests that LUCA had a genome of at least 2.5 Mb (range of 2.49 to 2.99 Mb), encoding around 2,600 proteins, comparable om size to modern prokaryotes. Authors’ results suggest LUCA was a prokaryote-grade anaerobic acetogen that possessed an early immune system…!! Although LUCA is sometimes perceived as living in isolation, authors infer that LUCA was a component of an established ecological system. The metabolism of LUCA would have provided a niche for other microbial community members, and hydrogen-recycling by atmospheric photochemistry could have supported a modestly productive early ecosystem.
Around ~2000, estimates of the LUCA’s age had ranged from 3.5 to 3.8 billion years ago in the Paleoarchean Era, a few hundred million years before the earliest fossil evidence of life. This present study (see attached pdf) pushes the origin-of-life time back to 4.0 to 4.3 billion years ago.
DwN