Abstract
Both the geologic and genomic records provide information on the earliest history of terrestrial life. Despite the challenges inherent to the fragmentary and highly metamorphosed earliest rock record, microfossils as old as ∼3.5 billion years (Ga) and potential morphological and isotopic traces in earlier rocks suggest the origin and diversification of life within the planet’s first billion years. This is consistent with a growing body of evidence for liquid water in Earth’s surface or near-surface environment by around 4.3 Ga, which provides at least one requirement of habitability. Genomic evidence further informs our understanding of early life, including some aspects of its environment, physiology, and metabolism. In addition, ancestral proteome reconstruction and molecular clocks provide clues to the nature and timing of the last universal common ancestor (LUCA), although the latter methodology remains challenging and highly uncertain. Improved molecular clock methods may be able to better constrain the timing of LUCA and the divergence of the major domains of extant life, while further interrogation of the earliest geologic and geochemical records may be able to reveal even earlier evidence for an inhabited planet.
1. Introduction: Challenges in the Search for the Earliest Terrestrial Life
Experimental studies of prebiotic chemistry aimed at determining how and where terrestrial life developed are complemented by efforts to reconstruct life’s earliest history from empirical evidence. Both the geologic and genomic records are available for this endeavor, although each has its own distinct challenges. As one moves back in time, evidence for life in the geologic record becomes scarcer and inferences of life’s evolution become more difficult. Understanding the inherent limitations of both records is necessary for effective hypothesis testing and identifying opportunities where integration of these approaches can improve their utility.
Challenges in the early geologic record
Earth’s rock record generally becomes sparser and more pervasively altered with age. Accordingly, much of Earth’s first billion years remain obscure due to the fragmentary and highly metamorphosed nature of surviving crust (e.g., Cloud, 1972). Well-preserved sections of the sedimentary record date back only to ca. 3.5 Ga (e.g., Lowe and Byerly, 2007). Evidence for microbial life in these ancient strata includes preserved cellular structures (e.g., Schopf et al., 2007) and macroscopic sedimentary structures associated with stromatolites (fossilized microbial mats; criteria by, e.g., Buick et al., 1981) along with organic carbon that shows the characteristic isotopic fractionation of biotic remains (e.g., House et al., 2000). Progressively higher metamorphic grades transform the amorphous carbon into kerogen and, eventually, graphite (e.g., Fries and Steele, 2018). Cellular structures and macroscopic features such as stromatolites, preserved in sedimentary and lower-grade metasedimentary rocks, are progressively erased by heat and strain. This leaves the first billion years (4.5–3.5 Ga) of Earth history with, at most, highly ambiguous evidence for a biosphere (e.g., reviews by Harrison, 2020; Javaux, 2019; Lepot, 2020), given the very sparse (meta)sedimentary record >3.5 Ga and the complete lack of known surviving rocks >4.03 Ga (Bowring and Williams, 1999; cf. O’Neil et al., 2008). On the other hand, despite the late 20th century consensus that earliest Earth was dry and inhospitable to life (e.g., Cloud, 1972), evidence from the detrital zircon record suggests the presence of liquid water in the surface or near-surface environment by at least 4.3 Ga (Mojzsis et al., 2001; Wilde et al., 2001), with marine sediments as early as 3.83 Ga (Nutman et al., 1997; Manning et al., 2006; cf. Fedo and Whitehouse, 2002). This provides an important necessary condition for an origin of life and a biosphere.
Challenges in the early genomic record
Similar to the geological record, comparative genomics and phylogenetics become more challenging as one moves further back in time. Informative genome sequence alignment sites for reconstructing the deepest divergences in phylogenies become increasingly scarce, due to fewer proteins being conserved across broader taxonomic groups (Crapitto et al., 2022; Theobald, 2010). In addition, multiple substitutions over long evolutionary distances overprint informative sequence substitution events and effectively erase earlier evolutionary information (Philippe and Laurent, 1998; Duchêne et al., 2022).
Ancestral state reconstruction and molecular clock studies similarly become more challenging and less informative as one works backwards in time. In the case of molecular clock studies, the lack of diagnostic microbial fossils before the Paleoproterozoic means that the deepest diversifications in the tree of life remain relatively unconstrained, with ages largely determined by the choice of evolutionary model (Roger and Hug, 2006). This significantly impacts molecular clock age estimates for the last universal common ancestor (LUCA), with correspondingly large uncertainties that are only bounded by the logical older-bound constraint of the Earth/Moon-forming impact (e.g., Betts et al., 2018; Moody et al., 2022; Moody et al., 2024). The lack of age constraints in evolutionary trees is compounded by the variable branch lengths observed for protein and RNA-encoding genes that trace back to LUCA from the archaeal and bacterial domains (Moody et al., 2022) and the long branch lengths associated with ancient protein duplications before LUCA (Mahendrarajah et al., 2023; Gogarten and Taiz, 1992; Fournier et al., 2011). These long branches are almost certainly indicative of high rates of evolution for many genes during the earliest history of life, which violates the underlying assumption of rate distributions in relaxed molecular clock models. As a result, current molecular clock models cannot reliably estimate ages for divergences earlier than the common ancestors of Bacteria and Archaea, respectively.
In dating the bacterial and archaeal crown ages, the general lack of diagnostic prokaryotic fossils is typically circumvented by using the eukaryal fossil record. In Bacteria, branches that represent Eukarya are present in the form of mitochondrial and plastid lineages within alphaproteobacteria and cyanobacteria, respectively (e.g., Muñoz-Gómez et al., 2022; Moore et al., 2019). In Archaea, the cytosolic eukaryal lineage is present as a group within Asgard archaea (e.g., Eme et al., 2023). When eukaryal fossil calibrations are placed on these groups, age estimates for even deeper splits within the Bacteria and Archaea are generated in relaxed molecular clock models, albeit with comparatively large uncertainties. Using this approach, the recovered crown ages are ∼2.75–3.5 Ga for Bacteria and ∼3.5 Ga for Archaea (Betts et al., 2018). Including additional calibrations from the cyanobacterial microfossil record and relative age constraints from horizontal gene transfer moves the bacterial crown age older (∼3.5–3.75 Ga for Bacteria, Fournier et al., 2011). In addition, age estimates for Archaea have been informed by phylogenies that include horizontal gene transfers to cyanobacteria. This permits cyanobacterial fossil calibrations to be directly applied to molecular clocks for Archaea, resulting in an estimated age of >3.7 Ga (Wolfe and Fournier, 2018). While different genomic sequence datasets, phylogenetic trees, and evolutionary models make direct comparisons and synthesis of results from different studies difficult, all of these efforts have consistently predicted the diversification of the major domains of life by the Paleoarchean.
In the case of reconstructing the sequences of the most ancient genes and proteins, the probabilistic inference of ancestral states at homologous sites within biological sequences is strongly dependent on many factors, such as model selection and alignment accuracy, that are complicated by long deep branches in phylogeny (Garcia et al., 2022). Phylogenetic information is lost along long branches due to multiple substitutions, so that inferred ancestral sites are more likely to reflect underlying compositional biases than an evolutionary signal of shared ancestry (Fournier and Gogarten, 2010). Furthermore, protein secondary and tertiary structures over long evolutionary distances often experience changes that current models do not take into account. The combined effect of these factors is to render inferred deep ancestral sequences highly uncertain and unreliable for overall sequence reconstruction. Even with these uncertainties, reconstructing the ancestral states of specific sites under strong functional selection, such as active sites for substrate binding, still likely provides a valuable source of information about the earliest life.
Perhaps one of the most inescapable challenges and limitations of reconstructing early life using the genomic record is the problem of coalescence. As we trace backward along tree branches that relate extant taxa, shared common ancestry results in fewer and fewer branches; hence, less of the diversity that was likely present at any given time is represented by these direct ancestors. In short, our knowledge of early lineages becomes increasingly biased by the criteria of direct ancestry, which may not reflect ecological significance or even be representative of the majority of life coexisting at any given point in time (Zhaxybayeva and Gogarten, 2004). To illustrate this bias, if our knowledge of Mesozoic fauna was only genomic, and thus limited to direct ancestors of extant taxa, we would have absolutely no knowledge of nonavian dinosaurs, pterosaurs, or marine reptiles (except sea turtles). We rely on the fossil record for our knowledge of these groups and any hope of accurate ecological reconstruction. When this same reasoning is applied to the microbial genomic record, it becomes apparent that, based on extant sequences, even an accurate phylogenetic history of the earliest ancestors of Bacteria, Archaea, or LUCA provides only a narrow window into the earliest life and biosphere. Yet even this narrow, biased sampling can potentially confirm the presence of specific biological properties and processes during the earliest history of the biosphere and provide a powerful means of evaluating hypotheses generated from evidence preserved in the geological record.
Strategies for Evaluating Potential Early Life in the Geologic Record
Demonstrating convincing geologic evidence for the earliest life on Earth requires establishing the age and biogenicity of a particular morphological structure or geochemical signal. Importantly, even when the biogenicity of an ancient biosignature is unambiguous, the structure may provide limited utility for testing evolutionary hypotheses; additional structural or physiological traits must be preserved for a fossil to be diagnostic of a specific group of organisms, a necessary condition for time-calibrating evolutionary narratives. For evaluating case studies in the Archean-Hadean geologic record, we will consider the following:
How likely is this feature to form abiotically? What is the host rock’s age (and how was it established)? Is the candidate’s biological feature syngenetic with and indigenous to the rock?
Many features of microfossils can be confused with abiotic mineral formations (e.g., Buick et al., 1981; Schopf et al., 2010; Allwood et al., 2018). Microfossils typically take the form of spheroidal, rodlike, or filamentous cell-like bodies, but small-scale mineral growth in metamorphic or hydrothermal systems can mimic some of these features (e.g., McMahon, 2019). Stromatolites, some of the prototypical macroscopic evidence for Precambrian life, are the remnants of subaqueous microbial mats that accreted sediment and typically consist of laminated carbonates in a variety of conical or domal structures (e.g., Buick et al., 1981). Many features of macroscale stromatolite morphology, such as cone and dome structures, can be mimicked by postdepositional disturbance or folding of sediments (Buick et al., 1981), although Awramik and Grey (2005) argue that a combination of morphological features unlikely to be formed abiotically may serve as evidence for stromatolite biogenicity.
Buick et al. (1981) and Schopf et al. (2007, 2010) advocate sets of criteria for evaluating the biogenicity of putative stromatolites and microfossils, respectively, based on a list of criteria unlikely to occur together in an abiotic system. In a similar vein, Allwood et al. (2006,2018) evaluate putative stromatolites in the Strelley Pool Formation (Pilbara Craton, Western Australia) and the Isua Supracrustal Belt (West Greenland) for biogenicity based on close examination of compositional, structural, and microscale features in the respective rocks. Schopf et al. (2010) consider that no single criterion, but instead multiple lines of evidence for biological morphology and chemistry, is required establish biogenicity. Schopf et al. (2007) argue that the occurrence together of (1) microbe-like structures (the reader is pointed to Schopf et al., 2010 for further details), which are (2) spatially associated with organic carbon, which has (3) an isotopic composition consistent with biological origins, is so unlikely in an abiotic context as to make the interpretation of fossilized life inescapable. The carbon isotopic composition is typically presented as
Host rock age and syngenicity of the biosignal
By their nature, most sedimentary rocks cannot be assigned an absolute age because it is rare that a mineral phase that forms syn-depositionally with the sediment can be radiometrically dated. Occasionally, datable authigenic minerals grow during metamorphism, which define a lower age limit (e.g., xenotime; Rasmussen, 2005). But generally, igneous features, such as dikes or plutons that intrude the sediment—that is, are demonstrably younger—are more readily datable and define the minimum age. If the sedimentary unit is stratigraphically above (i.e., younger than), below (older than), or interbedded with (similar age) a volcanic unit, this can also constrain the age. The greater preservation of the younger sedimentary record often permits correlations among units of large geographic extent, which helps constrain fossil ages. This becomes increasingly difficult with the more fragmentary and metamorphosed Archean record.
Examination of the larger context for the putative biosignal in its host rock may also help identify indigenous and syngenetic versus secondary features or features introduced by, for example, later external material exchange. For example, metamorphic strain affects all parts of a rock that formed before the strain was applied. Chemical features in the rock may have been deposited by later metamorphic or hydrothermal fluid flow, but these will typically be more uniformly distributed than primary geochemical features.
Case Studies in the Geologic Record
Microfossils and stromatolites reported in the Precambrian (meta)sedimentary record have varying morphology, and many reported occurrences are spatially correlated with reduced, isotopically light carbon reminiscent of modern kerogens (e.g., House et al., 2000; Osterhout et al., 2021). The confluence of multiple lines of evidence bolsters the case for their biogenicity. Several proposed occurrences of stromatolites or microfossils with isotopically light carbon as old as ca. 3.5 Ga are generally accepted as biogenic, while others are still subjects of active controversy. Older proposed stromatolites, microfossils, and isotopically light graphite occurrences lack the well-preserved spatial association of isotopically light carbon with identifiable cell-like microstructures, and their biogenicity is even more uncertain.
Reported Paleoarchean microfossils and stromatolites in Pilbara Craton
Several Paleoarchean sites in the Pilbara Craton (Western Australia) host features interpreted as stromatolites or microfossils. These are the most robust evidence for microbial life as early as nearly 3.5 Ga. Supporting evidence combines microfossil morphology with elemental chemistry and δ13CVPDB, which together suggest likely biogenicity.
The Strelley Pool Formation (East Pilbara Terrane) consists of chert interbedded with volcanics and was initially interpreted as a hydrothermal deposit (e.g., Lowe, 1980), although later geochemical results suggest instead a marine-like setting (van Kranendonk et al., 2008). Its age is constrained to 3.42–3.35 Ga by U-Pb zircon dates on underlying and overlying volcanics (Hickman, 2008). While the literature has differed over whether domal structures in the chert are stromatolites or abiotic hydrothermal features (e.g., Lowe, 1980,1994), reappraisal of some features—especially their spatial specificity in particular sedimentary petrofacies and paleoenvironments, along with their morphological diversity—suggests instead a biological origin as microbial mats (Allwood et al., 2006, 2007, 2009). The discovery of microfossil-like structures (e.g., Lepot et al., 2013; Sugitani et al., 2015) associated with isotopically light organic carbon—and texturally specific δ13CVPDB between microfossils and distributed carbon in the rocks—further reinforces the likely biogenicity of these structures (Lepot et al., 2013).
Hydrothermal chert deposits (van Kranendonk, 2006) within the 3.465 Ga Apex Basalt (near Marble Bar, East Pilbara Terrane) contain microstructures variably interpreted as either microfossils (Schopf et al., 2018; Schopf, 1993) or abiotic hydrothermal alteration features (Brasier et al., 2002,2015). The microstructures consist of cell-like compartments, are associated with organic carbon, and show a variety of morphologies interpreted by Schopf et al. (1993, 2018) as representing different taxa of microorganisms. Brasier et al. (2015) suggested that the mineralogy and carbon spatial distribution were more consistent with hydrothermal features, with carbon having migrated into the rock during multiple hydrothermal events. However, Schopf et al. (2018) demonstrated that the various morphologies contained distinctive δ13CVPDB similar to those of different microbial taxa on modern Earth and, in all cases, distinct from the distributed carbon in the chert. This is more difficult to explain by hydrothermal alteration and more consistent with the preservation of microfossils.
Finally, the 3.48 Ga (van Kranendonk et al., 2008) Dresser Formation within the North Pole Dome (East Pilbara Terrane) contains stromatolite-like domal features in a chert-rich member but is pervasively affected by hydrothermal alteration and weathering (van Kranendonk et al., 2008; Brown et al., 2006). Although conclusions about the biogenicity of these stromatolite-like structures have been greatly complicated by the outcrop’s advanced state of weathering, drill cores that have accessed less weathered material have revealed sulfide-rich stromatolite-like structures with apparent microbial remains, isotopically light carbon, and alternating patterns of trace element enrichment similar to modern stromatolites (van Kranendonk et al., 2008; Baumgartner et al., 2019, 2020). This provides a convincing case for biogenicity.
Isua proposed stromatolites: 3.7 Ga
The oldest proposed stromatolites occur in the Isua Supracrustal Belt of southern West Greenland, where supracrustal rocks are cross-cut by 3.7 Ga metaigneous units (Nutman and Friend, 2009), and in which Nutman et al. (2016) reported conical structures in finely laminated metacarbonates, which they interpreted as preserved stromatolites. Subsequent re-examinations of the outcrop by Allwood et al. (2018) and Zawaski et al. (2020) suggested that the apparently conical structures were instead elongated ridges—likely formed during later metamorphism—when viewed from another outcrop surface. It has been suggested that the apparent fine lamination reported in the original study may be a weathering effect, as it does not appear in the cores of ridges (Zawaski et al., 2020). While these later observations do not preclude biotic origins (e.g., Nutman et al. 2021), they cast substantial doubt on biogenicity.
Nuvvuagittuq Supracrustal Belt: 3.75–4.2 Ga (?) proposed microfossils
A series of Fe-encrusted filamentous microstructures in banded iron formation (BIF) of the Nuvvuagittuq Supracrustal Belt (NSB), northern Quebec, Canada, were reported by Dodd et al. (2017), who interpreted them as fossilized Fe-oxidizing bacteria, citing similar filaments formed by such organisms in the more recent geologic record. Elsewhere in the BIF, not directly associated with the filaments, isotopically light carbonaceous material and mineralogical structures consistent with metamorphism of carbon-containing metasediments are found (e.g., Papineau et al., 2011). The NSB consists of a supracrustal succession of metavolcanics and metasediments of uncertain age (cf. O’Neil et al., 2008; Boehnke et al., 2018) crosscut by 3.75 Ga orthogneisses (Cates and Mojzsis, 2007; Cates et al., 2013), which makes these filaments potentially the oldest identified microfossils. However, McMahon (2019) performed experiments showing that “chemical gardens,” which consist of Fe sulfides and solutions of several compositions, can produce similar structures abiotically in the laboratory. While an abiotic mechanism for producing these structures does not ultimately rule out their biogenicity, their abiotic production also becomes a reasonable alternative hypothesis.
Akilia: >3.83 Ga graphite inclusions
Mojzsis et al. (1996) reported the presence of ∼5 µm graphite inclusions encased in apatite grains within marine metasediments of the Akilia region in southern West Greenland. This graphite was isotopically light relative to modern abiotic carbon, with a range in δ13CVPDB similar to the biomass produced by several microbial metabolisms (Lepot, 2020) and the average for kerogens from 3.5 Ga to the present (e.g., Schopf, 2011). The isotopically light carbon suggests the possibility of terrestrial life at or before 3.83 Ga. The likely biogenicity of the light carbon isotope signature and the age and origin of the graphite-hosting metasediments subsequently became areas of controversy in the literature (e.g., van Zuilen et al., 2002; Nutman and Friend, 2006). This discussion follows the overview by Harrison (2020) to which the reader is referred for more detailed information.
Metasediment age
Following some discussion in the literature over the age of the 3.83 Ga tonalite cross-cutting the metasediments and providing age constraints (Nutman et al., 1997; Whitehouse et al., 2001; Mojzsis and Harrison, 2002), Myers and Crowley, 2000 and Whitehouse et al. (2009) argued that its relationship to the metasediment is tectonic rather than intrusive. However, a separate cross-cutting 3.82 Ga gneiss reported by Manning et al. (2006) does support an ancient age for the metasediment. In addition, Fedo and Whitehouse (2002) proposed a metasomatic rather than metasedimentary origin for the rock. However, the presence of mass-independent sulfur isotope fractionations characteristic of Archean supracrustal environments (Manning et al., 2006) is more consistent with metasedimentary origins (cf. Whitehouse et al., 2009).
Origin and location of graphite
Van Zuilen et al. (2002) suggested that isotopically light graphite in the rock could result from metamorphic disproportionation of carbonates, pointing to siderite crystals elsewhere in the region that reacted partially to graphite with a consequent lowering of δ13C in the graphite compared with the original siderite of ∼−6‰. Formation of the ∼−25‰ graphite reported by Mojzsis et al. (1996) would require carbonates already quite low in δ13C. Finally, McKeegan et al. (2007) re-examined the metasediments using three-dimensional Raman imaging and ion microprobe carbon isotope analysis following a study that failed to find graphite (Nutman and Friend, 2006) and demonstrated the presence of isotopically light graphite crystals within apatite in the quartz matrix of the rock.
While the interpretation of light δ13C in the Akilia graphite remains a subject of controversy in the literature (e.g., Van Zuilen et al., 2002; Whitehouse et al., 2009), subsequent work has demonstrated the preservation of the graphite within its enclosing apatite grains (McKeegan et al., 2007) and constrained the age of the metasediments that contain the graphite to the Eoarchean (Manning et al., 2006), establishing this as one of the oldest terrestrial carbon isotopic signatures yet identified.
Saglek Block: Saglek Block: >3.95 Ga (?) graphite
Tashiro et al. (2017) reported isotopically light graphite in metasediments from the early Archean Saglek-Hebron Block, northern Labrador, Canada. Several generations of supracrustal rocks here are intruded by several generations of igneous rocks with components that range as old as 3.95 Ga (Komiya et al., 2015,2017; Shimojo et al., 2016). Graphite in the metapelites (metamorphosed clay-rich sediments) has Raman-measured crystallization temperatures and δ13C values correlated with the metamorphic grade of the host rock, which suggests an origin of the graphite before the metamorphism rather than as later contamination (Tashiro et al., 2017). However, the field relations and complex geochronology of the study area render the age of the metasediments ambiguous (Whitehouse et al., 2019; Harrison, 2020) because >3.95 Ga metaigneous ages are confined to one outcrop (Shimojo et al., 2016; Whitehouse et al., 2019) of uncertain relationship to the graphite-bearing metapelites (Whitehouse et al., 2019). An apparent fault mapped between metapelites and the intruded gneisses near the graphite-bearing outcrop (Tashiro et al., 2017) suggests a tectonic rather than intrusive relationship between the 3.9 Ga gneiss and carbonaceous metapelites (Harrison, 2020). The age of the host rocks cannot, with the current information, be constrained to >3.95 Ga.
Jack Hills zircon: 4.1 Ga graphite inclusion
Detrital zircons from the Jack Hills (Western Australia) range from 3.0 to 4.4 Ga (e.g., Mojzsis et al., 2001; Wilde et al., 2001; Holden et al., 2009). On the basis of several geochemical criteria (e.g., Th/U, Rubatto and Hermann, 2007; Ti-derived crystallization temperature, Watson and Harrison, 2005), the zircons appear to be originally of igneous origin. They contain a variety of mineral inclusions (e.g., Maas et al., 1992; Hopkins et al., 2008, 2010) trapped from their source magma. Rare carbonaceous inclusions were identified by Raman spectroscopy, and two graphite crystals encased in one 4.1 Ga zircon were analyzed via ion microprobe for carbon isotopes (Bell et al., 2015). Their δ13CVPDB of −25 ± 5‰ is isotopically light compared with inorganic sources of carbon in the <3.5 Ga geologic record and much closer to the average for kerogens (e.g., Schopf, 1993), which suggests potential biogenicity. Enclosure in a concordant 4.1 Ga zircon suggests its age as ≥4.1 Ga.
The occurrence of this potential biosignature in igneous zircon requires explanation. Graphite occurrence in modern magmas typically requires a large metasedimentary source component (e.g., Vogt et al., 2023), given the often high concentration of organic carbon in sediments (e.g., Hunt, 1995). An origin in magmas that incorporate sediments is consistent with several separate observations about the zircons, namely, their elevated 18O/16O ratios (e.g., Mojzsis et al., 2001; Wilde et al., 2001), typical for sediment-derived magmas, and their peraluminous mineral inclusions (Hopkins et al., 2008,2010), also consistent with the incorporation of abundant (Al-rich) clays. The disordered nature of the graphite inclusions, as shown by Raman spectroscopy, appears inconsistent with graphite crystallization at igneous conditions (Alleon and Summons, 2019), although high spatial resolution X-ray CT imaging of the graphite inclusions within the host zircon before carbon isotope analysis (Bell et al., 2015) showed no association with cracks. One possible explanation lies in the ca. 100 ppm U in the host zircon (Bell et al., 2015), which should have irradiated the enclosed graphite to the point of some structural disorder in the absence of later annealing (Harrison, 2020).
Genomic Record Case Studies
Inferring the environment of earliest ancestors
Possible environments for life’s origins must contain sources of available energy and organic molecules and must facilitate concentrating mechanisms and synthesis reactions. In addition, these environments must be persistent so that there is sufficient time for chemical and/or Darwinian evolution to take hold (review in Westall et al., 2023). Given these constraints, the two main hypotheses for origin environments are deep-sea hydrothermal vents (e.g., Martin et al., 2008; Russell and Hall, 1997) and surface hydrothermal systems (e.g., Damer and Deamer, 2020; Van Kranendonk et al., 2008). It is also important to distinguish between environments for the origin of life versus the likely environment of LUCA and the earliest domain ancestors. These are likely substantially separated in both space and time (Cantine and Fournier, 2018), as LUCA merely represents the most recent common ancestor of extant life, rather than an origin of cells or any specific metabolism or physiology (see Fig. 3 in Forterre and Gribaldo, 2007; Gogarten and Deamer, 2016).
Perhaps the most direct genomic evidence for thermal constraints in the evolution of ancestral biota comes from the compositional bias within ancestral sequence reconstructions. Compositional analysis is robust to many of the uncertainties in reconstructed ancestral sequences, since it is a “bulk signal” integrated across thousands of individual amino acid or nucleotide sites, which can be informative even if the specific ancestral state at each site remains uncertain. At the time the earliest 16S tree of life phylogenies were constructed, the presence of many deeply branching thermophilic lineages was highly suggestive of a hyperthermophilic LUCA and origin of life (Stetter, 2006; Forterre, 1996). Subsequent ancestral sequence reconstruction analyses of conserved proteins and ribosomal RNA showed that these early ancestors had compositional biases similar to those observed in modern hyperthermophiles (Di Giulio, 2003; Akanuma, 2017). However, more recent analyses have refuted these earlier conclusions. Greatly expanded phylogenies and improved tree reconstruction methods show no such bias toward deeply branching hyperthermophiles (e.g., Martinez-Gutierrez and Aylward, 2021; Raymann et al., 2015), and more sophisticated ancestral sequence reconstruction methodologies recover ancestral protein and RNA compositions that are not indicative of hyperthermophily (Boussau et al., 2008; Groussin et al., 2013). Other genomic evidence for the nonthermophily of LUCA can be found in the phylogenetic distribution of enzymes necessary for hyperthermophily, such as reverse gyrase (Catchpole and Forterre, 2019). Phylogenetic studies have also led to the suggestion that other types of environments could have harbored extant life’s earliest ancestors. For example, evolutionary (Dombrowski, 2023) tree reconstructions based on the relatedness of UV damage repair enzymes, which have a history that traces back to at least LUCA (Kanai et al., 1997), are consistent with life having a common ancestry in surface environments where UV would have exerted selective pressure.
Inferring the metabolism of earliest ancestors
Energy metabolism is a fundamental feature of all living cells, which utilize the favorable chemical energy couples available in specific environments, either through chemotrophy or phototrophy (review in Spormann, 2023). Metabolic processes depend on substrate-specific enzymes to perform each reaction. Therefore, reconstructing the phylogenies of these metabolic protein families can inform which metabolic processes were present in early life and, by inference, the environments these ancestors inhabited. While inferences of the specific gene content of LUCA vary based on sampling, evolutionary models, and databases used (Crapitto et al., 2022; Martin et al., 2016; Koonin, 2003), these studies broadly agree that LUCA possessed an ATP synthase complex to generate ATP from proton gradients and was likely capable of glycolysis. While these same studies did not recover enough conserved gene data to infer more specific metabolic processes, additional phylogenetic analyses of the genes that form iron–sulfur clusters important in mediating electron transport indicate that their ancestry can be traced at least as far back as LUCA (Garcia et al., 2022). One limitation across these studies is the inability of phylogenomic reconstruction to identify ancestral enzymes and functions that may have been subsequently lost or replaced. In addition, they do not reconstruct all of the metabolic diversity that may have been present at the time, but only that within the lineage of shared common ancestry. One recent phylogenetic investigation to determine the likely proteome content of LUCA, based on protein families, revealed that the set of proteins with a high likelihood of being present in LUCA were most consistent with a heterotrophic metabolism performing acetogenesis (Moody et al., 2024). Since a heterotrophic lifestyle depends on the presence of fixed organic carbon substrates (the products of other metabolic processes), this inference also supports the view that LUCA existed within an ecological context with other microbes, presumably members of lineages that diverged before the time of LUCA.
Future Directions
The main barrier to investigating Earth’s earliest geologic record of life is the poor preservation of metasediments older than 3.5 Ga. Potential traces of life earlier than 3.5 Ga have been inferred, consisting of proposed macro- or microscopic microbial structures in sediments and separate traces of potentially biogenic isotopically light carbon, but such evidence is scant and often controversial. Future discoveries of plausibly biogenic structures >3.5 Ga in age that contain isotopically light carbon would be more convincing evidence of early life. This search may expand to other Archean materials, in particular, organic carbon incorporated in minerals that preserve carbon with minimal alteration—such as the zircon and apatite crystals described in Section 3. Statistical analysis of rare early carbon occurrences can help determine whether the origins of such carbon are abiotic or biotic: for example, do early organic carbon occurrences resemble the statistical distribution of δ13CVPDB of known microbial taxa or metabolisms (e.g., Thomazo et al., 2009; Schopf, 2011)? Alternatively, are these more similar to abiotic sources of fractionated carbon, such as carbonate rocks, hydrothermal reaction products, or various mineral phases from meteorites (see, e.g., Schopf, 2011; Marty et al., 2013; Kerridge, 1985)? Improved phylogenomics approaches also promise to increase our ability to resolve the nature and timing of LUCA and the earliest ancestors of extant life. More sophisticated models for reconciling the complex histories of horizontal gene transfers can potentially improve the confidence with which specific protein families and functions can be traced to LUCA, as well as correct potentially erroneous inferences. (Martin, 2008) Currently, these approaches consider the evolutionary history of each protein family individually, with models of expected frequencies of transfer and loss uniformly applied across the Tree of Life. However, these proteins may not necessarily evolve independently, and expected frequencies of transfer and loss events may vary for different clades. Therefore, the development of more sophisticated protein evolution models will likely result in more accurate inferences of the proteomic repertoire of LUCA.
Future directions to improve our timing of LUCA will require more careful evaluation of molecular clock sequence datasets and models. We cannot reasonably hope to find diagnostic fossils from the time of LUCA. Even if such fossils could be found, since we have no known outgroup to LUCA for comparison and no morphological knowledge of LUCA itself, it would be impossible to directly link LUCA to fossils in a way that would apply a meaningful calibration. A more hopeful expectation is to find fossils or biosignatures of early archaeal or bacterial life that can constrain the ages of these major domains—which, in turn, could provide more robust younger bounds on LUCA itself. Apart from this aspiration, efforts should focus on identifying protein family datasets that are consistent with evolutionary models, thereby highlighting those protein families that should be addressed independently in evolving models of molecular revolution.
While integrating Archean geology with phylogenomics is a difficult task, the continued search for non- or only lightly metamorphosed early Archean sediments, which preserve primary sedimentary structures, trace elements, or stable isotopic signatures, may provide some paths forward. For example, predictions from genomics regarding the metabolisms of various microbial ancestors may be testable using carbon or sulfur isotopic information, once candidate early microfossils have been identified (e.g., Thomazo et al., 2009). Information from trace elements, oxygen isotopes, and primary sedimentary structures can be used to test predictions about the physical environments early microbial ancestors inhabited, such as distinguishing shallow subaqueous environments from deeper hydrothermal vents. Both careful examination of the Archean sedimentary record and continued improvements in phylogenomics will be necessary for better constraining our knowledge of LUCA and potentially of the earliest terrestrial life.
Authors’ Contributions
E.A.B.: Writing—original draft preparation, reviewing, and editing. G.P.F.: Writing—original draft preparation, reviewing, and editing.
Footnotes
Acknowledgments
The authors thank Tim Lyons and the PCE3 leadership committee for the invitation to contribute to this special issue.
Author Disclosure Statement
The authors declare they have no conflicts of interest.
Funding Information
E.A.B. is supported by NSF EAR grant #
Associate Editor: Don A. Cowan