Early Microbial Life: Our Past, Present, and Future Project Report

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The site is secure. https:// Log in Show account info Close Account Logged in as: username Dashboard Publications Account settings Log out Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation Bookshelf Search database Books All Databases Assembly Biocollections BioProject BioSample Books ClinVar Conserved Domains dbVar Gene Genome GEO DataSets GEO Profiles GTR Identical Protein Groups MedGen MeSH NLM Catalog Nucleotide OMIM PMC Protein Protein Clusters Protein Family Models PubChem BioAssay PubChem Compound PubChem Substance PubMed SNP SRA Structure Taxonomy ToolKit ToolKitAll ToolKitBookgh Search term Search Browse Titles Advanced Help Disclaimer NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health. Early Microbial Life: Our Past, Present, and Future Project Report Project Report Washington, DC: American Society for Microbiology 2025 Copyright and Permissions © 2025 American Society for Microbiology. All rights reserved. This work is being shared with NCBI under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License Governors, American Academy of Microbiology GOVERNORS, AMERICAN ACADEMY OF MICROBIOLOGY Vanessa Sperandio, Ph.D., Chair Karen C. Carroll, M.D. Sean Crosson, Ph.D. Suzanne Fleiszig, O.D., Ph.D. Susan Golden, Ph.D. Jay T. Lennon, Ph.D. Melissa B. Miller, Ph.D. Susan E. Sharp, Ph.D. Alfredo Torres, Ph.D. Paul E. Turner, Ph.D. Susan Weiss, Ph.D. Henry Neal Williams, Ph.D. This project was supported by a grant from the Gordon and Betty Moore Foundation. PROJECT STEERING COMMITTEE Michael Lynch, Ph.D. (Co-Chair) Vaughn Cooper, Ph.D. (Co-Chair) Susana Coelho, Ph.D. Betül Kaçar, Ph.D. William Ratcliff, Ph.D. Paul E. Turner, Ph.D. PROJECT PARTICIPANTS Frank Aylward, Ph.D. Devaki Bhaya, Ph.D. Thibaut Brunet, Ph.D. Shelley Copley, Ph.D. Andrew Ellington, Ph.D. Laura Eme, Ph.D. Toni Gabaldón, Ph.D. Peter Gogarten, Ph.D. Holly Goodson, Ph.D. Georg Graf von Hochberg, Ph.D. Galen Halverson, Ph.D. Katrin Hammerschmidt, Ph.D. Matthew D. Herron, Ph.D. Julie Huber, Ph.D. Laura A. Katz, Ph.D. Eugene Koonin, Ph.D. Eric Libby, Ph.D. Purificación López García, Ph.D. Timothy W. Lyons, Ph.D. John McCutcheon, Ph.D. Elizabeth Ostrowski, Ph.D. Eva Pillai, Ph.D. Susannah Porter, Ph.D. Thomas Richards, Ph.D. Patricia Sanchez-Baracaldo, Ph.D. Julia Schwartzman, Ph.D. Arnau Sebé-Pedrós, Ph.D. Carl Simpson, Ph.D. Michael Travisano, Ph.D. Arvind Varsani, Ph.D. Paula V. Welander, Ph.D. Tom A. Williams, Ph.D. Peter Yunker, Ph.D. ACKNOWLEDGEMENTS This project is a product of the American Academy of Microbiology (Academy), a think tank at ASM. The project and this report were supported by a grant from the Gordon and Betty Moore Foundation to the Academy. A special thanks to Jon Kaye, Ph.D., Program Director in the Science Program at the Moore Foundation, for the partnership and insights on the project. This project and publication would not have been possible without the dedication, scientific leadership, and guidance of the Steering Committee. Especially, thank you to the co-chairs, Michael Lynch, Ph.D., and Vaughn Cooper, Ph.D., for the scientific vision and expertise to bring the project to completion. We also greatly appreciate the Steering Committee, who contributed to the design of the colloquia as well as to writing, reviewing, and editing of the report. We are very grateful for the contributions of the project participants who have collaborated with us on this project and written a section (or two) of the report. This report is based on the deliberations of experts who gathered to discuss a series of questions developed by the steering committee. All participants had the opportunity to provide feedback, and every effort has been made to ensure that the information is accurate and complete. The contents reflect the views of the participants and are not intended to reflect official positions of the Academy or of American Society for Microbiology. We greatly appreciated the support from Stefano Bertuzzi, Ph.D., MPH, ASM Chief Executive Officer, and Kimberly Shankle, Chief People and Change Management Officer, for this project. We would like to give special thanks to Nguyen K. Nguyen, Ph.D, MBA, Director, American Academy of Microbiology, Donalyn Scheuner, Ph.D., Program Officer, and Raemond Edwards, Academy Associate, for their expertise and efforts to develop the project and this report. We want to thank the ASM Communications Department for promoting the report. Finally, many people have advised and contributed to the colloquia and report, especially, thanks to Frank Rosenzweig, Ph.D. for scientific advice, Anthony Burnetti, Ph.D. for editing, and to Johnny Chang for designing the report. EXECUTIVE SUMMARY Life on Earth has evolved over the past 4 billion years, from simple, non-living chemical compounds to early unicellular life, culminating in the vast diversity of unicellular and multicellular organisms seen today. Understanding this extraordinary journey requires exploring how life first emerged, adapted, and diversified into increasingly sophisticated forms. In recent decades, advances in all of these areas, combined with new technologies for imaging and microscopy, isotopic analysis, single-cell sequencing, bioinformatics, artificial intelligence, computation, and deep-sea exploration, have positioned researchers to reach an unprecedented understanding of early microbial life (EML). The project aimed to critically examine what we know about the evolutionary trajectory of early microbial life by bringing together experts from diverse fields, including geobiology, paleogeology, oceanography, biophysics, bioinformatics, mathematics, microbiology, cell biology, virology, evolutionary biology, and evolutionary genetics. The endeavor uniquely emphasized the discoveries that might be promoted with an expanded role for microbiology in the collaborative network of scientists studying the origins of early microbial life. Furthermore, the conclusions of the project focused on projecting how advanced technologies and capabilities could be applied for groundbreaking progress in the future. Insights from microbial origins could inform biotechnology, environmental sustainability, climate science, and even planetary exploration, setting the stage to address many of our biggest human challenges. Supported by a grant from the Gordon and Betty Moore Foundation, the American Academy of Microbiology, a think tank at American Society for Microbiology, approached this project by bringing together experts from diverse areas of expertise around the world to meet at three colloquia. Given the immense scope of life's history, the discussions at each colloquium aimed to focus on one of the three key areas in the study of microbial evolution: (i) the origin of cellular life and the evolution of prokaryotic lineages, (ii) the development of cellular functions that led to the emergence of major eukaryotic groups, and (iii) the acquisition of multicellularity in both prokaryotic and eukaryotic organisms. Exploration of each transition was further divided into fundamental topics, allowing for an in-depth examination of recent breakthroughs, emerging technologies, and existing knowledge gaps. From pre-life to the tree of life The first EML colloquium centered on the origins of life on Earth, starting with chemical precursors and ending with the evolution of the last universal common ancestor (LUCA). This time span encompasses the transition from inanimate matter to self-replicating systems like modern prokaryotes, one of the greatest mysteries in all of science. For example, the protocell hypothesis was addressed and discussed; a central topic in the emergence of cellular life is the formation of protocells, primitive membrane-bound structures capable of housing biochemical reactions. One hypothesis is that protocells originated from micelles or vesicles formed spontaneously from lipids in hydrothermal environments, such as those present in the current-day deep ocean environments, although there are several other equally plausible hypotheses. Experiments have shown how small vesicles can spontaneously fuse and split, modeling rudimentary cell division (see, e.g., Chen 2006 Colloquium 1 concerned the various efforts to reconstruct the LUCA's key features, including genomic analyses that suggest the LUCA was far from primitive. For example, the LUCA likely had an impressive genome size of 1.6-2.7 Mb, perhaps encoded complex molecular machinery, including components of cellular defense such as CRISPR-Cas-like immune systems, and possessed the ability to thrive under anaerobic environmental conditions (Taib et al. 2022; Coleman et al. 2021 Witwinowski et al. 2022 From Colloquium 1, it became very evident that an interdisciplinary approach is vital to unlocking these mysteries and that it is desirable to better integrate tree-of-life studies with Earth-system models. To answer such complex questions, the colloquium emphasized integrating across myriad fields including paleobiology, geology, chemistry, molecular biology, and evolutionary biology. For example, Lyons et al. ( Lyons et al. 2024 Diversification and rise of eukaryotes The second EML colloquium investigated one of the most transformative events in the history of life on Earth: the emergence of eukaryotic cells. Eukaryogenesis and subsequent diversification introduced internal compartmentalization, organelles, and cytoskeletal dynamics that are associated with these modern relatively complex life forms when compared to prokaryotic species in the Bacteria and Archaea domains. Key topics discussed in Colloquium 2 included (i) the geological context of eukaryotic origins leading to a tremendous diversity of phylogenetic and morphological types, (ii) the molecular and cellular biology of organelle evolution and occurrence of greater protein complexity, and (iii) the evolutionary forces at work during eukaryogenesis, with considerations of population sizes, mutation rates, and energy constraints faced by these evolving systems. Colloquium 2 participants discussed the possibility of a eukaryotic big bang and whether the rapid diversification of eukaryotes, evidenced by both molecular and morphological differences compared to prokaryotes, was inevitable due to ecological pressures to exploit available niches at the time. Or was eukaryogenesis and subsequent diversification a passive outcome of specific eukaryote features such as their chromosomal architecture, gene duplication, and genome partitioning into nuclear and organelle compartments? Colloquium 2 also discussed other mysteries surrounding eukaryogenesis, bridging fields such as geochemistry and biochemistry. Whereas fossil eukaryotes such as Tappania Brocks et al. 2023 Colloquium 2 highlighted that understanding EML and the emergence of eukaryotes present profound implications for refining evolutionary theory. Eukaryogenesis challenges linear models of evolution. The emergence of eukaryotes represents a profound restructuring of the fundamentals of cellular biology, perhaps comparable in magnitude to the origin of life itself. Colloquium participants highlighted how this transformation complicates traditional approaches for inferring phylogenies and requires models that account for symbiotic mergers, horizontal gene transfer events, and shifts in cellular architecture. Evolution of multicellularity The third and final EML colloquium turned attention to one of biology's most profound puzzles: the causes and consequences of the origin of multicellular organisms. A dramatically different population-genetic context accompanies the emergence of multicellularity, changing the composition and arrangement of variation upon which natural selection and other evolutionary processes can operate. Does emergence of multicellularity reduce the range of possible beneficial mutations available for evolution by natural selection? Does it increase the possibility for deleterious mutations to accumulate via genetic drift? While multicellularity and its associated net benefits might seem inevitable in hindsight, its actual evolutionary history is sparse and highly contingent. Why does multicellularity evolve? Such questions probe whether complex organisms arose due to selection for key interactions, especially cooperation and division of labor, versus multicellular life emerging more passively, due to physical and genetic constraints. Is cellular and organismal complexity promoted by natural selection? If so, how do we explain morphological (but not biochemical) evolutionary stasis in bacteria, which have had the luxury of time to make such transitions (and in a few cases have done so)? Why has complex multicellularity evolved only a handful of times, even though it can readily emerge in laboratory selection experiments? The third EML colloquium revisited the “major transitions in evolution of life” framework. Participants grappled with ideas and models which posit that life evolves through cooperative mergers, especially cells giving up their individual autonomy to become parts of a larger whole. Multicellularity certainly seems to fit this pattern, as formerly independent cells became specialized units within larger organisms. Here it became evident that there is a need for more research across a greater variety of organisms to best understand the pros and cons of multicellularity. The comparative method could prove useful for drawing generalities across the tree of life; organisms such as holozoa and fungi remain poorly studied compared to other biological systems but exhibit clear multicellular architectures worthy of increased research attention. Plasmodial and cellular slime molds such as Dictyostelium Taken together, the three EML colloquia sought to synthesize expertise across traditionally siloed disciplines, with the ambitious goal to reshape the trajectory of microbial evolutionary studies. It is hoped that this effort will inspire a new generation of EML researchers to tackle foundational questions using modern tools and interdisciplinary frameworks, facilitating the merger of microbial science with evolutionary biology. By systematically addressing life's grand transitions, ranging from its origins to greater complexity, and the advent of cooperation among cell types, the stage is set for a deeper understanding of our biological roots. Microbial life underpins every ecosystem on Earth. Its history is our history. From the first metabolic reactions in Earth's cradle(s) of life to the rise of complex, multicellular organisms, the evolutionary story of microbes is also the story of life itself. I. INTRODUCTION: EVOLUTION OF MICROBES, A JOURNEY FROM PRE-LIFE TO MULTICELLULARITY Life on Earth has evolved over the past 4 billion years, transitioning from simple, non-living chemical compounds to early unicellular life, culminating in the vast diversity of unicellular and relatively complex multicellular organisms seen today. Understanding this extraordinary journey requires exploring how life first emerged, adapted, and diversified into increasingly sophisticated forms. Historically, the study of early microbial life has been approached from avenues that included (i) examination of microscopic fossils of bacteria and archaea preserved in ancient rock formations, (ii) analysis of ancient rocks for isotopic and geochemical signatures and biomarkers of biological activity, (iii) utilization of modern extremophile species as models of the first evolving microbes, (iv) simulation of early Earth conditions to mimic chemical evolution and emergence of microbial metabolism, (v) tracing the evolutionary relationships between organisms, and (vi) derivation of astrobiological insights from collection of planetary specimens and measurements of planetary conditions. In recent decades, advances in each of these fields, combined with new technologies for imaging and microscopy, isotopic analysis, single-cell sequencing, bioinformatics, artificial intelligence, computation, and deep-sea exploration, have positioned researchers to reach an unprecedented understanding of early microbial life (EML). One may wonder what may lie ahead and what scientific areas should be further investigated to advance the field and bring more knowledge to society at large. This project was started by our interest and curiosity to gain a deeper understanding of three of the primary evolutionary events in the history of life on Earth: the origins of life from simpler physical/chemical beginnings, the emergence of the eukaryotic lineage, and the evolution of multicellularity. All three issues have engaged the attention of scientists in many fields outside of biology, e.g., physicists, chemists, computer scientists, and geologists. Even within biology, most of the focus has been on identifying plausible physical/chemical settings for the major transitions, often (but not always) acknowledging that significant uncertainties exist on the ranking of alternatives. Guided by our understanding of how modern biochemistry and cell biology works, such an approach has helped inform our understanding of the peculiar features of biology, even though most of the hypotheses must be wrong. However, in these endeavors, remarkably little (generally, no) attention is given to the fine details of known evolutionary processes. Instead, we are confronted with an odd situation in which the primary subject material (evolutionary transitions) is largely uninformed by the principles of evolutionary biology itself. We worry about where the metals and cofactors that enzymes rely on came from, how the mechanisms of cellular bioenergetics came to be, and how life came to depend on lipids, a genetic code, RNAs with catalytic functions, and the case of the mitochondrion embraced an endosymbiont that might have initially been a parasite. Without direct observations from billions of years ago, the claims being made sometimes seem fantastical. Other times, they are made more plausible by experimentation on modern end points. But almost always, scenarios fail to take full advantage of the breadth of information and theory available. Part of the problem is that most evolutionary biologists are primarily engaged in trying to understand the features of modern lineages, with most attention being devoted to animals and land plants and timescales ranging from just a few hundreds to thousands of generations. Yet, guided by experimental validation, evolutionary theory has successfully achieved rigor comparable to other quantitative fields in the life sciences, such as biophysics. We know with certainty that evolutionary processes follow certain rules, dictated by the features of mutation, recombination, and random genetic drift, and we also know how to formulate expressions in ways that illuminate the probabilities of alternative outcomes, rates of transitions, etc. (Wright 1999; Kimura 1983 Charlesworth and Charlesworth 2010 Walsh and Lynch 2018 What follows is an incomplete listing of ways in which the integration of evolutionary thinking might help advance our understanding of the three major foci noted above. The first goal is to endow nonevolutionary biologists with an understanding of why the rules of evolution are just as pertinent to early-life hypotheses as the principles of chemistry and physics. The second goal is to inspire evolutionary biologists to appreciate that early-life (so-called macroevolutionary) transitions provide just as fertile (and in some cases simpler) grounds for study from a theoretical standpoint as do issues on microevolutionary timescales. We will place emphasis on all three open questions of the EML colloquia with the closing sections providing brief overviews of evolutionary theory being developed in evolutionary cell biology that are relevant to these issues and relevant key knowledge gaps. Supported by a grant from the Gordon & Betty Moore Foundation, the American Academy of Microbiology, a think tank at the American Society for Microbiology, has exercised its convening power to organize three colloquia to draw new insights into this topic. The project brought together experts from diverse fields, including geobiology, paleogeology, oceanography, biophysics, bioinformatics, mathematics, microbiology, cell biology, virology, evolutionary biology, and evolutionary genetics. The endeavor uniquely emphasized the discoveries that might be promoted with an expanded role for microbiology in the collaborative network of scientists studying the origins of early microbial life. Furthermore, the conclusions of the colloquia focused on projecting how advanced technologies and capabilities could be applied for groundbreaking progress in the future. Given the immense scope of life's history, the discussions at the colloquia were focused on three key areas in the study of microbial evolution: (i) the origin of cellular life and the evolution of the major prokaryotic lineages, (ii) the development of cellular functions that led to the emergence of major eukaryotic groups, and (iii) the acquisition of multicellularity in both prokaryotic and eukaryotic organisms. Each transition was further divided into fundamental topics, allowing for an in-depth examination of recent breakthroughs, emerging technologies, and existing knowledge gaps. The primary goals of the colloquia were threefold: (i) to identify critical knowledge gaps in microbial evolution, (ii) to evaluate existing tools and resources and propose future needs, and (iii) to explore the societal implications of microbiological discoveries. The effort also challenged the participants with diverse scientific expertise to think ambitiously. What might the field of microbial evolution achieve if it were granted resources comparable to those in high-energy physics or space exploration? Imagining such abundant resources encouraged researchers to speculate about a “moonshot” approach and encouraged their transformative thinking. With the right infrastructure, technologies, and international collaboration, it is plausible that scientists could recreate protocell environments, engineer evolutionary simulations at unprecedented scales, and apply deep learning models to millions of genomic records to identify unknown evolutionary patterns. Looking forward, the overarching premise is that elucidating the history of microbial evolution on Earth is not only a scientific challenge but also a cornerstone for addressing current real-world problems. Insights from microbial origins could inform biotechnology, environmental sustainability, climate science, and even planetary exploration, setting the stage to address our loftiest ambitions and perhaps to solve many of our biggest human challenges. II. PLANETARY, GEOCHEMICAL CONDITIONS, AND FOSSIL RECORD OF EARLY MICROBIAL LIFE A. Introduction: framing Earth's biological evolution in a geological context The evolution of life on Earth is fundamentally and intricately tied to the planet's dynamic geological history. Thus, it is essential to view the major transitions in the history of life, from its origins through the emergence of complex multicellularity, in the context of the evolution of the planet. This section provides a review of the geological framework for the origin and evolution of microbial life as a basis for exploring how Earth's changing physical and chemical conditions both enabled and constrained biological innovation over nearly 4 billion years. The emphasis of this review is on the Precambrian, which spans from Earth's origin about 4.56 billion years ago (Ga) to the beginning of the Phanerozoic Eon, about 540 million years ago (Ma), and which represents 88% of geologic time and perhaps more than 84% of life's evolutionary history. This extended interval encompasses the origin and early evolution of life, the establishment of the planetary biosphere including the earliest production and accumulation of oxygen, the emergence of eukaryotes, and the appearance of complex multicellularity. Invariably, our understanding of Precambrian history is limited by the fragmentary nature of the ancient rock record, becoming more fragmentary the further back in time we look. Our hope is that better integration of geological history with new discoveries from microbial and evolutionary biology will help overcome this limitation and yield new insights about the coevolution of life and Earth. B. Early Earth environments and the origin of life 1. Hadean Earth and the setting for life's emergence The Hadean Eon (4.56-4.0 Ga) represents Earth's earliest history, a period for which no rock record is preserved. Despite this absence, small but crucial fragments of evidence offer glimpses into this formative period. The oldest known rocks on Earth are found in the Acasta Gneiss in Canada's Northwest Territories, with an age of about 4.0 Ga. These metamorphic rocks formed through partial melting of pre-existing crust, indicating that continental crust had begun to form already prior to 4.0 Ga ( Bowring and Williams 1999 Wilde et al. 2001 Cavosie et al. 2007 Among these Hadean-aged zircons, some as old as 4.3-4.2 Ga preserve oxygen isotope signatures consistent with the presence of liquid water on Earth's surface ( Mojzsis et al. 2001 Moody et al. 2024 Doolittle 2000 Glansdorff et al. 2008 Weiss et al. 2016 2. Young sun paradox and early Earth's atmospheric composition Early Earth's habitability presents a fundamental paradox: while evidence suggests oceans of liquid water existed perhaps as early as 4.3 Ga, the early sun was only about 70% as luminous as today's sun, a dimness that should have resulted in frozen oceans. This “faint young sun paradox” necessitates atmospheric greenhouse gases sufficient to compensate for reduced solar luminosity. Carbon dioxide and methane have been proposed as the most likely candidates for this early greenhouse warming. The formation of Earth's core within the first tens of millions of years (reviewed in Carlson et al. 2014 The inward migration of iron to form the core set the stage for the development of Earth's magnetic field, which would help protect the nascent atmosphere, formed by magmatic degassing, from erosion by solar winds. However, the timing of our earliest dynamo and associated magnetic field remains debated ( Tarduno et al. 2015 Tang et al. 2019 2 Recent work has offered a potential solution to this problem, suggesting that large and frequent early asteroid impacts, if of the right size, composition, and trajectory, could have delivered enough metallic iron to the outer parts of Earth's interior to alter the redox balance of magmas and spawn transiently reducing atmospheric conditions ( Zahnle et al. 2020 Wogan et al. 2023 Wogan et al. 2023 Benner et al. 2020 The emerging model depicts an early atmosphere with high levels of CO 2 2 Halevy and Bachan 2017 Kim and Benner 2021 3. Elemental abundances and the origin of life The availability of nutrients, including metals, for life is controlled by and intertwined with the history of planetary accretion, chemical differentiation, tectonic evolution, and redox evolution of the atmosphere and oceans. Astrobiologists have a solid grasp of how selection shaped the use of macronutrients like C, H, O, N, P, and S by life, but the surface abundance of redox-active metals, which are sensitive to Earth's oxygenation, are less well constrained ( Anbar 2008 Lyons et al. 2014 Anbar and Knoll 2002 Understanding life's origins and metabolic evolution requires examining when metal cofactors, shaped by planetary geochemistry, are progressively being integrated into biology (Goldman and Kaçar 2021). Molybdenum (Mo), for example, plays a key role in modern biochemistry but was scarce in early oceans due to the redox environment. This apparent contradiction has fueled hypotheses that early metabolisms relied on more abundant metals like iron (Fe). However, new isotopic evidence suggests Mo-based nitrogen fixation may have evolved earlier than thought, despite low Mo availability ( Stüeken et al. 2015 In addition to metals like Mo and Fe, the availability and reactivity of specific elements posed crucial constraints on early life. Notably, phosphorus (P) stands out as a potential limiting factor in origin-of-life chemistry. Additions of P to organic molecules are critical in molecular transformations, such as the phosphorylation process that converts nucleosides to nucleotides required for RNA synthesis. Phosphate is the dominant form of P dissolved in water and is readily assimilated by modern life, but it is extremely unreactive during non-biological interactions with organic molecules. Consequently, laboratory experiments simulating abiotic phosphorylation typically employ phosphate concentrations orders of magnitude beyond those sustainable in natural waters due to mineral saturation constraints. Recent models have addressed this “phosphate problem” by invoking alkaline lakes with higher phosphate concentrations relative to ocean waters as the cradles of life ( Toner and Catling 2020 Walton et al. 2023 4. Emergence of land areas and origin of life settings The presence of emergent land areas early in Earth's history has important implications for origin-of-life scenarios. While large continents formed later, even small landmasses could have supported prebiotic chemistry, especially in “warm little pond” or “replication-first” models (see Sec. III). Terrestrial environments are favored by some researchers due to challenges posed by seawater salts during the formation of key biomolecules ( Damer and Deamer 2020 Powner et al. 2009 Song et al. 2024 Bada and Korenaga 2018 Korenaga 2021 C. First evidence of life and the Archean Earth 1. Pilbara Craton and earliest evidence of microbial life Much of what we know about early life comes from the Pilbara Craton, a geological formation in Western Australia, where 3.5-Ga stromatolites provide strong evidence for early microbial communities ( Hofmann et al. 1999 Allwood et al. 2006 Windley et al. 2021 Modern microbial mats are metabolically diverse, though they ultimately rely on an autotroph harnessing an energy source and forming the base of a microbial food chain. While microbial mats may be sustained by chemoautotrophs, in shallow water settings such as those inhabited by the ancient Pilbara examples, the autotrophs are presumed to have been photosynthetic. Given the anoxic atmosphere at this time and absence of cyanobacterial fossils, the Pilbara and other age-equivalent microbialites are typically inferred to record anoxygenic photosynthesis ( Noffke et al. 2013 Additional indirect evidence for microbial life from the North Pole Dome comes from very light carbon isotope (δ 13 Ueno et al. 2006 13 Garcia et al. 2021 13 34 Shen et al. 2001 2 Westall and Xiao 2024 Notably, rocks from the Apex chert in this same region also host purported micro-fossils that had long been regarded as the oldest direct fossil evidence of life on Earth at 3.3-3.5 Ga ( Schopf and Packer 1987 Brasier et al. 2002 Schopf et al. 2007 2. Development of oxygenated oases during the Archean era Despite abundant isotopic and mineralogical evidence that the Archean atmosphere was pervasively anoxic, various proxies, such as the isotopic ratios of Fe, Mo, and N, and enrichment of redox-sensitive trace elements suggest that local “oases” of oxygenated surface seawater developed in the Archean, beginning perhaps as early as 3.2 Ga and certainly by 2.8 Ga ( Olson et al. 2013 Catling and Zahnle 2020 2 Fig. 1 Sánchez-Baracaldo et al. 2022 A starkly different narrative is that oxygenic photosynthesis did not appear until the end of the Archean, at which time the resulting O 2 2 2 Fischer et al. 2016 2 2 2 Sánchez-Baracaldo et al. 2022 Bindeman et al. 2018 Blank and Sánchez-Baracaldo 2010 Sánchez-Baracaldo et al. 2022 2 D. Great Oxidation Event and its aftermath 1. Conditions and timing of the Great Oxidation Event The onset of the Great Oxidation Event around 2.4 Ga is marked by the disappearance of mass-independent sulfur isotope fractionation from the geologic record, signaling the rise of atmospheric O 2 −5 −7 Farquhar et al. 2000 Pavlov and Kasting 2002 13 Lyons et al. 2014 The onset of the GOE is closely associated with the first Paleoproterozoic (2.5-1.6 Ga) glaciation, which may have been a global (i.e., “snowball”) glaciation. The link between these events, which together would have dramatically changed the surface of the Earth, may have been a comprehensive reduction in atmospheric methane concentrations linked to the buildup of O 2 Fig. 1 2 Crockford et al. 2018 Hodgskiss et al. 2023 Lyons et al. 2014 Lyons et al. 2021 Anbar and Knoll 2002 FIG 1 Estimates for the evolving oxygen (O 2 2 (more...) 2. Environmental evolution and coevolution with microbial life Early microbial communities were shaped by both ecological interactions and environmental conditions, which in turn influenced evolutionary trajectories ( Goldman and Kacar 2021 2 Lyons et al. 2024 2 Stone et al. 2022 He et al. 2023 FIG 2 Coevolution of ocean chemistry and microbial metabolisms through time. Euxinic and ferruginous refer to anoxic waters rich in hydrogen sulfide and iron, respectively. This figure is reproduced from Lyons et al. 2024, which provides ample details about (more...) As oxygen rose in the atmosphere, dissolved oxygen in the oceans lagged far behind, with full oxygenation of the deep ocean perhaps having to wait until 0.4 Ga (reviewed in Lyons et al. 2024 Fig. 2 Falkowski et al. 2008 E. Rise of eukaryotes 1. Fossil evidence of eukaryotic life The oldest widely accepted eukaryotic fossils are organic-walled microfossils found in fine-grained shales. These are identified as eukaryotic based on features like large size, surface ornamentation, and signs of excystment ( Porter 2020 Valeria lophostriata, Tappania plana, Satka favosa Porter and Riedman 2023 Adam et al. 2017 Porter and Riedman 2023 Most middle Proterozoic eukaryotic fossils come from intracratonic basins, i.e., shallow, long-lived basins formed on continental crust. These environments, relatively common between ~1.7 and 1.4 Ga, were often isolated from the open ocean and may have been episodically restricted or even non-marine. As a result, the fossil record offers only a partial, biased view of eukaryotic life at the time. However, these basins are typically well preserved, unlike ocean-margin settings that often experience deep burial and deformation. If early eukaryotes evolved in shallow coastal or freshwater environments, as some suggest, intracratonic basins may offer our best fossil window into eukaryogenesis. 2. Proterozoic “boring billion” and eukaryotic diversification The ~1-billion-year gap between the origin of eukaryotes and the rise of complex multicellular life remains a major puzzle. This delay suggests that key innovations or environmental conditions were needed before multicellularity could emerge. Hypotheses include low oxygen levels, genomic constraints, Snowball Earth events, and tectonic or oceanic changes ( Knoll 2011 2 Sperling et al. 2013 Simpson 2012 F. Neoproterozoic era and the rise of multicellularity 1. Simple multicellular forms in the Precambrian record The dramatic shift near the Proterozoic-Phanerozoic boundary marks the transition from a largely microbial world to one accompanied by large, multicellular organisms with hard parts—shells, bones, and wood—that are readily preserved in the fossil record. This turning point reshaped the biosphere and left a far more visible mark on the geologic record (and is in fact the reason for the positioning of the boundary). However, this shift in complexity does not correspond to the first appearance of multicellular life. Simple multicellular forms, such as early cyanobacterial filaments and stromatolite-building communities, date back to the Archean ( Grotzinger and Knoll 1999 Schopf 2006 Trypanin spiralis Walter et al. 1976 Han and Runnegar 1992 Siphonoseptum bombycinum Riedman et al. 2023 Horodyskia Horodyski 1982 Rafatazmia chitrakootensis Ramathallas lobatus Bengtson et al. 2017 Zhu et al. 2016 Albani et al. 2010 Additional forms, some with differentiated cells, appear by the end of the Mesoproterozoic. Bangiomorpha pubescens Butterfield 2000 Butterfield 2015 Proterocladus Tang et al. 2020 Bicellum brasieri Strother et al. 2021 Maloney et al. 2021 Tappania Butterfield 2005 2. Cryogenian glaciations and the burst of multicellularity This early multicellular record is framed by two significant episodes of global change. At the onset of the Proterozoic, the Great Oxidation Event overlapped multiple glaciations, at least one of which was likely global. Near the end of the Proterozoic, a pair of Cryogenian global glaciations (the Sturtian and Marinoan Snowball Earth events) was followed by the shorter and less intense Gaskiers glaciation (~580 Ma). In the aftermath, macroscopic life expanded dramatically. Macroalgae, large protistan-grade organisms like Palaeopascichnus Xiao and Laflamme 2009 Seilacher et al. 2003 Grazhdankin and Gerdes 2007 The brief summary above highlights just some of the many examples of Precambrian multicellular life. Multicellularity evolved many times, in both prokaryotic and eukaryotic lineages, throughout the Proterozoic. Before the Neoproterozoic glaciations, multicellular organisms were typically small, relatively rare, and never extremely diverse. After the glaciations, large, complex multicellular organisms were abundant, globally widespread, and diverse. Thus, there seems to be a clear shift in Earth history with respect to the prevalence and importance of multicellular organisms. The key may have to do with the evolution of large size. Simple multicellularity is easy to evolve and easy to lose; it can come and go and did come and go in both prokaryotic and eukaryotic lineages throughout Earth history. More complex multicellularity, on the other hand, used here to refer to organisms characterized by a heritable macroscopic form in which only some cells are in direct contact with the external environment (compare Krause et al. 2022 Bonner 2004 3. Oxygen, predation, and other drivers of multicellularity One of the most popular explanations for the post-Cryogenian rise of complex multicellularity is increased oxygen availability. Oxygen levels appear to broadly correlate with organismal size over time, suggesting low O 2 Krause et al. 2022 Dahl et al. 2010 Sperling et al. 2015 Bozdag et al. 2021 2 Krause et al. 2022 Karhu and Holland 1996 One answer might be that while rising oxygen may have permitted the evolution of large size, ecological interactions and feedbacks were necessary to drive the shift and were slow to begin. Predation in particular is cited as a factor: one defense against predators is to get too big to handle, too big to eat, or too big to kill. Evidence of predatory drill holes and defensive structures in late Proterozoic rocks supports this view ( Porter 2016 Porter 2011 Crockett et al. 2024 Tang et al. 2024 G. Conclusion: Geological context as a framework for understanding life's evolution Earth evolution has both shaped and been shaped by life, from evidence of early habitability captured in Hadean zircons to the rise of complex multicellularity. However, it remains uncertain whether some biological milestones, like oxygenic photosynthesis and eukaryogenesis, align with major environmental shifts, highlighting the difficulty in linking the evolution of Earth's biosphere and environment. As geochemical and fossil records improve, integrating powerful tools of synthetic biology and molecular evolution with geology can help overcome these gaps, offering new ways to infer ancient microbial traits and metabolisms of early microbial life ( Kaçar 2024 Lyons et al. 2024 III. EARLY ORIGINS FROM ABIOTIC PROCESS TO CELLULAR ORGANIZATION AND HIGHER-ORDER MECHANISMS OF EVOLUTION A. Overview of early origins 1. Conditions to support the origin of life The origin of life can be considered both the most important and the hardest problem in biology if not in all of science ( Smith and Morowitz 2016 Mulkidjanian et al. 2012 2. Approaches to studying the origin of life Like many problems in evolutionary biology, the origin of life can be addressed through a bottom-up or a top-down approach, but combining both is essential. All life known to us is cellular, and there seem to be fundamental reasons for the universality of cellular organization in biology. Cells ensure the essential compartmentalization and concentration of the molecules that are involved in metabolic networks and comprise the building blocks for nucleic acid and protein synthesis (see Fig. 3 Babajanyan et al. 2023 Box Approaches to the origin of life. FIG 3 Non-cellular vs protocellular early evolution. The left panel shows a protocellular scenario whereby evolution started from lipid vesicles that enclosed multiple RNA segments, with coevolving viruses and membrane composition becoming more modern with (more...) The bottom-up approach strives to explain how the cellular level of complexity could have emerged from abiogenic molecules. One popular and likely productive concept is that at the earliest stage of pre-biological evolution, compartmentalization was provided by inorganic compartments that exist, in particular, in calcium carbonate or iron sulfide mounds in the vicinity of hydrothermal vents ( Martin et al. 2008 Mrnjavac et al. 2024 Schrum et al. 2010 Chen et al. 2004 Mansy et al. 2008 Mulkidjanian et al. 2012 The top-down approach involves comparative analysis of genomic sequences as well as protein and RNA structures from extant life forms aiming at the reconstruction of ancestral genome and cell compositions. With a continuously expanding collection of genome sequences from across the tree of life and improving phylogenomic methods, such reconstructions are becoming increasingly robust and informative. This was a major discussion topic of the colloquium (Sec. III C). Only about 100 RNA and protein genes are universal across the diversity of life forms ( Koonin 2003 Moody et al. 2024 Krupovic et al. 2023 Mahendrarajah et al. 2023 It might seem that the top-down approach can take us back no further than the LUCA. This is however not the case because the reconstructed gene repertoire of the LUCA includes many ancient paralogs, that is, genes that evolved through multiple duplications followed by diversification along the evolutionary path from the first cells to the LUCA ( Anantharaman et al. 2002 Aravind et al. 2002 Aravind et al. 2002 Pressman et al. 2015 Nissen et al. 2000 Bose et al. 2022 Wilson and Lilley 2021 Wolf and Koonin 2007 The origin-of-life problem is far from being solved. For some key steps, notably the origin of translation, no convincing hypothetical models have even been explored. Furthermore, one has to realize that detailed knowledge of the specific unique events that actually took place on Earth more than 4 billion years ago might not be achievable. The best that can be expected are convincing theoretical and experimental models and eventually, evolution of cells in the laboratory. Nevertheless, recent progress in the origin of life field has been tangible. From the consilience of bottom-up and top-down research efforts at the crossroads of different disciplines, increasingly tight constraints on the key processes required for the origin of life are emerging, paring down the range of realistic scenarios and fueling the hope for a breakthrough in a not so remote future. The gaps in knowledge and proposed future research that could be conducted to achieve groundbreaking progress are further discussed in Sec. VII. B. Cellularity: membranes, metabolism, and energy 1. Membranes and early origins of life a. Compartmentalization of catalytic reactions via a cell membrane structure The abiotic origins of microbial life have been studied extensively over the years with usually a focus on three areas: self-replication of genetic material, primitive metabolism and/or energy generation, and compartmentalization. While debates often ensue on the timing and connection of these three dimensions, it is accepted that all three are important for the formation of the first protocells and eventually the evolution of the last universal common ancestor ( Preiner et al. 2020 Szostak 2017 Sutherland 2016 Lane and Martin 2012 Jordan et al. 2019 Sithamparam et al. 2025 b. Function and structure of cellular membranes Membranes are an essential organelle of all microbial life providing a barrier against harsh environments, a scaffolding for proteins and their enzymatic reactions, and a framework for generating a proton-motive force and thus enabling high-energy metabolism. Overall, membrane lipid structure today is well preserved across all domains of life ( Singer and Nicolson 1972 Harayama and Riezman 2018 Subczynski et al. 2017 Fig. 4 Koga and Morii 2007 Archaea, however, harbor isoprenoidal-based alkyl chains that are ether linked to the G-3-P isomer glycerol-1-phosphate. Archaea also modify their membrane structures in unique ways, that is, linking their bilayers to form monolayers, which are then subsequently modified through methylations, cross-linking, and the addition of cyclopentane and/or cyclohexane rings ( Fig. 4 Schouten et al. 2013 c. Cellular membrane structure in the archaeal and bacterial domains of life The genetic pathways that encode the proteins for generating these different membrane structures in bacteria and archaea are quite distinct from each other ( Caforio and Driessen 2017 Cronan 2024 Lombard and Moreira 2011 Koga 2012 Tourte et al. 2022 FIG 4 Dichotomy of microbial membranes. Panel A highlights the chemical distinction between archaeal and bacterial bilayer membrane lipids adapted from (Summons et al. 2022). Panel B shows examples of how archaea further modify their membranes by linking their (more...) The dichotomy of membrane structure between bacteria and archaea raises a variety of questions about the chemical composition of the first protocell membranes, how these evolved to form the membrane structure of the LUCA, what the membrane structure of the LUCA was, and how the divergence between archaeal and bacterial membranes eventually occurred. Studies on the abiotic formation of cellular membranes have focused on the formation of primitive lipid micelles and coacervates (non-membrane-bound liquid phase-separated droplets formed by macromolecular interactions), elegantly demonstrating the formation of basic structures that can harbor some catalytic reactions ( Jia et al. 2014 O’Flaherty et al. 2018 Sahonero-Canavesi et al. 2022 Villanueva et al. 2017 Hoshino and Gaucher 2018 Moody et al. 2024 Weijers et al. 2006 Villanueva et al. 2021 Thermotoga Halamka et al. 2023 Hamerly et al. 2015 Lombard et al. 2012 Fig. 5 FIG 5 Mixed -membrane model, showing the divergence in membrane structure between archaea and bacteria. d. New frontiers in membrane biology To understand the potential ecological drivers, molecular mechanisms, and evolutionary pressures that led to the dichotomy that is the present-day microbial membrane, studies must continue to focus on both bottom-up synthetic approaches to the abiotic origins of membranes and top-down genomic and culture-based analyses of membrane structures found in archaea and bacteria known today. Environmental metagenomics have revealed an abundance of yet-uncultured archaea and bacteria in every imaginable ecological niche. To date, we do not fully comprehend the diversity of microbial lipids that are produced nor the biosynthetic pathways or regularity mechanisms that control membrane formation by the breadth of the microbial world. Exploring the diversity of membrane structures both through culture-independent (comparative genomics and heterologous expression) and culture-dependent (lipidomics) approaches is necessary to fully understand the membrane biology that is possible so we can model and predict the membrane biology that may have existed. 2. Emergence of life: cellularity and metabolism We know that life emerged on Earth within an astonishingly short time after the planet cooled enough to sustain liquid water at the surface. Isotopic evidence for life dates to 3.9 ( Mojzsis et al. 1996 Bell et al. 2015 Moody et al. 2024 a. Last universal common ancestor and what came before Bacteria and archaea were the major forms of life on Earth until the eukaryotic lineage arose within the Asgard archaea ( Williams et al. 2020 Vosseberg et al. 2024 Crapitto et al. 2022 Moody et al. 2024 Moody et al. 2024 Sousa et al. 2013 Mahendrarajah et al. 2023 The LUCA and earlier cells likely existed in communities of organisms that exchanged metabolites and genetic information, as cells still do today. Such exchanges may have been critical for pre-LUCA cells with small genomes and incomplete metabolic networks and could have allowed a community to thrive even if its individual components were incapable of independent existence. Further, exploration of novel functions in parallel in many different simple cells could have vastly sped up evolution of cells capable of independent growth. Gradual consolidation of genes and functions contributed by diverse cells, possibly from different environmental niches, could have led eventually to cells that were capable of independent autotrophic and/or heterotrophic growth. While all life that we know about on Earth descended from the LUCA, the progenitors of the LUCA may not have been the only organisms on the planet; there may have been alternative forms of early life that eventually were outcompeted by the lineage that led to the LUCA ( Moody et al. 2024 Forterre and Gribaldo 2007 Vetsigian et al. 2006 Hud et al. 2013 Hoshika et al. 2019 b. Where did life emerge? It is impossible to think about the origin of the first cells without first considering where those cells arose. One of the greatest controversies in the origin-of-life field centers on whether life emerged from ponds on the surface of the Earth supplied with pre-existing organics and driven by wet-dry cycling or other exogenous energy ( Pizzarello and Shock 2010 Chyba and Sagan 1992 Baross and Hoffman 1985 Martin et al. 2008 Miller 1953 Baross and Hoffman 1985 Martin et al. 2008 2 2 Catling and Zahnle 2020 Weiss et al. 2016 Martin et al. 2014 2 2 2 2 A second argument in favor of an origin of life driven by redox gradients at hydrothermal-vent systems relates to the complexity of organic compounds delivered from meteors and atmospheric discharges. The overlap between the compounds delivered to Earth from space and those used in life is relatively small. For example, 92 amino acids have been identified in the Murchison meteorite and hundreds more have been detected but not definitively identified ( Glavin et al. 2020 Copley et al. 2010 Although the origin-of-life field has not reached a consensus on where life emerged, recent experimental and computational works have made the hydrothermal-vent hypothesis a compelling scenario ( Goldford et al. 2017 Goldford et al. 2024 Preiner et al. 2020 Keller et al. 2016 Muchowska et al. 2017 Novikov and Copley 2013 Hudson et al. 2020 c. Emergence of compartmentalization The earliest cells must have had a mechanism for sequestering genetic information and valuable nutrients from being lost to the environment at large. The LUCA apparently had a lipid bilayer membrane, as reconstructions of its genome include membrane-embedded proteins ( Moody et al. 2024 Koonin and Martin 2005 Martin and Russell 2003 An intriguing possibility is that the first cells arose within the confines of hydrothermal vents in which amphiphiles were provided by Fischer-Tropsch synthesis. Long-chain alkanes, alkanols, and alkanoic acids are formed from CO 2 2 in situ McCollom et al. 1999 An additional mechanism for concentration of amphiphiles was demonstrated by Budin et al., who showed that fatty acids could be concentrated by a combination of convection and thermophoresis in bent capillary tubes meant to mimic structures in hydrothermal vents, leading to formation of vesicles that encapsulated a fluorescent dye ( Fig. 6 Budin et al. 2009 ). 10 15 Jordan et al. 2019 Martin and Russell 2003 FIG 6 (A) Vesicles formed by oleate concentrated at the bend in a glass capillary subjected to thermophoresis can (B) encapsulate a fluorescent dye. A temperature gradient of 30 K was imposed across the linear portion of the capillary. The inset in A indicates (more...) d. Emergence of metabolism A long-standing debate in the origin-of-life field centers on whether extant metabolic pathways evolved from prebiotic chemical networks ( Morowitz 1993 Lazcano and Miller 1999 Pross 2004 Orgel 2004 Yadav et al. 2020 The metabolism-first hypothesis has been criticized because known reactions that produce compounds like amino acids and adenine in high yields do not resemble biological pathways ( Orgel 2004 Harrison and Lane 2018 E. coli Sajed et al. 2016 A recent computational study provides an interesting perspective on the metabolism-first hypothesis ( Goldford et al. 2017 2, 2 3 2 2 4 2 2 2 2 Goldford et al. 2024 e. Critical role of catalysts Conversion of CO 2 2 Amend and Shock 1998 Shock and Schulte 1998 t 1/2 t 1/2 Wolfenden and Snider 2001 FIG 7 Hierarchy of catalysts. Beginning with minerals and ending with genetically encoded proteins would have enabled progressively more efficient catalysis of reactions in proto-metabolic and metabolic networks during the transition from abiotic to biotic (more...) FIG 8 A tiny oligoribonucleotide can catalyze aminoacylation of another oligoribonucleotide. A less-appreciated role for catalysts would have been (and still is) the pruning of complex reaction networks by accelerating particular reactions at the expense of competing reactions ( Copley et al. 2010 Extant protein enzymes accelerate chemical reactions by up to 26 orders of magnitude ( Edwards et al. 2012 2 2 Preiner et al. 2020 2+ Keller et al. 2016 0 2+ 3+ Muchowska et al. 2017 The products formed under hydrothermal-vent conditions would certainly have varied from place to place. An examination of the reactivity of pyruvate under simulated vent conditions showed that the suite of products formed depended upon the temperature, the mineral catalysts, and the presence or absence of NH 4 + Novikov and Copley 2013 Elaboration of proto-metabolic networks was likely enabled by a progression of improvements in catalytic efficiency and specificity as mineral catalysis led to the formation of organic compounds and those compounds became larger and more complex ( Fig. 7 The first organic molecules formed under hydrothermal-vent conditions could have contributed to catalysis, enabling the formation of new compounds. Catalysis by small organic molecules has been documented in thousands of journal articles since 1995 ( MacMillan 2008 Pizzarello and Weber 2004 List et al. 2000 Ramping up the level of complexity, small peptides (2-20 amino acids) can catalyze a range of reactions under aqueous conditions ( Metrano et al. 2020 Gorlero et al. 2009 Although nucleotides lack the functional diversity of amino-acid side chains, small RNA oligonucleotides could also have contributed to catalysis. Most naturally occurring and experimentally evolved ribozymes are large (more than 100 nucleotides), but catalysis can be provided by oligoribonucleotides as small as 5 nucleotides. The oligoribonucleotide CGGUG catalyzes aminoacylation of the substrate GCCU with a k cat Fig. 8 Turk et al. 2011 While this may seem like a very low reaction rate, the earliest days in the origin of life would have had the luxury of time. The RNA world hypothesis posits that RNAs (or other polymers akin to RNAs) were the first macromolecular catalysts. Large ribozymes can catalyze a range of chemical reactions, including ester hydrolysis ( Brackett and Dieckmann 2006 Saran et al. 2005 Zhang and Cech 1997 Fusz et al. 2005 Deng et al. 2022 k cat −7 −1 k cat K M −4 −1 −1 Fusz et al. 2005 E. coli −1 k cat K M −1 −1 Zgiby et al. 2000 Kavita and Breaker 2023 in vitro 2+ Tsukiji et al. 2004 The primacy of RNA in ancient biology is suggested not only by the obvious composition of the translation system (the ribosome, whose core protein-producing active site is composed of RNA, along with tRNA and tRNA-processing ribozymes), but by the fact that the most ancient and universal pieces of metabolic machinery, cofactors, are primarily derived from RNA (Benner and Ellington 1989; White 1979; Visser 1984 Tanaka et al. 2005 The largest leap in catalytic efficiency required the emergence of genetically coded proteins, arguably the most important evolutionary transition that ever occurred on Earth as it dramatically expanded catalysis and created heredity and the separation of genotype and phenotype as we know them today. Protein enzymes catalyze nearly every reaction in extant metabolism not directly involved in protein polymerization itself. The first protein enzymes would likely have been relatively inefficient and non-specific catalysts, but might have accelerated reactions by orders of magnitude, resulting in ever-more efficient synthesis of the amino acids and nucleotides required for growth and replication of early cells. By the time of the LUCA, the genome encoded hundreds of metabolic enzymes ( Moody et al. 2024 Moody et al. 2024 f. Summary of the origins of cellularity in the light of the hydrothermal-vent hypothesis Decades of research have explored a number of scenarios for the origin of life. Information from phylogenetic analyses, studies of hydrothermal-vent systems, investigations of catalysis by minerals and ancient enzymes that still use metal ion clusters, and computational studies is coming together to produce a coherent picture of how life might have emerged in a hydrothermal-vent setting. However, major gaps remain in our understanding of the emergence of the metabolism and membranes of the earliest cells. Progress in these areas could be greatly accelerated by the design and utilization of synthetic biology platforms. We need to better understand how long-chain fatty acids and alcohols could have accumulated in vent walls and generated primordial lipid membranes under hydrothermal-vent conditions. While a growing number of experimental studies have shown that key biochemical reactions can be catalyzed by minerals, few studies have addressed the substantial gap between the earliest mineral-catalyzed geochemical processes and the advent of macromolecular catalysts, initially RNAs with catalytic auxiliaries, and eventually proteins. The origins of the elaborate protein complexes that couple electron transfer to pumping of protons across membranes are obscure. Finally, we need to better understand the availability of phosphate on the early Earth and how phosphate esters were incorporated into early proto-metabolic networks. FIG 9 Overview of the tree of life in 2024, with some of the major lineages labeled. The root is thought to lie between the two main prokaryotic groups, Archaea and Bacteria. The origin of eukaryotic cells involved symbiosis between a member of the Asgard archaea (more...) C. Tree of life 1. History of the tree of life The modern understanding of a tree of life that relates life on Earth supplants the “ladder of being” of Aristotle and, somewhat later, Llull, in which humans occupied an intermediate position between the rest of the biological world on one side and angels and God on the other ( Gontier 2011 Woese and Fox 1977 Lake et al. 1984 Cox et al. 2008 Hug et al. 2016 Spang et al. 2022 The accumulation of microbial genomes over the past 30 years has subsequently demonstrated the power of horizontal gene transfer (HGT) as an evolutionary force, revealing that most, or perhaps all, genes have experienced HGT. Thus, there is actually no single tree that describes the evolution of all genes ( Doolittle 1999 Williams et al. 2024 Fig. 9 a. LUCA's position in the tree of life The current view is that the root of the tree lies between the Archaea and Bacteria, whose common ancestor, the last universal common ancestor, was a prokaryote-grade cell with many of the features common to extant Archaea and Bacteria, such as a cell membrane, a DNA-based genome, ribosome, ATP synthase complex that used proton gradients to capture chemical energy, and other familiar core cellular machinery ( Ouzounis et al. 2006 Weiss et al. 2016 Krupovic et al. 2020 Moody et al. 2024 Betts et al. 2018 Mahendrarajah et al. 2023 Martin et al. 2015 Roger et al. 2017 Eme et al. 2017 b. Archaeal and bacterial metabolism and the tree of life While there is some consensus on this high-level summary of the tree of life, the deep relationships within the Archaeal and Bacterial domains remain uncertain, in ways that have important implications for our understanding of early cellular evolution. For example, comparative analyses of archaeal and bacterial metabolism have tended to support the view that anaerobic, autotrophic metabolisms such as acetogenesis and methanogenesis may have provided the earliest means of carbon fixation and energy metabolism for cells ( Martin 2012 Sousa et al. 2013 Weiss et al. 2016 Moody et al. 2024 Garcia et al. 2022 Xavier et al. 2021 Coleman et al. 2021 c. Diversification of archaeal and bacterial lineages within the tree of life An additional difficulty relates to the apparently unequal biodiversity of the archaeal and bacterial domains. Phylogenetic analyses tend to suggest that, of the two prokaryotic lineages, there is substantially more genetic diversity within the Bacteria ( Hug et al. 2016 Cavalier-Smith and Chao 2020 Lake et al. 2009 d. Viral evolution and the role of viruses in shaping cellular evolution A key component of the biosphere not mentioned in the above treatment of the tree of life is the viruses, perhaps the most numerous and genetically diverse biological entities on Earth today. The understanding of viral evolution in deep time lags substantially behind that of cells, in part due to the difficulty of applying traditional phylogenetic methods to resolve evolutionary relationships among viruses due to their typically extremely high rates of sequence evolution. However, this picture is changing with major progress in recent years, including the proposal of evolutionary classifications and evolutionary trees for large groups of viruses ( Koonin et al. 2020 Aylward et al. 2021 Koonin et al. 2020 Moody et al. 2024 Garg and Hochberg 2024 Koonin 2016 Mi et al. 2000 Bell 2020 Takemura 2020 Garg and Hochberg 2024 Mi et al. 2000 Bell 2020 Takemura 2020 e. Revising and refining the prokaryotic tree of life Substantial progress on the tree of life has been made in the past 20 years. A major and widely celebrated advance in microbiology has been environmental genomics, and we now have a much better genome sampling of the natural microbial world than seemed possible 20 years ago ( Hug et al. 2016 Parks et al. 2017 Williams et al. 2020 Spang et al. 2022 f. Alternative perspectives to approach questions surrounding the origin of life One of the major themes of the colloquium was to ask whether principles from evolutionary biology might be able to stimulate progress on origin-of-life questions which are perhaps more typically studied from a chemistry perspective. The traditional limitation in extending evolutionary biology back towards the origin of life is, first, that all modern lineages coalesce at the LUCA, greatly narrowing the phylogenetic window that can be used to reconstruct more ancient evolutionary events. The second, and perhaps more fundamental, limitation is that evolution by natural selection is often held to require a high-fidelity hereditary material (such as RNA or DNA) because high-fidelity replication provides the correlation in fitness between parents and offspring that enables fitter types to increase in frequency over generations due to selection. A genetic system much like that in use today, involving a DNA genome and protein enzymes, was very likely already in place by the time of the LUCA. However, even the simplest such system, such as an RNA world of catalytic and replicating RNA molecules ( Bernhardt 2012 Babajanyan et al. 2023 Okasha 2006 The excitement of these ideas is that they offer the potential to bring more of the earliest phase of life's history into the realm of evolutionary biology, and there is clearly scope for much more conceptual work in this area. How these views might be put to the test remains an open question, to be discussed later in this document. One area that merits particular exploration is how neutral processes might have interacted with selection on early life and whether the drift barrier hypothesis ( Lynch 2024 Sung et al. 2012 Stoltzfus 1999 Brunet and Doolittle 2018 Kacar et al. 2017 D. Coevolution of infectious/parasitic relationships in early life 1. Viral classification After considering the status of prokaryotic phylogenetic research defining the relationships between Baltimore 1971 Koonin et al. 2020 Koonin et al. 2020 2. Viral evolution It has remained tantalizingly difficult to assess the timing at which major viral lineages emerged on Earth, as well as the roles they may have played in the early evolution of cells. Although evolutionary relationships between cellular lineages can often be inferred using molecular phylogenetic analysis of the core machinery involved in transcription and translation, viruses typically lack these genes, thereby hindering direct approaches for linking cellular and viral diversity. Moreover, the fast evolutionary rates of viruses often obscure phylogenetic signals and lead to rapid divergence between groups, making it challenging to assess deep evolutionary links. Nonetheless, structural analysis of key viral proteins involved in virion morphogenesis has shed light on the emergence of viral lineages. For example, the analysis of major capsid proteins suggested that viruses acquired these proteins from cellular homologs at least 20 distinct times ( Krupovic and Koonin 2017 Krupovic et al. 2019 The modular structure of viral genomes creates opportunities for evolutionary paths that are typically not found in cellular life, which creates further challenges for assessing their early evolution. Most viral genomes contain genetic modules for viral particle morphogenesis and genome replication that can evolve through dynamic mixing and matching over long evolutionary time frames, leading to a broad diversity of chimeric lineages ( Koonin et al. 2022 Over the course of their long evolutionary history, some viral lineages have acquired an impressive array of cellular machinery and evolved unusually large genomes. This phenomenon is particularly prevalent in the phylum Nucleocytoviricota Caudoviricetes) Warwick-Dugdale et al. 2019 Tian et al. 2024 Thompson et al. 2011 Tian et al. 2024 Johnston and McFadden 2003 Nucleocytoviricota. Moniruzzaman et al. 2020 Koonin et al. 2022 3. Viral-host coevolution Phylogenetic analysis of AMGs and other viral-encoded physiology genes generally supports the view of viral gene acquisition over the course of virus-host coevolution. For example, many bacteriophage AMGs share high nucleic acid identity with homologs in their hosts, indicating recent acquisition (Lindell at al. 2004). The virokines and virocepters encoded by herpesviruses and poxviruses were likely acquired during their early adaptation to vertebrate hosts ( Senkevich et al. 2021 Nucleocytoviricota, Iyer et al. 2006 Yutin et al. 2014 Da Cunha et al. 2022 Guglielmini et al. 2019 4. Viral integration into host genomes and evolution Many viral lineages integrate into the genomes of their hosts, creating ample opportunity for host-virus gene exchange that may shape the evolution of cellular lineages. In bacteria, proviruses derived from tailed bacteriophages are commonplace and many encode the toxin genes needed by their hosts to occupy pathogenic lifestyles (Brussow et al. 2004). This has been studied in a wide range of pathogens, including Vibrio cholerae, Shigella dysenteriae, and Clostridium botulinum, among others. In eukaryotes, viral integration and subsequent co-option of viral genes have been linked to many dramatic evolutionary innovations, such as the emergence of the placenta via the acquisition of viral fusion proteins ( Chuong 2018 Moniruzzaman et al. 2020 Zhao et al. 2023 Sarre et al. 2024 Delaroque and Boland 2008 FIG 10 Graphical summary of the early evolution of the translation system. The expansion of the genetic code occurred alongside the complexification of the ribosome from an ancestrally simple ribozyme. SIgns of an early simpler genetic code can be found in ribosomal (more...) 5. Evolution of antiviral defense strategies Some of the endogenous DNA viruses in eukaryotes have been shown to parasitize giant viruses and thereby act as a kind of inducible antiviral defense of the host ( Fischer and Hackl 2016 La Scola et al. 2008 Bellas et al. 2023 Barth et al. 2024 6. Future research on viruses and evolutionary history There are currently many gaps in our knowledge of how viruses have shaped the evolution of life on Earth, and much future work in this area is needed. We are only beginning to grasp the enormous extent of viral diversity on the planet, and further studies examining the timing and mechanisms through which viral lineages have emerged will be critical to examining their evolutionary history relative to cells. Given the vast timescales involved and the fast rates of viral evolution, it is likely that analysis of conserved protein structures will be a key element of these studies. In addition, key findings have highlighted specific examples in which the co-option of viral genes has led to dramatic evolutionary consequences for cellular life, but we still lack a broader view of the prevalence of these events and the overall extent to which cellular life has been shaped by viral gene acquisition. Finally, it is clear that viruses have exerted intense selective pressure on cellular lineages for billions of years, but the extent to which this has shaped the extant diversity and genome composition of both cells and viruses remains unclear. Studies examining coevolution of viruses and their hosts over shorter time periods have revealed extensive arms race dynamics involving changes to viral defense and anti-defense systems ( Hussain et al. 2021 E. Evolution of translation 1. Introduction Although there is tremendous variation in the form, metabolism, and function of present-day cellular organisms, a unifying characteristic is that they all use DNA to store genetic information, mRNA to transfer this information to the translation machinery, ribosomes made of RNA and proteins, tRNAs to carry amino acids to the ribosomes to be incorporated into genetically encoded peptides, and aminoacyl tRNA synthases to charge these tRNAs with their cognate amino acids. The uniformity in molecular biology is reflected in Monod's phrase “anything found to be true of E. coli must also be true of elephants,” which in his recollection he first uttered in 1954 (referenced in Friedmann 2004 Crick 1958 2. Vertical versus horizontal inheritance of genes encoding functions in translation A feature present in all three cellular domains is likely there because it was present in their common ancestor, the organismal cenancestor last universal common ancestor. Brochier et al. 2000 Wolf et al. 1999 Ibba et al. 1997 Brochier et al. 2000 Andam and Gogarten 2011 Wolf et al. 1999 Woese et al. 2000 Most gene transfers occur between closely related organisms, which as a consequence of the transfer appear even more closely related in molecular phylogenies ( Andam et al. 2010 Pace et al. 2012 Gogarten et al. 2002 In addition, highways of gene sharing connect unrelated organisms ( Beiko et al. 2005 Lapierre et al. 2014 Moody et al. 2024 Huang and Gogarten 2006 Williams et al. 2024 A popular hope has been that genes that encode pieces of a complex machinery such as a ribosome or rotary ATPase would more faithfully track organismal evolution because of their many obligate interactions between interdependent components restricting successful transfer ( Jain et al. 1999 Brochier et al. 2000 Yap et al. 1999 Gogarten et al. 2002 Yap et al. 1999 Igarashi et al. 2001 Olendzenski et al. 2000 Doolittle has proposed the “tree of cell divisions,” in contrast to the tree of life, as the tree that one could reconstruct if one had a perfect record of every cell division, including cell fusions and endosymbioses (discussion in Williams et al. 2011 3. Study of the pre-LUCA evolutionary history of the translation machinery While today's ribosomes consist of ribosomal RNA and ribosomal proteins, structural and mutation analyses reveal that ribosomal RNAs are at the core of ribosomal catalytic activity, suggesting that present-day ribosomes evolved from a simpler ribozyme ancestor ( Noller 2024 In today's ribosomes, formation of the peptide backbone is still mediated by the RNA component, demonstrating that the addition of ribosomal proteins over time ( Fournier et al. 2010 Brunet and Doolittle 2018 4. Compositional analyses of ribosomal proteins and ancient duplicated genes Ribosomal proteins that are shared between archaea, bacteria, and eukaryotes before HGT were added to the ancestral ribozyme over time. This gradual recruitment of proteins to the ribosome would plausibly have co-occurred with the expansion of the genetic code through the addition of genetically encoded amino acids. Thus, a protein that was added early to the ribosome, at the time of its addition, did not include amino acids that were later additions to the genetic code ( Fournier and Gogarten 2010 Wolf and Koonin 2007 Fournier and Gogarten 2007 Fournier and Gogarten 2010 1 Gogarten et al. 1989 Iwabe et al. 1989 Brown and Doolittle 1995 Gribaldo and Cammarano 1998 Boussau et al. 2008 Galtier et al. 1999 Catchpole and Forterre 2019 5. Molecular versus organismal cenancestors: the LUCA was not alone Phylogenetic trees for individual gene families trace a gene's evolutionary history back to common ancestors, also known as molecular cenancestors. Due to genetic exchange between lineages, these molecular cenacestors did not all coexist in the LUCA. Horizontal gene transfer events inferred from comparing the evolutionary history of individual genes to the consensus phylogeny can provide information about extinct lineages. The study of aaRSs is particularly instructive because these enzymes are extremely well conserved with slow evolution, transfer horizontally only infrequently, and are an integral part of the extant translation machinery linking tRNAs to their cognate amino acids. In many instances, the molecular aaRS cenancestors likely were present in the organismal LUCA or in organisms belonging to the same population of organisms. However, for some aaRSs, rare forms of the enzymes exist that constitute deeper branches (red dotted lines in Fig. 10 Fournier et al. 2009 6. Ancestral sequence reconstruction reveals an unknown tRNA charging system predated modern aaRSs FIG 11 Genetic information storage and transmission in viruses and mobile genetic elements. The classification of viruses by information transmission pathways was first established in the seminal work of Baltimore (1971 Koonin et (more...) The study of aaRSs provides another opportunity to study early evolution: all class I and class II aminoacyl tRNA synthases evolved and diversified from common ancestors through gene duplications. For example, the aaRSs for valyl (valRS) and isoleucyl (ileRS) diverged from a single pre-LUCA ancestral aaRS; the same is true for the tryptophanyl and tyrosyl aaRSs (trpRS and tyrRS). For the gene duplications leading to modern aaRSs with different amino-acid specificities, the similarity is sufficient to unambiguously conclude that within each class they all evolved from a common ancestor. However, the sequences are generally too divergent for reliable phylogenetic and ancestral sequence reconstruction deep into the tree using currently available approaches. Exceptions are the splits between the valyl and isoleucyl aaRSs, and the tryptophanyl and tyrosyl. For the latter pair the data are compatible with tryptophan not being genetically encoded at the time of the tyrRS and trpRS ancestor. In this case, the expansion of the genetic code to include tryptophan is reflected in the gene duplication that gave rise to the trpRSs ( Fournier and Alm 2015 Fournier et al. 2011 7. Retrodiction of the ancestral ribosome Ribosomal structures have been determined from many different organisms. The function of different structures is well established, e.g., the peptidyl transferase center, the tunnel through which the newly synthesized protein leaves the ribosome, tRNA binding sites, and the interface between the ribosomal subunits. Similar to Granick's hypothesis for the evolution of biochemical pathways (Granick 1965), the idea to reconstruct the pre-LUCA evolution of the ribosome focuses on the core onto which additional elements have been added or inserted. The accretion model ( Petrov et al. 2015 Petrov et al. 2015 Bowman et al. 2020 Noller et al. 1992 Agmon et al. 2005 Kawabata et al. 2022 Bose et al. 2022 Xu and Wang 2021 Agmon 2024 Patra et al. 2024 Douglas et al. 2023 F. RNA/DNA system of coding and heritability (Eugene Koonin, Ph.D.) 1. Universal genetic system Complementing the universality of translation [see the preceding section (III E)], the colloquium emphasized that all cellular life forms share a universal system of genetic information storage, replication, and expression and sought plausible hypotheses to describe how this evolved. The genomes of all organisms are large double-stranded DNA molecules that are semi-conservatively replicated by a dedicated enzymatic machinery and transcribed by a distinct enzymatic apparatus to yield a broad repertoire of structural and regulatory RNAs as well as mRNAs that are translated into proteins. The universality of the genetic pathways in cellular life forms implies that the genetic system was fixed, in its main features, at an early stage in the evolution of life, significantly antedating the LUCA. Moreover, among the approximately 100 genes that are conserved in all cellular life forms, nearly all encode RNA and protein components of the translation system, along with several core components of the transcription and replication machineries ( Koonin 2003 Koonin et al. 2020 Fig. 11 Defining the availability of the building blocks of a genetic system. Before discussing the origin and early evolution of the genetic systems and their primordial forms, it is necessary to define the setting for this stage of evolution as best we can. Obviously, before any sustained modern nucleic acid synthesis becomes possible, a steady supply of the essential building blocks, namely, activated nucleotides, is required. This appears impossible without, first, a proto-metabolic network capable of nucleotide synthesis, and second, compartmentalization that would ensure sufficient concentrations of these molecules. These considerations substantially constrain the conditions for the emergence and fixation of genetic systems so that the advent of genetics can be confidently inferred to postdate the origin of protocells harboring proto-metabolic pathways ( Babajanyan et al. 2023 2. RNA world concept The origin of genetic systems within such protocells is tightly linked to the RNA world concept ( Atkins et al. 2011 Joyce 2002 Wolf and Koonin 2007 Babajanyan et al. 2023 Joyce and Szostak 2018 FIG 12 Coevolution of primordial replicators and reproducers (Babajanyan et al. 2023 ) (more...) 3. Replication of genetic material in an early system A broad diversity of ribozyme activities has been reported ( Wilson and Lilley 2021 Papastavrou et al. 2024 Tjhung et al. 2020 Dhar et al. 2017 Briones et al. 2009 At present, the closest approximation of an efficient RNA-only self-replicating ribozyme appears to be a trinucleotide ligase-polymerase ribozyme ( McRae et al. 2024 The RNA world concept is also compatible with the existence of modern RNA replicators, namely, the highly diverse viruses with RNA genomes and viroid-like circular RNAs. The viroid-like agents are particularly notable because they include minimal replicators, circular RNA molecules of only about 200 nucleotides that recently have been demonstrated to be widely represented in diverse environments and to reproduce in bacterial cells ( Lee et al. 2023 Zheludev et al. 2024 4. Replication of RNA in the early protocell state A distinction must be drawn between a “reproducer” and a “replicator” in the context of the early evolution of genetic information. A reproducer constitutes an entire system capable of generating everything necessary for its own growth and multiplication, while a replicator is an individual pattern which can create more of itself in a particular context. In early life, compartments containing all necessary processes of a proto-metabolism would constitute a primitive reproducer, which would become subject to colonization by replicators. The emergence of the first replicators, that is, RNA molecules capable of templated synthesis, including self-replication by ribozyme replicases, within protocell reproducers would set the stage for evolution by selection on particular sequence properties combined with random drift. Selection in the reproducer-replicator system would operate at two distinct levels: (i) selection among replicators for replication efficiency in the context of the reproducer and (ii) selection for growth and division efficiency at the level of the entire reproducer-replicator systems (protocells) ( Fig. 12 Babajanyan et al. 2023 5. RNA-only translation in early evolution Explaining the emergence of an RNA/DNA system requires a hypothesis that explains the mechanics of the earliest forms of translation, and this was addressed in the discussions of the colloquium. There is far from a satisfactory understanding ( Wolf and Koonin 2007 Nissen et al. 2000 Hiller et al. 2011 Bose et al. 2022 Bokov and Steinberg 2009 Petrov et al. 2014 Turk et al. 2010 Evolution of the modern translational machinery. Aravind et al. 2002 Leipe et al. 2002 Fer et al. 2025 6. Origin of the modern coding system Apart from these indications on the nature of the early, RNA-based translation system, the origin of the coding mechanism remains enigmatic. Some form of physical affinity between amino acids and oligonucleotides or RNA structures might have played a role in the origin of coding, but so far experiments in this direction have been inconclusive ( Koonin and Novozhilov 2017 McRae et al. 2024 A key step in the evolution of genetic systems was the transition from RNA to DNA as the genetic material. A clue to this stage of evolution may be provided by viruses and other mobile genetic elements that use all forms of RNA and DNA as genomes including making DNA copies of RNA genomes as an intermediate stage in replication, via reverse transcriptase (RT) encoded by the respective MGEs ( Koonin et al. 2020 Koonin et al. 2021 Krupovic et al. 2019 Takeuchi et al. 2011 FIG 13 Scen ario for concomitant origin and early evolution of transcription and replication (Koonin et al. 2020). (A) Evolution of cellular (top) and viral (bottom) polymerases from a double-psi beta-barrel (DPBB) and RNA recognition motif (RRM)-containing (more...) 7. DNA replication machinery diverges across the domains of life Unlike the cores of the translation and transcription systems, the DNA replication machineries are not universal in the three domains of life although DNA chemistry itself is, suggesting a complex history ( Koonin et al. 2020 Raia et al. 2019 Sauguet 2019 Koonin et al. 2020 Fig. 13 Under this scenario, PolD was the original DNAP that was responsible for the genome replication in the LUCA. The primordial PolD remains the replication DNAP in most of the archaea, but was replaced by PolB, possibly, from a viral source, in some archaeal lineages, which was subsequently inherited by eukaryotes. In the common ancestor of bacteria, the ancestral PolD was replaced by an initially non-replicative DNAP. Notably, some other components of the replication apparatus, such as the sliding clamp, are conserved in all cellular life forms and appear to represent the heritage of the LUCA. The described sequence of events is obviously one of many possible scenarios for the origin and early evolution of genome replication, but it shows that structural comparisons of key proteins have the potential to provide insights into the deepest evolutionary past. 8. Summary To conclude, our understanding of the origins and early evolution of the pathways for biological information transmission, the genetic system, has progressed during the past few years, through theoretical modeling, comparative analysis of genomes as well as protein and RNA structures, and chemical experiments. However, key evolutionary steps remain highly problematic, above all the origins of replication and translation, despite some tantalizing clues emerging. Further research on these matters stands to drastically improve our understanding of the origins of life. IV. DIVERSIFICATION AND ORIGIN OF EUKARYOTES A. Overview of eukaryogenesis The second colloquium of this project aimed to expand on concepts explored in the first colloquium, outlined in Secs. III and IV, using them as a foundation to explore the origin of eukaryotes, known as eukaryogenesis. This was a unique evolutionary transition resulting in what can be broadly described as a substantial increase in cell complexity. Deciphering how eukaryotes first appeared has seemed to be an unattainable quest for a long time, remaining at the level of untestable hypotheses. However, recent advances in molecular phylogenomics, a better understanding of microbial diversity and their interactions in natural ecosystems, and the growing use of synthetic and cell biology approaches allow insights into cell function and evolution that create an opportunity for building a narrowed set of models for the origin of eukaryotes that are mechanistically, evolutionarily, and ecologically plausible and potentially testable. 1. Shifting views on eukaryogenesis Early models arising after the recognition of the prokaryote-eukaryote dichotomy postulated that eukaryotes evolved by complexification of simpler bacteria (reviewed in Lopez-Garcia and Moreira 2023). In the latter decades of the 20th century, initial molecular phylogeny approaches led to two important observations with implications for understanding eukaryogenesis: the discovery of archaea as an independent domain of life that appeared sister to eukaryotes ( Woese and Fox 1977 Woese et al. 1990 Schwartz and Dayhoff 1978 Gray and Doolittle 1982 Embley and Martin 2006 Cavalier-Smith 2002 Embley and Martin 2006 Williams et al. 2013 Eme et al. 2017 Imachi et al. 2020 Rodrigues-Oliveira et al. 2023 Zaremba-Niedzwiedzka et al. 2017 Fig. 14 FIG 14 Schematic scenario of eukaryogenesis and the early diversification of major eukaryotic lineages. Eukaryotes likely evolved ~2 billion years ago after the Great Oxidation Event from a symbiosis between one Asgard archaeon and at least the alphaproteobacterial (more...) 2. Asgard archaeal ancestry of eukaryotes Asgard archaea share many proteins exclusively with eukaryotes and phylogenomic analyses place the origin of the nuclear genome of eukaryotes within the Asgard clade, as closest relatives of the Heimdallarchaeia ( Zaremba-Niedzwiedzka et al. 2017 Liu et al. 2021 Eme et al. 2023 Zaremba-Niedzwiedzka et al. 2017 Spang et al. 2015 Valentin-Alvarado et al. 2024 Zaremba-Niedzwiedzka et al. 2017 Eme et al. 2023 Hatano et al. 2022 Tran et al. 2024 Lu et al. 2024 Stairs and Ettema 2020 + Liu et al. 2021 Spang et al. 2019 Candidatus C . Imachi et al. 2020 Rodrigues-Oliveira et al. 2023 3. Bacterial ancestry of eukaryotes After the endosymbiotic origin of the mitochondrion from Alphaproteobacteria was validated ( Schwartz and Dayhoff 1978 Gray and Doolittle 1982 Embley and Hirt 1998 Gray 2012 Roger et al. 2017 Lombard et al. 2012 Phylogenetic analyses and comparative genomics reveal a diverse bacterial genetic signature in the LECA beyond that of modern alphaproteobacteria. Intriguingly, bacterial genes seem more abundant than archaeal genes in the LECA, with non-alphaproteobacterial genes largely dominating over Alphaproteobacterial genes (Pittis and Gabaldon 2016; Bernabeu et al. 2025). However, a recent study suggests a higher archaeal gene abundance in the LECA (Tobiasson et al. 2025). Non-alphaproteo-bacterial genes frequently trace back to Deltaproteobacteria, Actinobacteria-Firmicutes, Planctomycetes, or Chlamydiae, among others (Pittis and Gabaldon 2016). Some of these genes encode cohesive functions, potentially suggesting that they were acquired in waves (Gabaldon 2018). Deltaproteobacteria include a great deal of diversity that is now split into several phyla in the Genome Taxonomy Database (Desulfurobacterota, Myxococcota, and Bdellovibrionota, among others) ( Parks et al. 2022 Imachi et al. 2020 Rodrigues-Oliveira et al. 2023 Hoshino and Gaucher 2021 FIG 15 Current models of eukaryogenesis based on the symbiotic merging of archaeal and bacterial partners (Lopez-Garcia and Moreira 2023). (A—C) Selection of scenarios lacking a specified basis for the symbiosis. (D-G) More detailed models postulating specific (more...) 4. Symbiotic origin of the eukaryotic cell Collective observations support a symbiogenic origin of the eukaryotic cell, whereby higher complexity evolved from the physical integration of prokaryotic cells accompanied by extensive gene exchange and genome shuffling ( Koonin 2015 Koonin 2015 Jain et al. 1999 Lombard et al. 2012 Fig. 15 Spang et al. 2019 Imachi et al. 2020 Baum and Baum 2014 Sousa et al. 2016 Imachi et al. 2020 2 Krupovic et al. 2023 Obviously, all of these models have limitations and none of them can fully explain the evolution of all eukaryotic traits. Some of these limitations will disappear as additional knowledge is generated, as discussed below. There is hope for a future consensus model of eukaryogenesis that explains the molecular and cellular mechanisms underlying this major evolutionary transition and the coordinate dating along with Earth and environmental conditions. However, it is important to concede that eukaryogenesis was a contingent event with a chance component that could be extremely difficult or impossible to reconstruct. B. Eukaryotic tree of life Eukaryogenesis culminated in the last eukaryotic common ancestor. From this LECA, a multitude of lineages rapidly diversified and colonized newly opened ecological niches. Establishing the phylogenetic relationships among those varied lineages and their relative branching pattern is essential to infer the evolution of particular eukaryotic traits following their diversification as well as for reconstructing the genome and phenotype of the LECA. However, reconstructing the phylogenetic tree of eukaryotes [the so-called eukaryotic tree-of-life (eToL)] is challenging because the major extant eukaryotic lineages appear to have radiated rapidly over a relatively short time period. 1. Diversity of eukaryotes and the eToL The majority of eukaryotes are a hugely diverse array of unicellular protists ( Adl et al. 2019 Burki et al. 2020 Burki et al. 2021 Adl et al. 2019 Burki et al. 2020 Fig. 14 Adl et al. 2019 Adl et al. 2019 Torruella et al. 2018 Worden et al. 2015 Burki et al. 2020 Al Jewari and Baldauf 2023 Roger and Hug 2006 Eme and Tamarit 2024 Nonetheless, significant progress has been achieved in recent years owing to the improvement of phylogenetic methods and to the generation of genomic and/or transcriptomic data from poorly studied or newly identified eukaryotes ( Burki et al. 2020 del Campo et al. 2014 Galindo et al. 2023 Ruiz-Trillo et al. 2023 incertae sedis, Brown et al. 2013 Opisthokonta and Amoebozoa were previously thought to share a uniflagellated ancestor ( Cavalier-Smith 2002 Richards and Cavalier-Smith 2005 Richards and Cavalier-Smith 2005 Derelle et al. 2015 Torruella et al. 2025 Palpitomonas), Torruella et al. 2025 Heiss et al. 2018 Lax et al. 2018 Mantamonas) Burki et al. 2020 Torruella et al. 2025 Brown et al. 2018 Trimastix, Carpediomonas) Burki et al. 2020 Derelle et al. 2015 Although the inclusion of phylogenomic data for many newly described flagellates has confirmed some eukaryotic supergroups, many lineages of heterotrophic flagellates are yet to find a home in the eukaryotic tree and remain incertae sedis. Atkins et al. 2000 Heiss et al. 2018 Torruella et al. 2025 Tikhonenkov et al. 2022 Ancoracysta Lax et al. 2018 Meteora Galindo et al. 2022 Eglit et al. 2024 Torruella et al. 2025 Eglit et al. 2024 2. Complex LECA As genome sequences for an increasingly wide diversity of eukaryotes became available, gene content comparisons started to point out to a rather complex LECA (Sec. IV A, Fig. 14 Koonin 2010 del Campo et al. 2014 Vosseberg et al. 2021 Newman et al. 2019 Richards et al. 2024 Koonin 2010 Newman et al. 2019 Derelle et al. 2015 Torruella et al. 2025 Suzuki-Tellier et al. 2024 Torruella et al. 2025 Suzuki-Tellier et al. 2024 Williamson et al. 2025 Naegleria) 3. Knowledge gaps and future research on eToL Although significant progress has been made over the past 15-20 years, the eToL is not yet resolved. The eToL is not fully populated inasmuch as new phyla are still being discovered (e.g., Provora and Meteora) Williamson et al. 2025 Al Jewari and Baldauf 2023 Richards et al. 2024 4. Resolving and rooting the eukaryotic tree of life There are two major approaches to resolving the eToL. One is improving taxon sampling. The inclusion of a more balanced diversity across eukaryotes in phylogenomic trees can help stabilize relationships among taxa. Many eukaryotic lineages, especially parasites, evolve rapidly. Therefore, including more slowly evolving representatives of different clades can alleviate phylogenetic reconstruction biases caused by differences in evolutionary rate across lineages. Favoring genome sequencing of free-living, slower-evolving eukaryotes from clades previously only represented by parasites or otherwise fast-evolving species can help overcome this problem. In addition, it is important to continue exploring the diversity of eukaryotes and incorporating newly identified eukaryotic lineages in phylogenomic analyses, which may in some instances consolidate unstable areas in the eToL. Unfortunately, many newly detected lineages encompass predatory protists, which are difficult to culture and remain undersampled. This problem can be illustrated by apusomonads (Karpov and Mylnikov 1989, WoRMS - World Register of Marine Species - Apusomonadidae), for which the use of specific 18S rRNA gene primers revealed a broad diversity of these gliding flagellates in freshwater and marine benthos across the globe ( Torruella et al. 2017 Torruella et al. 2025 In general, culturing predatory protists is challenging for several reasons. They depend on specific bacterial or eukaryotic prey, which in turn are not necessarily easy to identify and maintain in co-culture. Also, being predators, they are not abundant in their native ecosystems and are hence underrepresented in metagenomic data. If assembling MAGs from metagenomes is already difficult for relatively abundant protists due to the compositional divergence of coding versus non-coding regions, it becomes virtually impossible for rare protists. In this case, either cultivation or single-cell genomics/transcriptomics are reasonable options. Of course, having protists in culture allows for the study of otherwise inaccessible features such as cell and molecular biology, ultrastructure, ecology, and the life cycle (although establishing life cycles is highly challenging even with cultures available). From this perspective, promoting classical protistology studies of the sort that was more common in the days before research focused almost exclusively on molecular biology, and combining this with modern genomics/transcriptomics as well as high-resolution microscopy approaches to study ultrastructure (e.g., SEM, TEM, cryoTEM, and expansion microscopy) is necessary. Alternatively, promoting these kinds of studies from single cells can help complement single-cell genomic/transcriptomic approaches, especially if “omics” and ultrastructural studies from the same single cell could be combined. In terms of environments that remain poorly studied and likely host a still undescribed diversity of eukaryotes, suboxic settings and especially freshwater and marine sediment surfaces and soils are the most promising. Sediment-dwelling heterotrophic flagellates remain largely understudied despite their long-appreciated ecological role as grazers (Fenchel 1986). Several deeply branching lineages of protists with key positions in the eToL have been obtained from benthic ecosystems ( Torruella et al. 2025 Lax et al. 2018 Tikhonenkov et al. 2022 Eglit et al. 2024 The other crucial way to resolve the eToL is methodological and involves improving the quality of phylogenetic markers used to reconstruct the tree as well as the methods of phylogenetic reconstruction. An important need for the community of evolutionary protistologists is to develop and maintain a properly curated database of genomes and/or transcriptomes that represents the full diversity of eukaryotes in a balanced way, mirroring the ambition of the Genome Taxonomy Database for prokaryotes. Eukprot ( Richter et al. 2022 https://evocellbio.com/eukprot/ Typically, clustering genes and comparing them with those of a eukaryotic database such as BUSCO (Simao et al. 2015) or using phylogeny-informed pipelines oriented to deal with massive data and make the process transparent and replicable ( Katz et al. 2024 In addition, refining methods of phylogenetic reconstruction to cope with compositional biases and differences in evolutionary rates across marker alignment sites and across lineages ( Roger and Hug 2006 Eme and Tamarit 2024 Resolving the eToL is also challenging due to the complications from horizontal gene transfer (HGT) during eukaryotic evolution. Although not as massive as it is in prokaryotes, eukaryotes can import genes either from prokaryotes or from other eukaryotes (Sibbald 2020). Genes can be gained from other members of the ecosystem and from viruses, but also especially from (endo) symbionts. Deciphering the extent and the impact of HGT and (endo)symbiotic gene transfer across the eukaryotic tree and how these transfers impact the eToL inference should be an important goal of research in coming years. Some of these transfers may be useful for reconstructing the eToL via reconciliation approaches, but others, especially ancient or frequent transfers, might hamper its inference. In this regard, it will be extremely useful to study symbioses in protists and determine the patterns of gene transfer and genome evolution that accompany them. Comparing those patterns may allow inferring the general fate of symbionts and more broadly their genetic information ( Husnik and Keeling 2019 A final challenge is rooting the eToL. This could in principle be achieved using either an external outgroup ( Derelle et al. 2015 Al Jewari and Baldauf 2023 Williamson et al. 2025 Williams et al. 2024 5. Inferring the LECA and other ancestral states Having a resolved and rooted eToL should allow inferring the genotypes and phenotypes at different ancestral nodes, including the LECA stage. This can be achieved by mapping genes/proteins and observed traits onto a resolved eToL and applying some form of maximum parsimony or other approaches for ancestral trait inference to identify the earliest node at which particular traits were first observed. This can include complex traits such as the presence and number of flagella, the occurrence of pseudopodia, the ploidy, or the coupling of karyokinesis and cytokinesis. Identifying the origins of such traits in the eToL should uncover crucial steps that conditioned lifestyles and subsequent major evolutionary trends along the natural history of eukaryotes. Unfortunately, different confounding factors, including HGT and convergence (e.g., independent recruitment of homologous proteins and similar mechanisms to build a specific trait) can blur these inferences. Reconciliation approaches are progressively being used for the reconstruction of ancestral states ( Williams et al. 2024 Chernikova et al. 2011 Eme et al. 2014 Brocks et al. 2023 In summary, resolving the eToL and inferring the genotype and phenotype of ancestral deep nodes, including the LECA, will require a combination of exploratory, descriptive, and molecular phylogenomic approaches. There is an urgent need not only to explore the full extent of eukaryotic lineage diversity and delineate their (pan) genomes/transcriptomes/proteomes but also to integrate these data with morphological, cellular, molecular, and ecological information—including cell cycle dynamics—within a phylogenomic framework. FIG 16 (a) Representation of the six virus realms in bacteria, archaea, and eukaryotes. (b) Host ranges, at the domain of life level, of the six realms of viruses. Virus diversity in each realm is illustrated by images of the corresponding virions. The fractions (more...) This can be achieved through revival and encouragement of classical cultivation and microscopy studies and combining these with modern techniques to obtain information on single cells in natural ecosystems as well as specific labeling and high-resolution microscopies. The improvement of bioinformatic methods to methods to efficiently analyze large numbers of genes/proteins, establish orthology, and reconstruct phylogenomic trees is essential. Resources are needed to sustain and foster more classical characterization approaches and take advantage of long-accumulated knowledge in this sense, as well as to sequence, assemble, curate, and reliably annotate genomes and transcriptomes of free-living protists across the eToL. Developing and maintaining a freely accessible, reliable, and curated database for eukaryotic omic data across the eToL is highly desirable as well. C. Eukaryotic virome 1. Viral taxonomy and the LECA virome Viruses and other mobile genetic elements (MGEs) infect all life forms and can assume a parasitic or mutualistic role. The current viral taxonomic framework, as of 2025, categorizies most known viruses into seven realms based on nucleic acids and characteristic proteins: Adnaviria Duplodnaviria Monodnaviria Riboviria Ribozyviria Singelaviria Varidnaviria https://ictv.global/vmr 2. Viromes of prokaryotes and eukaryotes Several researchers have tried to address the open question on the LECA virome and perhaps the role viruses have played in eukaryogenesis ( Irwin et al. 2022 Kazlauskas et al. 2019 Koonin and Dolja 2014 Koonin et al. 2015 Koonin et al. 2020 Koonin et al. 2023 Krupovic et al. 2023 Krupovic et al. 2024 Wolf et al. 2018 Fig. 16 Varidnaviria Duplodnaviria, Riboviria Monodnaviria Krupovic et al. (2020) 3. Bacterial origins of the eukaryotic virome Bacterial origins for the eukaryote-infecting viruses from Riboviria, Monodnaviria, Duplodnaviria, Varidnaviria Krupovic et al. 2023 The deepest branch of the kingdom Orthornavirae Lenarviricota) Amabiliviricetes Howeltoviricetes), Amabiliviricetes Howeltoviricetes An ancestral reverse-transcribing virus evolved by recruiting and repurposing cellular proteins to function as capsid proteins and also viral proteases during a pre-LECA eukaryogenesis phase. Cressdnaviruses (realm Monodnaviria, Cressdnaviricota) Viruses in the phylum Preplasmiviricota, Ancestral members of the phylum Nucleocytoviricota The structural gene module of mirusviruses is derived from tailed bacteriophages or archaeal viruses in the class Caudoviricetes. Horizontal gene transfer of virus-derived genes to eukaryotes has likely played a role in eukaryogenesis. A comprehensive study on horizontal gene transfer between viruses and their eukaryotic hosts highlighted the high frequency of gene transfer with the majority of transfers involving double-stranded DNA viruses, particularly nucleocytoplasmic large DNA viruses ( Irwin et al. 2022 Irwin et al. 2022 4. Summary Current evidence points to a primarily bacterial origin of the LECA virome and extensive identification of viral-derived genes in eukaryotes, particularly single-cell protists, suggests their role in their early evolution and subsequent diversification. Analyses of the evolution of currently known viruses point to the origin of most of the eukaryotic virome from bacterial viral ancestors. However, deciphering the archaeal virosphere, particularly that of the Asgard archaea, will help further clarify steps in the evolution of eukaryote-infecting viruses. Perhaps unraveling the diversity of single-cell eukaryote-infecting viruses will provide novel insights into the viral-host dynamics of this ancient evolving system, coupled with novel insights into virus and host evolution. Research knowledge from the latter may lead to biotechnology innovations spanning therapeutics to environmental adaptation for microbial-based ecosystem remediation. FIG 17 Cellular features inferred to be present in the LECA. This schematic follows on from Richards et al. (2024) and summarizes the cellular features discussed in the section titled “What organelles were possed by LECA?” (and references therein). (more...) D. Cell structure and function: organelles, endosymbiosis, and energetics 1. Eukaryotes possess uniquely intricate subcellular organization A defining characteristic of eukaryotic cells, in contrast to prokaryotic cells, is their extensive compartmentalization. This organization features numerous membrane-bound organelles, such as the nucleus, mitochondria, and plastids, which contain particular cellular processes. While some eukaryotic cells have simplified their intracellular organization, such as the loss of mitochondria in Oxymonadida ( Novák et al. 2023 Murat et al. 2010 Gabaldón 2021 López-García and Moreira 2015 Gabaldón and Pittis 2015 2. Cellular features inferred to be present in the LECA When discussing the origin and evolution of “organelles,” it may be useful to note that this term is imprecise and has been previously used to refer to membranous subcellular structures (e.g., mitochondria or the Golgi apparatus) as well as to complex multi-protein structures (e.g., the ribosomes and the cytoskeleton). In addition, due to the intrinsic complexity and functional modularity of some organelles, they are often subdivided (e.g., Golgi apparatus, the endoplasmic reticulum, and the nucleus are all parts of the broader endomembrane system). This, together with variations of shape and function of organelles across eukaryotic organisms (e.g., hydrogenosomes and mitosomes are highly derived forms of mitochondria), adds to the difficulty of defining the boundaries of what an organelle is and what could be its origin in the context of eukaryogenesis. 3. What organelles were possessed by the LECA? Despite these challenges, numerous complex cellular structures can be traced back to the last eukaryotic common ancestor. This can be achieved through parsimonious inference based on the cellular structures of different eukaryotic supergroups or by computational reconstruction of the ancestral LECA proteome from extant sequences ( Richards et al. 2024 Fig. 17 4. Origination of organelles When discussing the mechanisms by which organelles can originate, the fundamental distinction between endogenous (also known as autogenous) or exogenous origins can be useful ( Gabaldón and Pittis 2015 Sagan 1967 Gabaldón 2021 de novo Roger et al. 2017 The origin of other organelles is less clear. While several complex endosymbiotic hypotheses have been put forward for the origin of other LECA organelles such as the flagellum ( Sagan 1967 López-García and Moreira 2006 de Duve 2007 Baum and Spang 2023 Raval et al. 2022 López-García and Moreira 2023 Forterre and Gaïa 2016 5. Timing of appearance of eukaryotic organelles A contentious aspect in the field of eukaryogenesis is the specific order in which the different organelles appeared in the LECA, particularly with respect to the timing of the acquisition of mitochondria relative to the origin of other organelles. Initial ideas depicting a complex, protoeukaryotic host for the mitochondrion, i.e., the archaeozoon, which contained most eukaryotic features with the exception of this organelle, were based on initial cell biology and molecular phylogeny results suggesting the existence of extant amitochondriate eukaryotes branching before the diversification of mitochondrial-bearing eukaryotes ( Cavalier-Smith 2009 Koonin 2010 Martin and Müller 1998 López-García and Moreira 2020 6. Early acquision of mitochondria Many hypotheses for the origin of other organelles rely on an early mitochondrial symbiosis, as the presence of this endosymbiont is suggested to have provided the selective force driving the origin of other organelles ( Raval et al. 2022 Force et al. 2005 Pittis and Gabaldón 2016 Gabaldón 2018 Vosseberg et al. 2021 A mitochondrion-first scenario is also defended by some with the argument that subcellular complexification would not be possible if not for the additional energy provided by mitochondria ( Lane and Martin 2015 Lane and Martin 2015 Lynch and Marinov 2016 Roger et al. 2017 Novák et al. 2023 Spang et al. 2015 Vosseberg et al. 2024 7. Summary and future directions of organelle research We can reconstruct the organellar repertoire that was likely present in the LECA, and there is consensus that this was an already complex organism with a subcellular complexity similar to that of extant free-living unicellular eukaryotes. However, beyond an endosymbiotic origin of mitochondria from an alpha-proteobacterial related organism and a likely Asgard archaeal origin of the cytoskeleton, little is known about the sources, mechanisms, selective triggers, or relative timing of the origin of all other organelles. During the colloquium, ideas were discussed about how the field could move forward. As for potential endosymbiotic origins or organelles, there was general agreement that the study of more recent endosymbioses such as the plastid ( Sibbald and Archibald 2020 Paulinella Gabr et al. 2020 McCutcheon 2016 Husnik et al. 2021 Coale et al. 2024 Giger et al. 2024 As with endogenous origins, there is a general lack of more recent examples of new organelles derived from previously existing ones that could be used as a model to study how a new organelle can be generated from previously existing structures. These examples should not be mere transformations of existing organelles, such as mitochondria into mitosomes or hydrogenosomes during adaptation to anaerobic conditions ( Roger et al. 2017 Diversification of the endomembrane into different subcompartments by duplication of key organellar biogenesis proteins, or the possible origin of peroxisomes from the endoplasmic reticulum ( Gabaldón 2010 Dunkley et al. 2004 Many ideas discussed during the colloquium underscored the need to support active exploration of the Earth's microbial diversity. Untargeted DNA-sequencing approaches such as shotgun metagenomics of environmental samples have opened our eyes to a vast microbial diversity and its distribution across ecosystems. However, beyond the taxonomic description of microbial diversity and the analysis of genomes, there is very little information regarding their physiology, intracellular structures, or ecological interactions. Closing this gap would be instrumental in answering the question of how eukaryotes may have emerged from prokaryotes. E. Evolution of protein structure during eukaryogenesis and after 1. New protein complexes and functions and causality in eukaryogenesis Eukaryogenesis involved the evolution of a number of new protein complexes and functions that are thought to be closely tied to the cellular properties that differentiate eukaryotes from their prokaryotic ancestors. These novelties are thought to be causally responsible for eukaryotes’ much more complex cellular biology. Examples of this are the nuclear pore complex and the components of the eukaryotic cytoskeleton. Much of the general problem of how eukaryotes came to be can be restated as understanding the origins of these new biochemical components. This is exemplified by the discovery of Asgard archaea, which was so significant because their genomes contained close homologs of proteins that were previously thought to be novel to eukaryotes, thereby removing those components from the list of novel eukaryotic components and changing the question to one of the reasons for them producing eukaryogenesis in one particular lineage. The goal of studying new proteins or molecular functions during eukaryogenesis is to find the list of biochemical components and functions that originated over the branch leading to the LECA, as it may allow us to assign causality in the evolution of eukaryotic cellular biology to these components. Is this hunt for causal biochemical novelties productive, and how should we go about it? Examples from other evolutionary transitions in which we also seek to find biochemical novelties that cause a change in cellular or organismal complexity are instructive. The first phenomenon for which we can draw such an analogy is the fact that the list of eukaryote-specific proteins has been continually shrinking. The biggest reduction in this list came from the discovery of Asgard archaea, but other examples exist. ESCRT proteins, for example, which are involved in membrane re-modeling, have recently been discovered and characterized in multiple prokaryotes ( Liu et al. 2021 Souza et al. 2025 This situation is very analogous to recent advances in the study of the origin of metazoa, where evolutionary biologists are similarly focused on an ever-shrinking list of animal-specific proteins that are believed to help explain what exactly caused the evolution of multicellularity. In this case, the branch leading from the closest relatives of metazoa to their last common ancestor is shorter, and increasingly available genomes from close outgroups have led to the identification of several components that were initially thought to be exclusive to animals ( Ros-Rocher et al. 2021 Ruiz-Trillo et al. 2023 Gao et al. 2024 This is a common phenomenon in the study of novelty. When we interrogate dramatic evolutionary transitions in cellular complexity that at first seem to be associated with an equal explosion of novelty at the biochemical level, we often find that biochemical novelty tends to dissolve into many examples of pre-existing components being recruited into new (for example, eukaryote-specific) cellular functions. However, this just puts off confronting a difficult issue: if the emergence of new kinds of cellular biology cannot be pinned to the emergence of their most prominent components (like actin for the eukaryotic cytoskeleton or Sox proteins for animal pluripotency), then what else would be the decisive change? 2. Mechanisms of new eukaryotic-specific functions The answers may lie in the emergence of new interactions between pre-existing components, which in turn may result in new eukaryote-specific functions. Such interactions could evolve between proteins that did not interact yet in the LECA's archaeal ancestors. Or new functions could evolve through duplication and functional diversification of pre-existing components. Indeed, eukaryotic protein complexes that have identifiable relatives in prokaryotes often contain more duplicate paralogs. Where the prokaryotic versions may have multiple copies of one protein in a complex, eukaryotes can have one separate paralog for each copy in the prokaryotic complex. This greater topological and genetic complexity of eukaryotic assemblies may be key to their different cell biology, if it indeed allows for new kinds of functions. 3. Entrenchment along the route from prokaryotic to eukaryotic protein functions If changes in interactions are causally important for the much greater cellular complexity in eukaryotes, we are presented with a much greater challenge. One complication is that exactly this kind of structural complexity can in principle arise and then persist for long periods of time through entirely neutral processes ( Schulz et al. 2022 Archibald et al. 2001 Zhang et al. 2010 Liou and Willison 1997 4. Assigning causality on the road to the LECA to changes in protein-protein interactions and biochemistry To truly understand if additional eukaryotic protein-protein interaction is causally responsible for a eukaryote-specific function, we ideally need to understand if those new interactions arose at the same time as the new functions. However, it is much harder to predict how a component interacts and functions in an ancestral organism than to simply infer that it existed. Our understanding of what all components predicted to be present in the LECA and its predecessors do is very limited, for several reasons. First, our understanding of eukaryotic biochemistry is often mostly informed by the situation in model organisms such as yeast and in animals, which can be unrepresentative of the broad diversity of eukaryotes. One example for this problem is the RNA exosome. This is an RNA degrading machine, which also participates in the maturation of ribosomal RNAs. The exosome is present in archaea, where its core comprises a ring of six proteins, made from three heterodimers of two paralogs, capped by another homomeric ring ( Lorentzen et al. 2005 Kilchert et al. 2016 Fromm et al. 2017 Sikorska et al. 2017 This example illustrates the difficulty we have in inferring the LECA's detailed biochemistry, which requires structural and functional characterization across the tree of eukaryotes. This, however, is a challenging proposition. First, purifying and assaying large multi-protein complexes (as was necessary to realize that the plant exosome does not require peripheral RNAases) from just one organism is a very significant undertaking. It is hard enough in organisms that are easy to cultivate or even have tractable genetics such that affinity tags can be introduced. It is much harder in difficult-to-culture single-cell eukaryotes with no genetic tools available. On top of this, our own experience with sampling simple protein complexes across bacteria suggests that the more one looks, the more one finds a profound variation in structure for proteins with ostensibly very basic functions ( Sendker et al. 2024 The same difficulties apply to the LECA's Asgard archaeal relatives, which are very hard to culture and whose proteins are not necessarily easy to purify recombinantly, meaning that it can be challenging to confirm what exactly Asgard-archaeal homologs of eukaryotic proteins do. Nevertheless, there are success stories. Loki-profilins and gelsolins, both important regulators of dynamic actin cytoskeletons in eukaryotes, for example, have been produced recombinantly, purified, and characterized, showing them to be remarkably similar to their eukaryotic cousins ( Akıl and Robinson 2018 Akıl et al. 2022 Overall, our biggest challenge in the study of the biochemical innovations that may have played a significant part in eukaryogenesis is to move from gene content predictions for the LECA and its predecessors to more detailed predictions about the interactions and functions of those components in ancestral organisms. To make these inferences, we need either much more data on the sequence-structure-function relationship across the archaeal and eukaryotic trees or more direct ways to assay the function of the LECA's biochemistry. F. Sex and molecular evolution of eukaryotic genomes The concluding topic addressed by the eukaryogenesis colloquium revolved around the molecular evoution of eukaryotic genomes and population genetics. The evolution of sex and the resultant genetic variation in eukaryotic genomes is a cornerstone of life's complexity and diversity. Sexual reproduction is generally defined as the fusion of genetic material from two distinct individuals, producing offspring with a unique combination of genes. This process generates genetic variation, which is crucial for a species’ ability to adapt and evolve in response to changing environments ( Fu et al. 2019 Heitman 2015 Lenormand et al. 2016 1. Genome remodeling to facilitate adaptation Sexual reproduction uniquely reshapes genomes through extensive recombination. Recombination, which occurs during meiosis, involves the exchange of genetic material between homologous chromosomes, leading to novel gene combinations. This genetic shuffling not only disrupts linkage disequilibrium (the non-random association of alleles at different loci) but also introduces novelty into populations in the form of previously unseen combinations of alleles. Recombination can “clean up” the genome by breaking up harmful gene combinations and promoting beneficial ones, a process known as genetic purging. This purging eliminates deleterious mutations, contributing to genome stability and reducing genetic load over successive generations. While mutations are often harmful, they are ultimately the source of genetic diversity. Sexual reproduction can mitigate the negative effects of mutations by ensuring they coexist with unmutated versions of the same locus, a process known as masking. This phenomenon occurs when recessive deleterious mutations are hidden in the presence of a dominant healthy allele. Moreover, mutations can give rise to new traits that may be advantageous, enabling adaptation through natural selection. In this sense, sexual reproduction acts as both a creator and an organizer of genetic variation, facilitating adaptation in eukaryotic species. However, while en route to fixation, selection can also break up adaptations, so there is a sweet spot ( Lynch and Abegg 2010 FIG 18 Repeat sequences cause the failure of LGT (Colnaghi et al. 2022). (A) The total gene loss rate ΔM=Δt (more...) 2. Costs and benefits of sexual reproduction However, sex comes with evolutionary costs, notably the «twofold cost of sex.» In many sexually reproducing species, only half of an individual's genes are passed to offspring, which is less efficient than asexual reproduction, where an individual transmits its entire genome. Despite these costs, the benefits of sexual reproduction in maintaining genetic diversity and adaptability are believed to outweigh the disadvantages, particularly in environments subject to frequent changes. The genetic shuffling provided by sex may allow populations to respond more rapidly and effectively to selective pressures, such as pathogens, climate shifts, and ecological competition, compared to clonal populations. The evolution of sex has significantly influenced genome structure, with specific genomic features evolving to facilitate recombination and safeguard genetic integrity. For example, centromeres, telomeres, and repetitive elements in eukaryotic genomes may have coevolved in the context of sexual reproduction and recombination, playing critical roles in genomic stability. 3. Sexual phenotype of the LECA leading to modern eukaryotic sexual cycles There is broad consensus that the last eukaryotic common ancestor was sexual. Evidence supporting this includes the shared set of genes related to meiosis that is found across all modern eukaryotes, suggesting that sexual reproduction originated only once, prior to their most recent common ancestor. The earliest form of sexual reproduction likely involved an increase in ploidy, followed by a rudimentary parasexual cycle that returned to a haploid state through concerted chromosome loss. Two potential scenarios for the transition from «one to two» have been proposed. The first suggests that diploidy occurred accidentally through replication of the nuclear genome without subsequent cell division, and the return to the haploid state was selected for as a corrective mechanism. The resulting life cycle may have resembled the parasexual cycles seen in some modern fungi, though other eukaryotes also exhibit somatic ploidy changes depending on growth stage or environmental stimuli. The second scenario posits that proto-meiosis evolved as a response to the fusion of two haploid cells, a process seen in modern eukaryotic sexual cycles. This form of syngamy may have been favored because it allows recessive deleterious mutations to be masked in diploid organisms. FIG 19 Diagram of the potential paths underlying the origins of eukaryotic sex. 4. Why only one origin for meiosis/syngamy and so many ways to determine mating types/sexes ? What purpose do “mating types”/”sexes” serve? Meiosis is a crucial step in sexual life cycles and it also represents a cellular and genetic bottleneck at the critical transition between the diploid and haploid phases. The range of permissible mating partners is regulated by categories known as mating types or sexes, which vary across different lineages. Several hypotheses have been proposed to explain this variation, including the regulation of cytoplasmic mitogenome conflicts, the maintenance of uniparental inheritance of organelles, the avoidance of inbreeding, and the activation of developmental switches ( Bachtrog et al. 2014 Perrin 2012 5. Did meiotic sex contribute to the success of the LECA? One hypothesis is that meiosis originally evolved as a mechanism for repairing DNA damage, particularly damage caused by increased oxidative stress due to rising atmospheric oxygen or endosymbiosis. The ability to repair DNA effectively could have been crucial in the development of complex cellular structures. This scenario presupposes that DNA maintenance is inefficient in the absence of meiosis; however, prokaryotes (including archaea) have efficient repair mechanisms that involve recombination but not meiosis ( Hörandl and Speijer 2018 Lenormand et al. 2016 Speijer et al. 2015 Sexual recombination upon meiotic crossover allows rapid gene reshuffling, quickly both eliminating bad combinations and finding good ones. This diversity may have given early eukaryotes an evolutionary advantage. However, prokaryotes also have efficient ways to promote diversity; homologous recombination evolved long before meiosis, as it occurs in all domains of life and involves proteins that share strong homology. All organisms must allow recombination between their genomes and external DNA at least occasionally, lest Muller's ratchet and clonal interference cause a mutational meltdown. One attractive hypothesis proposed recently is that meiotic pairing and extensive homologous recombination in meiosis evolved to avoid the burden and consequences of ectopic recombination in the large, repetitive genomes of early eukaryotes, which became unable to reliably recombine with exogenous DNA without triggering large deletions ( Fig. 18 Colnaghi et al. 2020 Insights into this process arise from computational modeling of mutation and selection in a population undergoing lateral gene transfer (LGT) via transformation with increasingly repetitive DNA. Mutational decay can only be resisted with recombination to erase deleterious «hitchhiker» mutations, templated by homology along extended sequences of DNA. As repeats expand, longer and longer stretches of homology are needed to avoid ectopic recombination until eventually the entire genome must be homologous. Then, only the closest relatives are capable of recombination. The model highlights a tradeoff between the benefits of LGT (greater genetic variance, enhancing purifying selection) and its cost (loss of genetic information through ectopic recombination). This leads to the view that the transition to meiotic sex was driven by the need for purifying selection in the expanding and repeat rich genomes of early eukaryotes, which could not be met by stepwise increases in recombination length or LGT rate. A second possibility is that recombination arose by the spread of self-promoting genetic elements exploiting the machinery of DNA repair and associated gene conversion (reviewed in Goodenough and Heitman 2014 6. What drove the transition to meiotic sex? What selective scenario (if there is one) led to evolution of meiosis (and by extension to eukaryogenesis)? What selective pressures (if any) drove the evolution of meiosis and, by extension, the emergence of eukaryogenesis? Uncovering the underlying evolutionary scenario requires examining several key factors (see Fig. 19 Lenormand et al. 2016 7. Conclusion Eukaryogenesis research is at a critical juncture, with exciting opportunities for conceptual and technological breakthroughs. By leveraging interdisciplinary approaches and cutting-edge methodologies, the scientific community can unravel the complexities of eukaryotic origins and refine our understanding of the evolutionary processes that shaped complex life. V. EVOLUTION AND MECHANISMS GOVERNING MULTICELLULARITY A. Overview of multicellularity The concept of “multicellularity” seems straightforward and intuitive, often understood through basic reasoning. However, a closer look reveals considerable variation in its use in current research ( Rose and Hammerschmidt 2021 Grosberg and Strathmann 2007 1. Frequency and process for evolution of multicellularity The transition to multicellularity is widely accepted to have occurred multiple times throughout the history of life, though determining the exact number of transitions remains challenging. It is clear that multicellularity has evolved independently at least 50 times and likely more across diverse lineages, including plants, animals, fungi, and various groups of algae, bacteria, and protists. These independent transitions suggest that multicellularity is not a singular event but rather a recurring phenomenon that emerged in diverse evolutionary contexts. It is thus useful to conceptualize multicellularity as part of an evolutionary process that involves multiple steps. Consequently, multicellular organisms accumulated “multicellular” traits, such as cellular specialization and interdependence, over evolutionary timescales. It is plausible that the progression of this phenomenon may have manifested to varying degrees, rather than as discrete events, across diverse lineages. Factors contributing to this variation in the “degree of multicellularity” may include distinct evolutionary trajectories, temporal differences in lineage origins, and the impact of diverse environments. While not all lineages necessarily progress towards more complex multicellularity, the evolutionary processes and sequence of events leading to multicellularity resemble those of other evolutionary transitions in individuality or major transitions in evolution ( Maynard Smith and Szathmáry 1995 2. Multicellular groups, individuals, and organisms The transition to multicellular organisms can be defined as beginning with multicellular groups and then progressing to multicellular individuals, which can evolve into multicellular organisms ( Fig. 20 Rose and Hammerschmidt 2021 The advantages of using the framework of evolutionary transitions in individuality are many. It redefines multicellular as an adjective modifying “group,” “individual,” and “organism,” which helps avoid definitional ambiguities, since classifications of “groups,” “individuals,” or “organisms” are typically clearer. This approach unifies different manifestations of multicellularity, thereby enabling comprehensive comparative analyses, such as identifying commonalities in early multicellular evolution or in later stages such as tissue-level organization. It also allows comparisons between multicellularity and other evolutionary transitions in individuality, such as the origin of cellular life, eukaryotes, and superorganisms. FIG 20 Multicellular entities are defined as multicellular groups, multicellular individuals, or multicellular organisms, reflecting the stages of the evolutionary transition from single cells to multicellularity imbedded within the hierarchical structure of (more...) The transition to multicellularity involves complementary approaches across various disciplines. Traditionally dominated by paleontology and philosophy/theory, recent technological advances have enabled the integration of comparative and experimental approaches. It has become clear that interdisciplinary collaboration is essential for advancing our understanding of the evolution of multicellularity. 3. Environmental conditions and fossil record of multicellularity Paleontological research offers insights into the geological context and timeline of the evolution of multicellular life and provides key constraints for comparative analyses across the different occurrences of multicellularity evolution (described in additional detail in Secs. II F and V B). Current data suggest that multicellularity emerged in bursts, such as extreme glaciations (e.g., Snowball Earth) during the late Neoproterozoic (1000-541 Ma). However, it remains unclear whether these so-called bursts represent multiple independent origins of multicellularity from potentiated ancestors or the rapid diversification of single multicellular lineages. This uncertainty makes it difficult to identify the key environmental driver(s) of multicellularity. A major challenge in this field is the limited data from the Proterozoic, which leads to a sampling bias, where the observed diversity depends on the number of formations examined ( Porter et al. 2025 However, there is increasing evidence that multicellularity has evolved across domains, including archaea ( Rados et al. 2025 Tang et al. 2023 Schwartzman et al. 2022 Tang et al. 2023 McShea 1996 The differences between prokaryotic and eukaryotic multicellularity may be due to variations in membranes ( Rados et al. 2025 Valentine 2007 Lotharukpong et al. 2024 Bingham and Ratcliff 2024 Olivetta et al. 2024 Petroff et al. 2010 Persat et al. 2015 Simpson 2021 Crockett et al. 2024 Simpson 2021 Halling et al. 2024 Crockett et al. 2024 4. Prokaryotic versus eukaryotic multicellularity Recent data demonstrate that prokaryotic multicellularity is more variable than eukaryotic multicellularity, challenging classical views of multicellularity (see Sec. V C). In addition to clonal multicellularity, as seen in organisms such as actinobacteria or cyanobacteria, prokaryotes frequently engage in aggregative multicellularity, as seen in myxobacteria. In both cases, differentiation in the form of terminal division of labor (with cells becoming unable to alter their function while taking part in the function of the group) can be observed. While prokaryotic multicellular life cycles can be complex ( Tang et al. 2023 Barka et al. 2016 Futo et al. 2021 Tang et al. 2023 Mizuno et al. 2022 Geesink et al. 2024 Huelsmann et al. 2024 Black et al. 2020 Doulcier et al. 2024 Hammerschmidt et al. 2014 5. Approaches to understand the mechanisms underlying multicellularity It was originally thought that the transition to multicellularity required the evolution of many novel genes. However, recent comparative genomics studies, particularly of animal multicellularity, show that many genes once thought to be animal specific, such as adhesion proteins, signaling receptors, and transcription factors, are also present in their close unicellular relatives ( Sebé-Pedrós et al. 2017 McShea 2016 Early theoretical approaches to the evolution of multicellularity focused on how cooperating groups of cells overcome the risk of destruction by “cheating” types, lineages which evolve to take advantage of the “public goods” created in a multicellular group context without contributing anything towards their maintenance, thus gaining an advantage over their hosts within the group context. While the role of conflict in shaping multicellular biology remains unclear, it is thought that aggregative multicellularity, where different parts come together to form a multicellular individual, is more susceptible to conflict than clonal multicellularity. This has been suggested to be the reason for the repeated origin of development from a unicellular bottleneck that limits diversity and excludes potential cheaters from being incorporated into new groups, which is in some lineages combined with germline sequestration and policing strategies. Initially, selfish behaviors in aggregative multicellularity were thought to be controlled by kin recognition. However, it is becoming increasingly clear that not all ecologies of aggregative multicellularity favor strong kin recognition. Similarly, clonal organisms are not always purely clonal, as seen in human microchimerism, where fetal and maternal cells are exchanged ( Boddy et al. 2015 More recently, models have shifted the focus to understanding multicellularity through evolutionary construction, emphasizing life cycles and tradeoffs that shape different evolutionary paths to multicellularity ( van Gestel and Tarnita 2017 Libby and Ratcliff 2021 New model systems, including unicellular species, such as yeast, algae, protozoa, and bacteria, have successfully been used in experimental evolution studies to investigate the establishment and evolution of early stages of multicellularity ( Hammerschmidt et al. 2014 Ratcliff et al. 2012 Brunet et al. 2019 Kapsetaki and West 2019 Lotharukpong et al. 2024 Bourdareau et al. 2021 Coelho and Cock 2020 FIG 21 Ten selective drivers of simple multicellularity: predation avoidance, stress resistance, improved metabolism, faster sedimentation, increased motility, chimeric benefits, cross-feeding and division of labor, competitive overgrowth, resource utilization, (more...) The evolution of multicellularity is largely studied under the premise that it is an adaptive process solely shaped by natural selection ( Fig. 21 Bingham and Ratcliff 2024 Lynch et al. 2023 Lynch 2024 Lynch et al. 2023 6. Biophysics and multicellularity Further, many traits of emerging multicellular life cycles arise naturally from the physical properties of groups, and this was a topic of active discussion for the colloquium participants (see Sec. V E). The snow-flake yeast model system has been particularly informative in this regard. In this system, group reproduction and heredity emerge from the way yeast cells are physically packed into clonal groups ( Jacobeen et al. 2018 Day et al. 2022 Jacobeen et al. 2018 Day et al. 2022 Milinkovitch et al. 2013 Despite progress, the origin of multicellularity is far from being universally understood. A key challenge is the quantification of complexity, which requires standardized metrics beyond simple counts of cell types. Another uncertainty is timing, for example, the late emergence of multicellular eukaryotes, which requires new conceptual frameworks that can be experimentally tested. Future research also needs to integrate more accounts of prokaryotic multicellularity. Combining studies of the fossil record with experimental evolution, synthetic biology, and theoretical models will be important for understanding constraints on multicellular evolution. Open questions include why not all organisms became multicellular, the role of parasites in complexity, and the impact of (morphological) constraints on evolutionary trajectories. B. Insights from comparative biology 1. Comparative biology to distinguish simple versus complex multicellularity After reflection on the Earth conditions and causal forces underlying multicellularity, the colloquium participants continued to examine approaches that can be used to explain the mechanisms of evolution of multicellularity. Across all domains of life, multicellular organisms have evolved independently many times. Among this set, they express a wide range of form and organization, from biofilms to bison. Yet despite this vast diversity and disparity of multicellular life, there are similarities and differences among unrelated organisms that hint at processes that drive the evolution of multicellularity that can be discovered by comparative means. Two frameworks have emerged to make sense of this. The first focuses on how multicellular organisms form ( Bonner 1998 Queller 2000 Tarnita et al. 2013 Knoll 2011 Buss 2016 Michod and Herron 2006 Knoll 2011 These frameworks are both so broad that they each divide all multicellular life into two categories. It should therefore be easy to categorize any multicellular organisms cleanly into the categories. Yet many organisms are now being discovered that either blur these categories or otherwise upset the frameworks. For example, the colonial choanoflagellate Choanoeca flexa Brunet et al. 2019 Ros-Rocher et al. 2024 Vibrio splendidus Schwartzman et al. 2022 Combining these frameworks into a single four-category classification (e.g., Simpson 2011) highlights another limitation. Most multicellular life is simple, and neither aggregative nor clonal development seems to dominate in terms of this diversity. Complex multicellularity (defined as multicellularity with tissue differentiation mediated by networks of regularity and developmental genes), on the other hand, is rare with only five clades containing this trait (some with multiple origins), all of which are clonal. These frameworks highlight a just few groups as requiring special consideration, but they do not provide any insight into how the most complex multicellular organisms originated or evolved. Nor do they provide insight into why simple multicellular organisms remain simple. 2. Comparative analysis of multicellular organisms from the three domains of life To gain insight into the drivers of multicellular evolution, it may be useful to go back to basics and consider the similarities and differences in multicellular organisms from the three domains of life, the archaea, the bacteria, and the eukaryotes. Until recently the only example of archaean multicellularity was from the sarcina-forming group the Methanosarcinae exemplified by Methanosarcina Mayerhofer et al. 1992 Rados et al. 2025 Rados et al. 2025 Haloquadratum M. maripaludis Within bacteria, multicellular group formation is essentially ubiquitous ( Shapiro 1998 Aguilar et al. 2007 Rosenberg 2009 Lyons and Kolter 2015 Allwood et al. 2006 Allwood et al. 2009 Nostoc Vibrio splendidus Schwartzman et al. 2022 Nisbet and Fowler 1999 Bosak et al. 2013 Despite this complexity of form and deep time evolutionary history, there remains a sense in the scientific community that bacterial and archaean multicellularity is fundamentally constrained to low complexity in some fashion (Bonner 2001; Bingham and Ratcliff 2024 Crockett et al. 2024 Tang et al. 2023 Multicellularity in eukaryotes is not ubiquitous, despite the fact that it has evolved at least 45 times ( Grosberg and Strathmann 2007 Herron et al. 2013 Niklas and Newman 2013 Lamża 2023 Tang et al. 2024 Yang et al. 2016 Bykova et al. 2020 Tang et al. 2024 Erwin et al. 2011 Dohrmann and Wörheide 2017 Tang et al. 2024 Choi et al. 2024 Herron et al. 2009 In total, five groups of eukaryotes evolve large, complex multicellularity, with clonal development and cellular differentiation. The tally of groups, specifically red algae, green algae, brown algae, fungi, and animals, underestimates the number of origins of complex multicellularity within most of these groups ( Lamża 2023 Yang et al. 2016 Umen 2014 Umen and Herron 2021 Nagy et al. 2018 Choi et al. 2024 Erwin et al. 2011 This laundry list of origins does little to highlight the striking pattern that the origin and diversification of macroscopic complex multicellularity within red and green algae and animals appear pulsed, occurring in the late Tonian through Cryogenian Periods (800-635 million years ago) ( Erwin et al. 2011 Yang et al. 2016 Nagy et al. 2018 Choi et al. 2024 Tang et al. 2024 3. Resource transport as a defining trait of complex multicellularity One neglected attribute Knoll ( Knoll 2011 Persat et al. 2015 Schwartzman et al. 2022 Hunter and Vogel 1986 4. Comparative analysis using resource transport as a differentiator to discern mechanisms governing multicellularity This physical perspective on the mechanisms of resource acquisition and transport is a potent means of comparing and contrasting multicellularity among all groups. Though it does not include explicit evolutionary and developmental mechanisms (such as mechanisms of inheritance and development of phenotypes), it helps us demarcate bounds in which evolution can act. Do to their small size and slow swimming speeds, bacteria and archaea rely solely on diffusion to acquire resources from the environment and they use diffusion to transport those resources within individual cells and thoughout any multicellular structures they produce (Vogel 1996; Petroff et al. 2011; Persat et al. 2015 Persat et al. 2015 Fernandez et al. 2019 Słomka et al. 2023 −3 −2 Simpson 2021 In stark contrast, eukaryotes frequently generate their own flows and have larger sizes such that their Péclet numbers are 1-5 or more orders of magnitude higher than for bacteria ( Simpson 2021 Simpson 2021 Crockett et al. 2024 Internal transport in eukaryotes can be different too. Though they use diffusion to move resources short distances, cytoskeletal networks can aid in active transport over larger internal cellular distances ( Agrawal et al. 2022 Knoll 2011 Buss 2016 5. Comparison of prokaryotic and eukaryotic multicellularity based on genomic properties Another perspective that distinguishes between the styles of prokaryotic and eukaryotic multicellularity comes from a comparison of the regulatory structure of their genomes and how their genomes respond to drift ( Bingham and Ratcliff 2024 Kuo and Ochman 2009 Bobay and Ochman 2017 Lynch and Conery 2003 Lynch 2007 Lynch 2010 Bingham and Ratcliff 2024 In conclusion, there may be fundamental differences between prokaryotic and eukaryotic multicellularity stemming from the basic differences between the eukaryotic domain and others, based on their size and their population genetics. The larger eukaryotic cell is less diffusion limited internally than bacteria due to active transport on cytoskeletal pathways, but also less diffusion limited externally due to its larger size and ability to introduce large bulk fluid flows in its environment, enabling the creation of multicellular adaptations that circumvent limitations that prokaryotes face in multicellular groups. This same larger organismal size also alters their population genetic environment, potentially altering the forms of mutation selected against and allowing for constructive neutral evolution that is all but prevented in the prokaryotic context of very large population sizes. C. Prokaryotic multicellularity From the first attempts to cultivate microbes in more naturalistic conditions than dense populations grown in flasks, it has been clear that the ability to physically interact with other cells is a widespread property of microbial life. Physical interaction is a motif that appears in contexts ranging from tissue infection ( Whiteley et al. 2017 Remus-Emsermann and Schlechter 2018 Baker et al. 2024 Michielsen et al. 2024 Metcalfe et al. 2021 Baker et al. 2024 McCallum and Tropini 2024 Pfreundt et al. 2023 Prasad et al. 2023 FIG 22 Properties that shape prokaryotic multicellularity. (a) The modularity of metabolism shapes how cells interact to form obligate syntrophic consortia (top) or to differentiate into subpopulations to perform distinct metabolic tasks (bottom). (b) The plasticity (more...) The discovery that prokaryotes can co-ordinate behavior through true signals ( Boedicker and Nealson 2016 West et al. 2007 Shapiro 1988 West et al. 2007 Hallatschek et al. 2023 Hengge 2020 1. How do microbes divide metabolic labor? A key feature of prokaryotic systems is their metabolic diversity. Metabolic divisions of labor are a core motif both for multicellular prokaryotes ( Evans et al. 2020 Gralka et al. 2020 Fig. 22 Recent work in microbial community ecology has revealed how the structure of metabolic networks provides scaffolds for interactions between cells. This principle extends from communities to multicellular groups. For marine microbial food webs that decompose organic matter, a few “specialist” taxa mobilize initially recalcitrant resources, providing scaffolding for community assembly by excreting metabolites that are both more diverse and more generally metabolizable ( Gralka et al. 2020 Wilbanks et al. 2014 Metcalfe et al. 2021 Chlorocrhomatium Liu et al. 2013 Libby et al. 2019 Schwartzman et al. 2022 2. How does cellular plasticity shape prokaryotic multicellularity? The ability to differentiate into cell types that express subsets of genes and to evolve new regulation that coordinates the expression of these distinct phenotypic states shares some similarities with the programs of gene expression that coordinate the development of multicellular eukaryotes. However, there are few known examples of terminal cell differentiation in prokaryotes, meaning that prokaryotic multicellular systems typically retain the ability to reproduce and readily change state, that is, they remain phenotypically plastic. Recent studies of the regulatory architecture underlying bacterial collective behavior reveal strategies to coordinate collective behaviors in dynamic environments ( Bridges et al. 2022 One ubiquitous type of conflict that must be dealt with is the evolution of social cheating. This is a well-studied area of collective behavior and one that can be solved many ways, including evolving regulatory architecture that ties individual and collective fitness together, such as the metabolic prudence mechanism of Pseudomonas aeruginosa in which public goods are only produced when they are not overly costly and provide a group benefit ( Whiteley et al. 2017 Claessen et al. 2014 Poltak and Cooper 2011 Hammerschmidt et al. 2014 Fig. 22b Conflict can also arise through the incorporation of new genetic content through lateral gene transfer into a subset of group members. Mobile elements, including plasmids, phage, and integrative conjugative elements, can disrupt genetic architecture through loss of function or regulatory shifts, and they can also transfer functional genes such as signal synthases and metabolic genes ( Weisberg and Chang 2023 Smith et al. 2023 Schaible et al. 2024 Chlorochromatium. Doré et al. 2024 Fig. 22b 3. How does physical structure emerge in multicellular prokaryotes? An attribute of prokaryotic cell systems is the ability to generate patterns through localized variation in growth, death, and cell packing. Emergent patterns include “broccoli” formed by Escherichia coli Martínez-Calvo et al. 2022 Vibrio cholerae Hallatschek et al. 2023 Fig. 22c Flärdh et al. 2012 Haloferax volcanii Rados et al. 2025 Jo et al. 2022 Jo et al. 2022 Schwartzman et al. 2022 Tecon and Leveau 2016 Work on bacterial biofilms reveals a key role for the physical interactions in co-ordinating the metabolic exchange in a multicellular group ( Fig. 22c Evans et al. 2020 D’Souza et al. 2018 Chou et al. 2022 Wielgoss et al. 2019 4. Three current barriers for the field of prokaryotic multicellularity a. We are early in the sampler's curve for our understanding of prokaryotic multicellularity. While analogy with multicellular eukaryotes has guided the discovery of multicellular forms in bacteria ( Lyons and Kolter 2015 b. Cultivability Part of the challenge with identifying forms of prokaryotic multicellularity is that outside of cyanobacteria, myxobacteria, and actinobacteria, terminal cell differentiation is not widespread (Van Gestel 2015), which suggests that most prokaryotic multicellularity is dependent on environmental context. This means that we must develop methods to detect hidden multicellularity “in the wild” and foreground ecological interactions into the frameworks that we use to understand the drivers of multicellularity. Some forms of prokaryotic multicellular capacity may be inaccessible or unobservable until we can overcome barriers to sampling and create observation chambers. Devices such as the in situ chemotaxis assay ( Clerc et al. 2020 Gao et al. 2018 c. Metabolic complexity Prokaryotic communities contain far more complex metabolism than eukaryotic communities, in terms of the substrates that can be respired or catabolized. As highlighted above, an emerging paradigm for multicellularity across domains is the ability to coordinate tasks. A great success of microbial ecology is predicting “missing” metabolic pathways that should be feasible through an understanding of thermodynamic constraints ( Li et al. 2025 Peterson et al. 2024 Kaçar 2024 D. Evolutionary construction of multicellularity This section examines the types of multicellular complexity that can be constructed from the building blocks of single cells and highlights the questions addressed by the participants surrounding the evolution of multicellular life cycles, fitness and adaptation, division of labor, and genetic conflict. The focus is on the process of construction itself, analyzing how multicellular structures emerge and evolve from initially unicellular organisms. By adopting a bottom-up approach, the aim is to understand how simple cells transition to cooperative groups and ultimately complex multicellular life forms. A benefit of this approach is it elucidates the diversity of possible evolutionary trajectories, which can lead to the identification of constraints that may have shaped or limited what was realized by evolution. It also highlights differences between the earliest stages of multicellularity and later, more established forms, which may experience distinct evolutionary processes and dynamics. 1. Life cycles in multicellularity The process of evolutionary construction is informed by theoretical studies of evolutionary transitions in individuality, which describe the emergence of new types of individuals or higher-level units of selection (Michod and Nedelcu 2003; Michod 2007 West et al. 2015 Kauffman 2011 De Monte and Rainey 2014 From this perspective, two fundamental features define a multicellular life cycle: (i) the mechanism by which a group is initially formed and (ii) the pathway by which groups give rise to daughter groups (Libby and Rainey 2013). Examining the various combinations of these features provides deeper insight into the stability and possible evolutionary trajectories of multicellularity ( Ratcliff et al. 2017 Staps et al. 2019 Libby et al. 2016 2. Mechanisms of group formation and life cycles Empirical studies of microbial model systems reveal that there are many ways that simple groups of cells can be formed ( Ratcliff et al. 2012 Tong et al. 2022 Chavhan et al. 2023 Herron et al. 2019 Tarnita et al. 2013 Dictyostelium discoideum, Schaap 2011 Ratcliff et al. 2012 Pentz et al. 2020 The two main modes of group formation are usually incorporated into distinct life cycles ( Staps et al. 2019 Ratcliff et al. 2017 Pichugin et al. 2017 Isaksson et al. 2023 3. Consequences of group formation and life cycles for fitness and adaptation The differences in group formation and life cycles can have consequences for adaptation and the evolutionary stability of multicellularity ( Ratcliff et al. 2017 Pentz et al. 2020 Pentz et al. 2023 Dictyostelium discoideum Dubravcic et al. 2014 Tarnita et al. 2015 Ratcliff et al. 2017 Pentz et al. 2023 Since clonal life cycles eschew the unicellular phase, they are better able to select traits that are costly to individual cells but beneficial to groups ( Márquez-Zacarías et al. 2021 Libby et al. 2016 Libby et al. 2014 Libby and Ratcliff 2014 Márquez-Zacarías et al. 2021 Pentz et al. 2020 Ratcliff et al. 2017 4. Emergence of functional specialization A major challenge for clonal multicellularity is generating cellular diversity, particularly division of labor, in the absence of genetic diversity. A hallmark of complex multicellularity is the ability to regulate the production of cells with diverse functions, often organized into specialized tissues. However, early clonal multicellular life cycles contain genetically and phenotypically identical cells, making the emergence of functional specialization unclear. One possibility is that since single-cell organisms often have life cycles that incorporate multiple distinct phenotypic states, the evolution of multicellularity could require translating such patterns of diversification in time to diversification in space (Nedelcu and Michod). For this to occur, groups must be large enough to generate sufficient spatial heterogeneity to drive diversification. Yet as groups grow larger, they face resource limitations, in terms of either cell crowding or restricted diffusion of essential nutrients ( Libby et al. 2014 Duran-Nebreda and Solé 2015 Bozdag et al. 2021 5. Genetic conflict and group formation Another key difference between aggregative life cycles and clonal relates to genetic conflict (Strassman and Queller 2011; Márquez-Zacarías et al. 2021 Dao et al. 2000 Pentz et al. 2020 Nicotra 2019 Hirose et al. 2011 Ostrowski 2019 Foster and Ratnieks 2000 Ratnieks et al. 2006 Dao et al. 2000 While there are strong arguments against aggregative life cycles leading to complex multicellularity ( Márquez-Zacarías et al. 2021 Spribille et al. 2016 Grube and Berg 2009 de La Torre et al. 2010 Ros-Rocher et al. 2024 Figure 23. Biophysics in multicellularity. a) Mechanical signals encountered by cells include compressive, tensile and shear forces, hydrostatic and osmotic pressure, and environmental stiffness and viscoelasticity. b) Cellular contractility, adhesions and interfacial (more...) E. Biophysical basis of evolutionary construction 1. Biophysics and group formation A multicellular organism is composed of many cells that stay connected and coordinate their activities, allowing them to function as a unified whole. Understanding the biophysics of these intercellular connections is thus crucial, as these connections likely played important roles in the early stages of multicellular evolution, offering some important multicellular traits, such as group-level reproduction and heredity, “for free” before specialized biological mechanisms evolved ( Yanni et al. 2020 Pokhrel et al. 2024 Bozdag et al. 2023 Kalziqi et al. 2018 Zamani-Dahaj et al. 2023 Yanni et al. 2019 Yanni et al. 2020 Day et al. 2022 Day et al. 2022 Jacobeen et al. 2018 Jacobeen et al. 2018 Hallatschek et al. 2023 2. Biophysical principles govern the key processes of multicellularity Cells are subject to a plethora of physical forces from their environment ( Fig. 23a 3. Biophysics and cell division The multicellular state, particularly in clonal multicellularity, begins with cell division. Cell division is an inherently mechanical event, involving forces generated by the cytoskeleton, molecular motors, and cellular structures that drive physical changes in cell shape, chromosome movement, and ultimately the separation of two daughter cells ( Taubenberger et al. 2020 Dayel et al. 2011 Ratcliff and Travisano 2014 Hanschen et al. 2016 Suga et al. 2013 Sebé-Pedrós et al. 2013 Parra-Acero et al. 2020 Cell division can also drive group-level reproduction. In branched tree-like clusters of cells, such as snowflake yeast, multicellular reproduction emerges naturally from the mechanical consequences of multicellular growth, without requiring specialized genetic mechanisms. Snowflake yeast evolved in the laboratory from a single-cell baker's yeast that underwent daily selection for large size. As these multicellular clusters grow, permanent “unreformable” bonds between mother and daughter cells lead to the formation of branching multicellular structures. As clusters continue to grow, cells collide with each other, increasing the stored mechanical stress within the cluster ( Delarue et al. 2016 Day et al. 2022 Jacobeen et al. 2018 Jacobeen et al. 2018 Okasha 2005 4. Biophysics and morphogenesis This repurposing of mechanisms from unicellular ancestors extends beyond adhesion to include morphogenesis, the process of shape change. In unicellular organisms, means of shape change, such as the use of cytoskeletal dynamics, surface tension, and internal turgor pressure are crucial for motility, engulfment, and the formation of protrusions. These same mechanical principles are leveraged in multicellular contexts to drive tissue organization and form complex structures. For instance, the regulation of contractile forces and the modulation of cell shape, once critical for a cell's motility and environmental response [such as during the formation of a yeast's mating appendage, the schmoo ( Huberman and Murray 2014 Goldenbogen et al. 2016 Internal mechanical stresses represent an evolutionarily novel phenomenon that acts over length scales too long to be relevant for single cells ( Jacobeen et al. 2018 Boudaoud 2010 Jacobeen et al. 2018 Jacobeen et al. 2018 Physical forces not only dictate the organization of cells in morphogenetic processes by governing the physical shape and organization of cells, but also influence the differentiation of cell types. For instance, the stiffness of the extracellular matrix has been shown to influence stem cell fate, with softer substrates promoting neurogenic differentiation, while stiffer ones enhance osteogenic differentiation ( Engler et al. 2006 Bergert et al. 2021 5. Biophysics and group-level heredity Emergent multicellular physics also supplies a mechanism for group-level heredity. The inheritance of group-level traits presents a challenge in the evolution of multicellularity: for simple groups of cells that lack multicellular developmental control, how can offspring reliably resemble parents? After all, development is itself a multicellular adaptation. Surprisingly, when simple physical constraints (cells cannot overlap or groups have defined volumes) are combined with roughly random cell positioning, the distribution of space within groups becomes predictable using statistical physics. In snowflake yeast, the distribution of space follows a so-called maximum entropy distribution, as predicted by physical models of how sand grains pack ( Day et al. 2022 Aste and Di Matteo 2008 Srinivasan et al. 2024 Understanding the role of biophysics in the evolution of multicellularity is essential for our understanding of this pivotal transition. This emergent physics perspective on multicellular evolution suggests that many early steps in the evolution of multicellularity did not require the evolution of specialized biological mechanisms ( Westbrook et al. 2011 Prakash et al. 2021 6. Integration of biophysics and evolution Currently, our knowledge on this topic is limited by a lack of integration of biophysics and evolution, especially regarding multicellular and large-scale tissue evolution. While biochemical and genetic approaches have significantly advanced our knowledge of molecular evolution, incorporating mechanical principles provides another perspective and a more complete understanding of how cells coordinate and organize into complex entities. Going forward, biophysical traits that emerge for free may serve as a null model of multicellularity, from which organisms diverge as they evolve further multicellular complexity. Understanding how multicellular development evolves, in part, involves co-opting cell traits (e.g., adhesion or motility) that are shaped by physical constraints and genetic flexibility. Progressing on these critical problems necessitates the use of diverse and integrated approaches. For example, experimental evolution, synthetic biology, and computational modeling represent bottom-up approaches that offer a broader understanding of the interplay between physics and the evolution of multicellularity, despite potential differences between experimental conditions and natural processes. Conversely, while studying the multicellular fossil record and extant multicellular organisms may bias our understanding of the earliest steps of the evolution of multicellularity, these approaches provide valuable insights and, importantly, are grounded in reality. Thus, understanding the role physics plays in the evolution of multicellularity requires both bottom-up and top-down approaches, each complementing the other to provide a comprehensive view of how multicellular organisms evolved. F. Early evolution of multicellular eukaryotes and possible escape from virus infection The evolution of single-cell eukaryotes very likely occurred through symbiogenesis, specifically as an ancient archaeon gained a bacterial cell as a component through the process of endosymbiosis. Subsequently, the evolution of multicellularity in eukaryotes profoundly changed how these cellular organisms interacted with their environments, suggesting some plausible immediate benefits. Multicellular organisms are necessarily larger than unicellular life, which should provide expected advantages ( Bozdag et al. 2023 Tong et al. 2022 Fisher et al. 2020 1. Viral infection as a ubiquitous environmental pressure affecting evolution of multicellularity Based on the overwhelming evidence that viruses and virus-like selfish genetic elements are associated with all current-day cellular life forms and are the most abundant biological entities on Earth ( Koonin and Dolja 2013 Krupovic et al. 2020 Duplodnaviria Varidnaviria, Krupovic et al. 2020 Was it possible that the early evolution of multicellular eukaryotes was somehow advantaged by the ability to escape or mitigate the mortality caused by infectious parasites? This intriguing question has not received abundant attention (e.g., Koonin 2016 2. Cellular specialization can protect against generalized virus infection In the evolution of early multicellular eukaryotes, cellular differentiation for the purposes of division of labor and specialization of cells could have incidentally produced a more complex and challenging landscape for invading parasites such as viruses. In general, viruses tend to be specific to infecting only certain cells/tissues within multicellular hosts ( Elena et al. 2009 Medzhitov and Janeway 2000 When considering the differentiation of cells into specialized types for reproduction, the separation between somatic and germline cells can provide a barrier to infection by restricting viruses from accessing reproductive cells precious for moving genetic information across generations. Germline cells can be protected from viral infection via physical barriers and/or molecular mechanisms, such as epigenetic modifications and immune responses that prevent the propagation of viruses and transposons within germline cells ( Kohlrausch et al. 2022 These observations in modern multicellular eukaryotes and their interactions with infecting viruses suggest that the evolutionary advent of specialized cells and tissues in early multicellular eukaryotes may have coincided with a tendency for only a subset of cells to become infected, preventing the entire organism from being compromised. An intriguing avenue for future research would be studies that manipulate the extent of cell surface variability and intracellular defenses among cells of multicellular eukaryotes, to test various hypotheses concerning the inherent advantages of cell differentiation for precluding virus spread. 3. Benefits of extra- and intracellular defenses in evolution of multicellular eukaryotes An evolutionary advantage provided by multicellularity could be the development of physical barriers to infection, which coincide with changes in cells; e.g., multicellular organisms have cellular junctions and extracellular matrices whose structural nature can limit both viral entry and spread. The tight junctions between epithelial cells in animals form a physical barrier that prevents pathogens from infiltrating tissues. Moreover, different cell types expose different external envelope structures, which viruses must penetrate to initiate the infection cycle ( Poranen et al. 2002 As viruses experience selection to specialize on different types of unicellular host cells and on differing cell types in multicellular hosts, we can expect that the required intimacy of virus-cell interactions should present automatic protections for specialized cells within multicellular organisms. However, assuming that all types of differentiated cells are crucial for organismal fitness and some degree of cell vulnerability is detrimental to the overall fitness of the organism, it still becomes essential for evolving cellular life to possess robust intracellular defenses as well, which is consistent with the widespread evolution of immunity in biological systems. Prokaryotes possess myriad cellular defenses to thwart infection by viruses and other mobile genetic elements, where a single population of bacteria may present an impressive variety of mechanisms that reduce probabilities for infection ( Bernheim and Sorek 2020 Morehouse et al. 2020 Wein and Sorek 2022 Ishikawa and Barber 2008 Burdette et al. 2011 Morehouse et al. 2020 Nevertheless, evolution of a more complex immune system over time seems to be an important step in the development of multicellular-host resistance to parasitic infections and it appears that as multicellularity evolved, so did immune strategies. Perhaps one of the earliest immune systems to evolve in multicellular organisms was an innate immune system that differs from known features of prokaryotic innate immunity, to provide general protection of multicellular organisms against a wide range of pathogens. For example, pattern recognition receptors, such as Toll-like receptors, detect common viral components ( Takeuchi and Akira 2010 Medzhitov and Janeway 2000 Waldron et al. 2018 FIG 24 Brown algae development follows a molecular hourglass pattern. Multicellular species of animals and plants differ in form but look similar when their body plan is established, described as an hourglass-like pattern of development. This feature has independently (more...) 4. Summary The early evolution of multicellular eukaryotes likely provided several advantages that helped these organisms escape or mitigate infections from viruses and other parasites. The development of cellular specialization, the evolution of immune systems, and physical barriers to infection afforded by divergence in cell structure may have been contributors to avoidance of parasitic infection in early multicellular eukaryotes. The transition to multicellularity may have allowed eukaryotes to evolve new strategies for defending against pathogens commonly found in their environments, providing the foundation for the development of more complex immune systems in higher organisms that are prevalent today. This dynamic process of coevolution between viruses and their hosts during the history of life of Earth remains fertile territory for empirical and theoretical studies, suggesting that a crucial next step in elucidating the evolution of early microbial life would be expanded effort to understand the consequences of virus infection for evolution of eukaryotic multicellularity. G. New model systems 1. Brown algae model system While multicellularity arose independently multiple times during eukaryotic evolution, complex multicellularity emerged only a few times. The mechanisms and evolutionary forces underlying this key transition remain unclear. Brown algae (Phaeophyceae) have emerged recently as a key model system for investigating the emergence and evolution of complex multicellular development due to their phylogenetic position, range of developmental complexity, ecological importance, and the existence of model organisms, Ectocarpus in Batista et al. 2024 Coelho 2024 Coelho and Cock 2020 Brown algae are a diverse group of predominantly marine organisms, classified within the Stramenopiles, which also include diatoms and oomycetes ( Bringloe et al. 2020 Batista et al. 2024 Arun et al. 2019 Arun et al. 2019 Godfroy et al. 2023 a. Brown algae cellular differentiation and complex morphology Many brown algae have complex multicellular body plans that arise from a single zygote or spore. This involves a series of coordinated cell divisions that ultimately lead to differentiation of dozens of cell types in the adult individual ( Lotharukpong et al. 2024 Liesner et al. 2025 Badis et al. 2021 Coelho 2024 Coelho et al. 2011 Luthringer et al. 2024 Fig. 24 The transition to multicellularity requires not only the coordination of cellular differentiation but also the organization of cells into a cohesive structure. In brown algae, this process is facilitated by organized cell-division patterns that ensure proper tissue formation. Brown algae exhibit a variety of unique architectures, such as pseudoparenchymatous and parenchymatous tissues, that are key to their structural complexity ( Batista et al. 2024 b. Identifying mechanisms of growth, development, and differentiation Brown algae provide an opportunity to expand the study of comparative multicellular genetic regulation. The new availability of brown algae genomes and transcriptomes ( Denoeud et al. 2024 Badis et al. 2021 Farnham et al. 2013 Batista et al. 2024 Denoeud et al. 2024 Finally, one of the most interesting features of brown algae is their complex life cycle, which includes alternation of generations between multicellular haploid and diploid stages with very different sizes of the haploid stage between different lineages ( Bourdareau et al. 2021 2. Model systems for study of multicellularity a. Choanoeca flexa: In recent years, Choanoeca flexa Brunet et al. 2019 C. flexa C. flexa Ros-Rocher et al. 2024 The structural dynamics of C. flexa Brunet et al. 2019 Fung et al. 2023 C. flexa Brunet et al. 2019 C. flexa Reyes-Rivera et al. 2022 A key advantage of C. flexa C. flexa's Ros-Rocher et al. 2024 Unlike many other unicellular relatives of animals that are difficult to isolate from nature, C. flexa Brunet and King 2017 C. flexa Brunet et al. 2019 Ros-Rocher et al. 2024 C. flexa Brunet and King 2017 Ros-Rocher et al. 2024 b. Multicellularity long-term evolution experiment Progress in understanding the evolution of multicellularity has been hindered by the ancient nature of multicellular lineages and the extinction of virtually all transitional forms. This challenge is compounded by the “problem of looking backward in time,” that is, the difficulty in discerning which elements of modern multicellular organisms were relevant to their origins. The Multicellularity Long Term Evolution Experiment (MuLTEE) was initiated in early 2018 to address this fundamental challenge and connect the microevolutionary dynamics of this major transition to the macroevolutionary history of multicellular life on Earth. The MuLTEE offers an unprecedented opportunity to watch one of life's major transitions unfold in real time, allowing direct observation of how single cells evolve into increasingly complex multicellular organisms. The transition to multicellularity can be conceptualized through three key stages: (i) the formation of multicellular groups through cellular adhesion or aggregation, (ii) the emergence of these groups as Darwinian individuals capable of evolving multicellular adaptations, and (iii) the transformation of these groups into functionally integrated organisms with specialized cell types. While this conceptual framework provides a road map for understanding the transition, evolutionary biologists have debated which stages represent the most significant barriers to multicellular evolution. It is important to acknowledge that the MuLTEE is not a universal model for multicellular evolution; such a system cannot exist given the dozens of independent origins of multicellularity across the tree of life, evolving from different unicellular ancestors with different unique properties under different ecological conditions. Rather, it serves as a powerful experimental system illustrating how a lineage can make the transition to multicellularity, and we believe that some of these insights hold broad significance. The experiment uses snowflake yeast, a strain of the unicellular yeast Saccharomyces cerevisiae with a single mutation (Δace2) that causes daughter cells to remain attached after division, forming fractal-like branching clusters. This simple starting point, just one mutation away from unicellularity, provides an ideal system for studying how multicellular complexity emerges from the simplest possible beginning. The experimental design employs daily selection for rapid settling through liquid media ( Fig. 25a c. Major discoveries: illuminating the three stages of multicellular evolution The MuLTEE has yielded transformative insights into all three stages of the transition to multicellularity, revealing unexpected solutions to seemingly intractable evolutionary challenges. Stage 1: formation of multicellular groups. Ratcliff et al. 2015 FIG 25 Multicellularity Long Term Evolution Experiment. (a) Selective regime of the MuLTEE. Each day we select for larger size. (b) Snowflake yeast have an emergent multicellular life cycle, growing until strain resulting from growth breaks a cell-cell bond, (more...) Stage 2: rise of groups as Darwinian individuals. The MuLTEE has revealed that physics plays a crucial and unexpected role in providing scaffolding for the origin of multicellular Darwinian individuality. Snowflake yeast possess an emergent life cycle that arises not through genetic adaptation but through inevitable physical consequences of their growth pattern. As they grow, mechanical strain from cellular packing eventually ruptures cell-cell bonds, producing multicellular propagules that maintain the snowflake growth form (Ratcliff 2015; Jacobeen 2018) ( Fig. 25b and c Even more surprisingly, these groups display high heritability of novel multicellular traits through purely physical mechanisms. The arrangement of cells within clusters follows predictable patterns based on maximum entropy considerations, ensuring that offspring clusters reliably inherit the structural properties of their parents ( Day et al. 2022 Zamani-Dahaj et al. 2023 Stage 3: group transformation into functionally integrated organisms. Fig. 25d Fig. 25e Bozdag et al. 2023 Bozdag et al. 2023 Montrose et al. 2024 The MuLTEE has revealed another remarkable innovation: snowflake yeast overcome size-related diffusion constraints through the generation of self-induced hydrodynamic flows ( Narayanasamy et al. 2024 Fig. 25g Perhaps most remarkably, preliminary data from an ongoing single-cell RNA sequencing have revealed the evolution of nascent cellular differentiation, a key feature of complex multicellularity. After ~5,000 generations, three distinct transcriptional states emerged from an initially homogeneous ancestor: cells focused on growth, cells specializing in cell wall biosynthesis, and cells undergoing programmed cell death. This might represent a spontaneous evolution of a primitive form of division of labor, where different cells within the cluster perform different functions to benefit the whole. Beyond illustrating how groups transition into organisms, the MuLTEE has yielded several other insights into the evolution of multicellularity. The experiment has revealed complex relationships between environmental conditions and multicellular evolution. Large size readily evolved in both anaerobic and high-oxygen conditions, but not under intermediate oxygen levels, challenging simplified views of oxygen's role in multicellular evolution ( Bozdag et al. 2021 Pineau et al. 2024 The MuLTEE has provided the first direct experimental evidence for the role of whole genome duplication in multicellular evolution, with tetraploidy spontaneously arising and being maintained for thousands of generations due to selection for increased size ( Tong et al. 2025 Future outlook. H. Hypothesis testing with evolutionary synthetic biology Synthetic biology aims at applying engineering principles to re-create or modify biological systems. Its aims range from purely fundamental understanding (Feynman's “What I cannot create, I do not understand”) Zarkesh et al. 2022 In the past few years, diverse developmental mechanisms (patterning modules) have been engineered from scratch in cell cultures and micro-organisms (reviewed in Martínez-Araet al. 2022). Yet the dream of building a functioning organ, let alone a full organism, from scratch remains elusive. This section will be inspired by a thought experiment: what would it take to deeply understand develepmental processes using established model systems (or an adult multicellular organism) from scratch, starting from a unicellular microbe? What molecular pathways would need to be invented, modified, recycled, and rewired, and how? We will discuss in turn (i) engineering of biochemical signaling pathways, (ii) engineering of cellular mechanical properties, and (iii) insights from comparative studies that “pinpoint” mechanisms of real-life transitions to multicellularity, either by comparing multicellular groups to unicellular relatives or by experimentally evolving multicellularity in the laboratory. 1. Engineering signaling pathways a. Gene regulatory networks for patterning and differentiation Ever since the discovery of the principles of bacterial gene regulation ( Jacob and Monod 1961 Britten and Davidson 1969 King and Wilson 1975 Carroll 2008 Hoekstra and Coyne 2007 Synthetic genetic networks have played a key role in experimental manipulations restricting gene expression to cell types or stages of interest, for example, the UAS-Gal4 system ( Duffy 2002 Elowitz and Leibler 2000 Zhu et al. 2022 As the fundamental design principles of genetic circuits for differentiation are better understood and as more model organisms are becoming genetically tractable, engineering multicellular differentiation into novel (or evolutionarily relevant) models becomes an envisionable prospect. b. Cell-cell communication Gene regulation in multicellular organisms does not occur in a vacuum, but is under the control of intercellular cues that ensure proper spatial and temporal patterning. Thus, engineering cell-cell communication is key to re-creating embryogenesis. Cell-cell communication long predated multicellularity and is, for example, widespread in bacteria that can sense population density via quorum sensing. Indeed, manipulating quorum sensing has allowed engineering of new, custom density-dependent responses in bacteria, such as biofilm formation or dispersal ( Hong et al. 2012 Embryonic cell fate relies on both paracrine (long-range, diffusion-dependent) and juxtacrine (short-range, contact-dependent) signaling (sometimes referred to as shouts and whispers, respectively). The importance of paracrine signaling in development was famously predicted on theoretical grounds by Wolpert's so-called French flag model, which envisions patterning of several distinct stripes of cells by a single morphogen diffusing from a point source. Although multiple such morphogens have been discovered by developmental biologists (BMP, Wnt, Hedgehog, etc., reviewed in Barresi and Gilbert 2023 Stapornwongkul et al. 2020 Toda et al. 2020 Greber and Fussenegger 2010 Sekine et al. 2018 Matsuda et al. 2015 Morsut et al. 2016 2. Engineering biophysical building blocks a. Engineering cellular mechanical properties and mechanical activity Morphogenesis is ultimately a mechanical process, as cells move and exert forces on each other during development. Over the past few years, several engineered systems have allowed researchers to gain experimental control over the mechanical properties of cells. For example, synthetic membrane-cytoskeleton attachment proteins have been used to fine-tune the mechanical properties of the cell surface ( Lembo et al. 2023 Valon et al. 2017 Xu et al. 2014 Izquierdo et al. 2018 Guglielmi et al. 2015 b. Engineering differential adhesion Cell sorting in embryos is largely achieved by expression of differential adhesion molecules, as initially hypothesized by Holtfreter ( Townes and Holtfreter 1955 Halbleib and Nelson 2006 in vitro Cachat et al. 2016 Toda et al. 2018 3. Feedback between mechanics and signaling Mechanotransduction is key in development ( Bailles et al. 2019 Wozniak and Chen 2009 Caldarelli et al. 2024 Sloas et al. 2023 4. Forward and reverse engineering of the unicellular-to-multicellular transition: experimental evolution and comparative functional genetics What was the pre-multicellularity molecular toolkit? King et al. 2008 Suga et al. 2013 Grau-Bové et al. 2017 Abedin and King 2008 Sebé-Pedrós et al. 2010 Linden and King 2021 Levin et al. 2014 brachyury de Mendoza et al. 2013 Impressively, functional substitution experiments have shown that these homologs from unicellular relatives can sometimes partly or totally rescue loss-of-function mutations for these genes if animals. For example, the brachyury ortholog in the amoeba Capsaspora Sebé-Pedrós et al. 2013 Gao et al. 2024 What function do these “multicellularity genes” perform in unicellular relatives? Although some of these relatives can engage in facultative multicellularity, “animal-like genes” are also found in some strictly unicellular ones, raising the question of their function in this context. For example, cadherins in unicellular choanoflagellates have been proposed, based on immunofluorescence localization, to function in prey capture ( Abedin and King 2008 Capsaspora Parra-Acero et al. 2020 Brunet and King 2017 Young et al. 2011 Brunet and King 2017 TABLE 1. Cell cycle and ECM genes from diverse eukaryotes regulate the unicellularity/multicellularity switch. In these studies, the most remarkable feature of the pre-metazoan “multicellularity toolkit” is perhaps its unremarkableness: the genes involved (transcription factors, adhesion proteins, extracellular matrix, etc.) belong to functional categories that are broadly distributed among eukaryotes ( de Mendoza et al. 2013 Linden and King 2021 Stoy et al. 2024 5. Re-running the tape of life: recreating the origin of multicellularity by experimental evolution Testing extrinsic factors in the origin of multicellularity: selective pressures. Lynch et al. 2014 Cornwallis et al. 2023 The simplest selection pressure one might envision is selection for size, as multicellular colonies must (all other things being equal) be larger than their component single cells. Although not necessarily ecologically relevant, this can provide a robust and efficient pipeline for experimental evolution. Indeed, selection for settlement speed (used as a proxy for size) allows robust evolution of multicellularity in budding yeast (snowflake yeast) in the laboratory within a few months ( Ratcliff et al. 2012 Dudin et al. 2022 E. coli Chavhan et al. 2023 Selection pressures more similar to potential natural conditions include size-selective predators, which might favor the evolution of large clusters which cannot be easily captured. Indeed, predation has been shown to select for multicellular forms in the (normally) unicellular green algae Chlorella Boraas et al. 1998 Chlamydomonas Herron et al. 2019 Finally, other extrinsic factors may be used to mirror important geological events. For example, ocean oxygenation has classically been hypothesized to have facilitated the evolution of multicellularity in the early Cambrian (but see Cole et al. 2020 Bozdag et al. 2023 Wong et al. 2025 Simpson 2021 Halling et al. 2024 6. Combining genetic manipulation and experimental evolution Perhaps one of the most interesting new opportunities in the study of multicellular evolution is the use of synthetic biology to kickstart directed evolution experiments and understand particular events in the history of real lineages. Laboratory evolution experiments have one fundamental limitation: time. In the time available for study, while interesting regulatory changes and functional alterations to extant genes are accessible, larger events such as the origins of entirely new genes or biochemical functions are largely out of reach. Genetic manipulation allows researchers to circumvent this, for example, the aforementioned work transferring oxygen-binding proteins that originated at the base of the bilateria into the genomes of snowflake yeast, which altered their relationship to aerobic respiration. Researchers are just beginning to explore what can be accomplished by providing model systems of these major molecular adaptations that occurred over the evolution of multicellularity and allowing them to evolve in the context of these new capabilities to see how their evolutionary trajectories are altered. 7. Testing intrinsic factors in the origin of multicellularity: genetic changes How does a single cell become many? The mechanisms that maintain multicellularity in modern-day animals and plants are complex and partly redundant, making it difficult to pinpoint what specific genetic change first initiated the switch to multicellularity. Thus, study of facultatively multicellular organisms (in which single mutations can suffice to cause a complete loss of multicellular phenotypes) and of experimentally evolved multicellularity (where individual causal mutations can be pinpointed) is crucial to understand the principles of these very early steps. In the past years, studies of yeast, of choanoflagellates, and of multicellular algae have identified a few such “multicellularity genes.” Interestingly, these tend to regulate either one of two main biological processes: extracellular matrix secretion (potentially providing “stickiness” between cells) and abscission, i.e., cell separation after division [incomplete abscission is a common mechanism keeping cells connected ( Chaigne and Brunet 2022 Table 1 8. Conclusions and perspectives Is the re-engineering the evolution of multicellularity in key taxa such as animals, fungi, algae, and plants a distant dream? On the one hand, much (and perhaps even most) of the necessary toolkit now exists. We can engineer cell fate, cell-cell signaling, and cell shape at the scale of single cells and of entire tissues. On the other hand, these approaches have largely been restricted to animal embryos (already multicellular), animal cell cultures (extracted from a multicellular organism and studied in very artificial in vitro Booth et al. 2018 Booth and King 2020 Parra-Acero et al. 2018 Suga and Ruiz-Trillo 2013 VI. EVOLUTIONARY THEORY AS A GUIDE TO UNDERSTANDING THE PLAUSIBILITY OF EARLY-LIFE SCENARIOS This colloquium series focused on three of the primary evolutionary events involving life on Earth: the origins of life from simpler physical/chemical beginnings, the emergence of the eukaryotic lineage, and the evolution of multicellularity. Much of the focus has been on identifying plausible physical/chemical settings for the major transitions, often (but not always) acknowledging that significant uncertainties exist on the ranking of alternatives. Guided by our understanding of how modern biochemistry and cell biology works, such an approach has helped inform our understanding of the peculiar features of biology, even though most of the hypotheses must be wrong. However, in these endeavors, remarkably little attention is given to the plausibility of arguments from the standpoint of known evolutionary mechanisms and processes. Instead, we are confronted with an odd situation in which the primary subject material (evolutionary transitions) is largely uninformed by the principles of evolutionary biology itself. We worry about where the metals and cofactors that enzymes rely on came from, how the mechanisms of cellular bioenergetics came to be, and how life came to depend on lipids, a genetic code, RNAs with catalytic functions, and in one case embraced an endosymbiont (now the mitochondrion) that might have initially been a parasite. Without direct observations from billions of years ago, the claims being made can sometimes seem fantastical. Other times, they are made more plausible by experimentation on supposed end points. However, almost always, evolutionary scenarios fail to take account of known fundamental evolutionary principles. Part of the problem here is that most evolutionary biologists, theoreticians in particular, are primarily engaged in trying to understand the features of modern lineages, with most attention being devoted to animals and land plants and timescales ranging from just a few hundreds to thousands of generations. Yet, guided by experimental validation, evolutionary theory has achieved a status every bit as rigorous as other quantitative fields in the life sciences, such as biophysics. We know with certainty that evolutionary processes follow certain rules, dictated by the features of mutation, recombination, and random genetic drift, and we also know how to formulate expressions in ways that illuminate the probabilities of alternative outcomes, rates of transitions, etc. ( Wright 1969 Kimura 1983 Charlesworth and Charlesworth 2010 Walsh and Lynch 2018 What follows is an incomplete listing of ways in which the integration of evolutionary thinking might help advance our understanding of the three major foci noted above. The first goal is to endow nonevolutionary biologists with an understanding of why the rules of evolution are just as pertinent to early-life hypotheses as the principles of chemistry and physics. The second goal is to inspire evolutionary biologists to appreciate that early-life (so-called macroevolutionary) transitions provide just as fertile (and in some cases simpler) grounds for study from a theoretical standpoint as do issues on microevolutionary timescales. Primary emphasis will be placed on some of the key mysteries regarding the origin of life, as this is where novel applications of theory are most badly needed, with the closing sections providing brief overviews of theory being developed in evolutionary cell biology that are relevant to issues central to the molecular and cellular diversification of post-LUCA life. The key point is that the basic principles of population genetics that we apply to today's organisms are general features of all self-replicating forms of life. Advocacy for the integration of population-genetic thinking into origin-of-life research is not meant to imply that solutions will be forthcoming on the precise setting for life's emergence, any more than geochemical study can lead to such pinpointing. However, like the latter, evolutionary theory not only can contribute to our understanding of the plausibility of alternative early-life scenarios, but in doing so may enhance our general understanding of modern-day biology and its applications. For example, evolutionary theory can help clarify the rapidity at which alternative paths to the establishment of life might occur, potentially informing agendas for synthetic-biology studies. A. Origin of life As outlined in previous sections, a major and perhaps insurmountable challenge in understanding the origin of life on Earth is the absence of a historical record of the early events. Given the lack of such information, all we can do is concoct what seem like reasonable scenarios based on estimates of early-Earth chemical, geological, and physicals settings. What is less acceptable is the lack of attention given to the basic evolutionary processes that lead to the success or failure of alternative origin pathways. Just as chemistry and physics follow certain rules, so does evolution. 1. Avoidance of mutational meltdown Among today's life forms, genomes contain hundreds to thousands of protein-coding and noncoding-RNA genes, the products of which carry out diverse tasks, including nutrient acquisition, environmental sensing, metabolism, cell division, and pathogen avoidance. To maintain such genomic repertoires, mutation rates must be low. Anything much above one deleterious mutation per genome per generation generally puts a lineage on the road to extinction via “mutational meltdown” or “error catastrophe,” as evolutionary stability requires the removal of such mutations at a rate equal to the rate of input. With more than one deleterious mutation per genome per generation, this would require lethality of every newborn and this thus represents a hard limit on the maintenance of genetic information ( Lynch and Gabriel 1990 Lynch et al. 1993 −8 Lynch et al. 2023 In the earliest stages of life, error rates were likely much higher, on the order of 10 −2 −1 Johnston et al. 2001 Attwater et al. 2013 Zhang et al. 2013 Wachowius and Holliger 2019 Eigen 1971 Eigen and Schuster 1977 Bull and Wilke 2008 Chen and Shakhnovich 2009 Although attempts to identify critical genome sizes are informative, they do not address the key issues. How could early genomes establish and expand in the presumed absence of genome-repair processes? Were the earliest molecules of life constructed in ways that could function at reasonable levels in the face of unusually high numbers of errors? Did successful parental molecules template enough progeny molecules to ensure the rare stochastic production of some error-free offspring to carry on the line? How could improved replication fidelity, essential to genome-size expansion, evolve if this in turn required an increase in genome size to harbor the agents of accuracy? Theory from population genetics provides the tools for addressing these problems. Natural selection operates on the mutation rate in an indirect manner, via selection against the damage inflicted on nucleotide sites linked to the locus responsible for fidelity ( Kimura 1967 Lynch 2008 Lynch 2011 Shah et al. 2019 Despite the well-known problem of error catastrophe for the origin of life, remarkably little attention has been given to the joint evolution of the mutation rate and other functional genotypic features. All of the relevant theoretical machinery to do so has been developed for studies on mutation-rate evolution in contemporary populations ( Lynch 2011 2. Evolution of individuality A key to adaptive evolution is the presence of discrete individuals. Products that influence fitness derive from genomes consisting of polymers of nucleotides, which might have been RNA in the earliest stages of life. However, if those products are dissociated from the genomes that generated them, any benefits will be distributed to other members of the population to a degree that depends on product diffusion, as just noted for the case of replication fidelity. This can substantially reduce the efficiency of natural selection as suboptimal genotypes reap the benefits of products constructed by others, while also encouraging the emergence of cheaters that harvest public goods without any production at all. Owing to the extraordinarily high mutation rate in the RNA world and the regular production of shortened molecules by premature termination of replication, parasitic molecules were almost certainly a major factor from the very onset of life. To minimize the consequences of such perpetual threats, some sort of mechanism ensuring a strong link between genomes and their products must have been an essential first step in the establishment of efficient processes for adaptive evolution ( Maynard Smith and Szathmáry 1995 Michod 1998 Takeuchi and Hogeweg 2012 Shah et al. 2019 In these earliest stages, the materials for lipids (assuming these to be the building blocks of primordial membranes) must have been supplied by external environmental factors or non-RNA-driven proto-metabolism, and there are numerous settings in which such availability might have been naturally ensured, even in association with nucleotides ( Budin and Szostak 2011 Black et al. 2013 Deamer and Georgiou 2015 Again, whereas substantial work has already been done on the relative advantages of completely encapsulated individuals, as evolution is generally a gradual process, a desirable approach would be to model the transitions to individuality with the conventional machinery of population genetics. This might be approached in several ways. For example, for the earliest stage in which environmental lipids (or another means of individualization) were relied upon, one could simply model a genome's degree of isolation as a continuous trait, from completely open to completely closed. Associated with such a composite trait would be (i) an increase in fitness resulting from the privileged use of self-produced (or imported) metabolites, (ii) reduced susceptibility to molecular parasites, as lineages with excess parasites are selected against, and (iii) a protective setting for the development of expanded genomes endowed with novel functions, perhaps operating in a coevolutionary loop with replication fidelity (as noted above). Given these joint benefits, unless there were substantial associated costs, e.g., reduced access to an external pool of ribonucleotides, this early stage of individualization may have been very rapid, conditional on the availability of the molecular constituents of primordial membranes. 3. Evolution of genes and chromosomes Individuality and the degree to which it enables further expansion of functional genomes raise the issue of the evolution of chromosomes containing multiple linked genes and indeed the matter of the emergence of genes themselves. Life may have initiated in a world in which different unencapsulated RNA molecules carried out different functions, possibly in collaboration with each other, or in a setting in which two or more ancestral RNAs were contained within the same envelopes. The latter condition eventually became the status quo, but given that an organized equal partitioning of each molecule to daughter cells (mitosis) did not arise until a billion or more years later, there would have been a selective incentive for linkage of the previously separate genomic components into a single multifunctional chromosome to avoid stochastic gene loss. In principle, extended chromosomes may have had the capacity to yield functional components with individual specialized tasks in the RNA world, using a ribozyme capable of fragmenting the parental chromosome. Such an idea is made plausible by the existence of introns that self-splice at the RNA level in many bacterial and eukaryotic organelle genomes ( Zimmerly and Semper 2015 Forterre 2005 Wolf et al. 2018 Transitions to chromosomes containing linked genes could be readily modeled using a standard population-genetics framework, with or without exchange between alternative cell types (i.e., asexuality or primitive sexuality/HGT). Assuming that primordial cells divided by random fission, a primary cost of nonlinked genes would be random segregation of single-gene fragments and the resultant aneuploidy in subsets of progeny cells. Such problems might be mitigated by fusion of cells with different patterns of genic imbalance, although fusion can also create less favorable complementation groups and, more significantly, opens up opportunities for the spread of molecular parasites (Vig-Milkovics et al. 2018). Linked genomes avoid the problem of assortment imbalance, although there would presumably be some physical costs of processing functional strands into their constituent parts. The largest challenge associated with multigenic chromosomes is the impact on the evolutionary process, most notably the side effects of strong linkage on the efficiency of selection, particularly in a high-mutation-rate world. Because most mutations are deleterious, the linkage of beneficial mutations to contaminated backgrounds substantially compromises the ability of natural selection to promote individual mutations on the basis of their unique features ( Charlesworth 2013 Devi et al. 2023 Lynch and Menor 2025 Gabriel et al. 1993 Matuszewski et al. 2017 How does the accrual of novel genes proceed? Genome-size expansion can occur exogenously via horizontal transfer from cells of unrelated lineages or endogenously via within-lineage gene duplication ( Lynch 2007 Although the latter two conditions are generally in conflict with the interests of the host genome, they are nonetheless extraordinarily common in today's organisms with DNA genomes, and as noted above, parasitic RNAs were likely present from the very outset of life. Indeed, there are compelling arguments that viruses played a key role in the molding of the basic genomic features of the LUCA and even in the ancestors of the bacterial and archaeal/eukaryotic domains ( Forterre 2002 Gadelle et al. 2003 Wolf et al. 2018 Charlesworth and Charlesworth 1983 Charlesworth and Langley 1986 Langley et al. 1988 Although the genomes of today's organisms are the culminations of more than 3 billion years of genome expansion, and the de novo Lynch et al. 2001 Lynch and Marinov 2015 7 −6 Together, this indicates that at the earliest stages of life's emergence, both mutation rates and selection strength on mutations (both positive and negative) were exceptionally large. Although population-genetic theory provides all of the tools necessary for such analysis, the field has historically focused on parameter regimes in which both mutation rates and selection coefficients are quite small as in modern biology, so there is considerable need for extension of the theory to evaluate early-life issues. 4. Emergence of protein-coding genes and translation Following a starting point of just RNA, potentially supplemented with noncoded cofactors or peptides, the next major transitions en route to the LUCA were the emergence of functional coded protein and the switch to a DNA-based genome. Given that most of the central players in the translation system of today's cells consist of RNAs (e.g., transfer and ribosomal RNAs), the most likely scenario seems to be an initial transition to an RNA-protein world prior to the emergence of DNA-based genomes. If this view is correct, the use of key functional RNAs across the tree of life is a dramatic example of a historical artifact, so deeply entrenched that there is essentially no possibility of elimination. To be sure, in the earliest days of life's establishment, with ribonucleotides apparently the only coded building blocks that could be used to construct molecules capable of catalysis and replication; functional RNAs would have undergone adaptive evolution. However, catalytic speeds and efficiencies of solitary ribozymes are orders of magnitude below that of proteins, and eventually many key functions were supplemented with hundreds to thousands of accessory proteins, now encoded in DNA. In this view, the genetic code emerged at least in part in the RNA world, prior to the establishment of the ribosome and a DNA-based genome. In today's cells, the code is read off of triplet codons contained in intermediate messenger RNAs, with anticodons in transfer RNAs serving as the interpreters for delivery of appropriate amino acids. However, today's sophisticated system could hardly have been the starting point. One possibility is that the primary function of primordial tRNAs was to deliver specific triplets for genome replication via ligation, with the delivery of amino acids possibly serving only as a secondary activation function (Szathmary 1993; Gordon 1995 Poole et al. 1998 Agmon et al. 2021 Müller et al. 2022 Whatever the mechanism of association between tRNAs and their amino-acid cargoes, the earliest (pre-ribosome) proteins would have been laden with errors. The initial code would have been highly redundant, as the entry of all 20 amino acids into the protein world would have been incremental, and prior to the emergence of specific amino-acyl tRNA synthetases, promiscuity in substrate utilization would have been high. This lead Woese ( Woese 1965 Lynch 2024 Although much thought has been given to things like the optimality of the structure of the now near-frozen genetic code (e.g., Freeland et al. 2000 Force et al. 1999 Lynch et al. 2001 To get to a full-fledged RNA-protein world, we ultimately need an explanation for the evolution of translation. This means not just the origin of the genetic code and associated tRNAs, but the emergence of the universal machine for translating messenger RNAs, the ribosome, in particular its ribozyme components. If the ribosome emerged after life had already embraced the use of functional proteins, then we must resolve how an earlier mode for constructing proteins was converted into or displaced by an invading ribosome. If, on the other hand, the primordial ribosome preceded the utilization of proteins, then it must have had a different function prior to transforming into a necessary protein-making machine. In the absence of compelling answers here, it is again reasonable to entertain the possibility that instead of being propelled by mechanisms that were adaptive for the host cell, the protoribosome was initially a genomic parasite, dependent on the host cell for replication. To become successfully established in this manner, there would need to be a feature that ensured a stable coexistence of the protoribosome and its host cell. This could happen, for example, if colonization of the protoribosome coincided with the loss of a key host-cell function. For example, it has been suggested that the protoribosome served as an RNA polymerase or ligase stringing together codon-like triplets, which in principle may have ensured the replication of its host, albeit at a reduced rate ( Poole et al. 1998 The protoribosome could also have promoted some sort of selfish toxin-antitoxin scenario, as is common in today's bacterial plasmids (although based on proteins). Such invaders ensure their own existence by imposing a serious problem on the host cell but also delivering a solution. If the antidote dissipated more rapidly than the toxin, stable coexistence of both participants would be enforced. Moreover, even if such a protoribosome was selectively disadvantageous to the host cell, complete takeover would be possible at the population level if it horizontally transferred into other individuals at a sufficiently high rate. Given the likelihood that RNA viruses were present from the beginning of life, this kind of scenario may not be too far-fetched. Once fully established, such a protoribosome would then be free to become an integral part of the host cell and might then be gradually converted into a protein-making machine, further enhancing the fitness of its essential vehicle. The outcomes of scenarios like this could readily be pursued with models of intermolecular coevolution of populations of hosts and multiple parasites, with each imposing reciprocal pressures on the other. For example, in addition to the parasite striving to maintain a stable host environment, there would be selective pressure on the host to obtain mechanisms for neutralizing parasite toxicity. It must be emphasized, however, that the preceding idea is just one hypothesis for the enigmatic origin of the ribosome, which remains one of the greatest mysteries associated with the emergence of the predecessors of the LUCA. 5. Emergence of DNA-based genomes Today's nucleic acids operate in a two-step fashion. Cellular genomes are constructed out of DNA, which houses information but otherwise has no function, but to do anything else useful DNA must be transcribed into RNA. Today's cells minimally require proteins dedicated to two tasks: the turning on of genes (transcription factors) and the synthesis of complementary single-stranded RNAs using DNA-dependent RNA polymerases. In a minority of cases, the RNAs are the end products, serving as ribozymes with key functions (e.g., ribosomal RNAs and transfer RNAs), whereas in other cases, they serve as intermediate messages (mRNAs) that must be translated into proteins. Translation is a collaborative effort of transfer RNAs, amino-acid loading tRNA synthetases, and a large complex of RNAs and proteins called the ribosome. Thus, a key to understanding the origin of modern-day genetics is to determine (i) how DNA came to be the repository of genomic information, (ii) how several key catalytic functions encoded in that DNA were retained by the transcribed RNAs, (iii) how some transcribed RNAs became intermediate vehicles for the downstream production of proteins, and (iv) how the small remaining subset of catalytic RNAs came to depend on accessory proteins to carry out their functions. As noted above, single genes might have been strung together in chromosomes in the RNA world, but this still leaves unanswered all of the preceding questions. Additional challenges to consider here are the errors associated with transcription and translation. In modern-day organisms, such rates are 10 5 7 −5 −3 Lynch et al. 2023 In a population consisting entirely of RNA molecules, how could a DNA-based genome become incrementally established, eventually harboring the blueprint for transcription of all remaining ribozymes in all cells across the tree of life forever more? As with any other key transition associated with the origin of life, one has to start with the realization that evolution does not proceed instantly, with an entire population being converted to a new state together. All evolutionary changes initiate within single mutant individuals and to become fully established must experience progressive changes leading to fixation at the population level, generally with minimal negative changes in mean population fitness during such navigation. The conventional approach to understanding the transition to DNA-based genomes is to assume that DNA takeover was driven by some form of natural selection, with an unusual RNA molecule somehow orchestrating the establishment of a profitable DNA-RNA collaboration. DNA is substantially more chemically stable than RNA, so one possibility is promotion via the benefits of enhanced stability of genomic information. As noted above, for this to be effective, the cells involved would almost certainly need to have been isolated from other such lineages to ensure that any advantages remained linked to the anti-mutator (the DNA-based genome) and that the machinery necessary to maintain cells with joint RNA and DNA molecules remained together. It is commonly argued that because cytosine (C) deamination to uracil (U) is one of the most common forms of mutation, the usage of thymine (T) instead of U in DNA yields an advantage because a C → U change is undetectable as a mutation in RNA. However, this seems unlikely to have been the driving force in the establishment of DNA-based genomes, as this would require both a change in the sugar backbone and nucleobase side chain simultaneously to allow thymine to have a function while preserving the function of uracil in RNA. The change in backbone chemistry, however, has functional significance in stabilizing the molecule on its own. Thus, DNA genomes would have likely initially used U instead of T, with wholesale conversion of U to T requiring the later evolution of a thymidylate synthase, and detection and removal of U further requiring the evolution of uracil glycosylase. An alternative possibility is that DNA was not a benevolent invention of the RNA world but instead was introduced by a parasite driving itself through the RNA world by selfish survival and/or replication mechanisms. For example, the first U-DNA organism might have been a virus, with its unique genome conferring immunity to destruction by host cells ( Forterre 2001 Forterre 2002 Although this view may not be very appealing for those who view viruses as foreign agents unworthy of being designated as life forms relevant to cellular lineages, molecular phylogenetic analyses of key molecules involved in nucleic-acid metabolism support the idea of independent viral takeovers in different basal branches of the tree of life. For example, there are at least three classes of ribonucleotide reductases that have little sequence similarity and use different cofactors ( Stubbe 2000 Poole et al. 2002 Myllykallio et al. 2002 Leipe et al. 1999 Moreira 2000 Filée et al. 2002 Forterre 2002 Gadelle et al. 2003 These disparities are rampant enough to suggest to some that the LUCA itself was an RNA-based genome that succumbed to independent transitions to DNA in the basal lineages leading to the two major domains of life. An alternative view, that the LUCA harbored redundant mechanisms for DNA metabolism with coordinated losses of divergent pathways occurring on the branches leading to bacteria vs archaea or that repeated takeovers continued replacing machinery after the LUCA, still does not rule out the involvement of drive-like processes associated with DNA-based viral genomes as vital. To date, however, all such hypotheses are purely verbal in nature, and there is a clear need for the development of mechanistic models for evaluating the relative ease of alternative scenarios for the progressive entry into the DNA world. Placed in a population-genetics framework, models from epidemiology might be very useful here. 6. Pace of early adaptation Given that the age of the Earth is ~4.56 Ga, the recent suggestion that the last universal common ancestor dates to 4.1-4.3 Ga ago and had a genome size of ~2.5 Mb ( Moody et al. 2024 6 Lynch 2007 6 We now turn to the matter of the refinement of features of individual genes. Evolution by natural selection requires variation at the genomic level, and we can be sure that there was plenty of that given the error rates noted above. Roughly speaking, the rate of establishment of a particular kind of adaptive mutation can be viewed as the product of the number of individuals (N), the rate of origin of the mutation per generation (u), and the probability of fixation. Provided the selective advantage s exceeds the power of genetic drift, the probability of fixation is minimally equal to 2s(N e Ne N e 2suN e In modern organisms, with sexual reproduction and low mutation rates, we generally think of adaptations being progressively built from the accumulation of single mutations in a sequential manner. However, with the extraordinarily high mutation rates in the RNA world, the dynamics of molecular evolution would likely have been much different. For example, ribozymes almost always have secondary structures consisting of stems and loops, with the former held together by complementary base pairs (A:U and C:G) and the latter avoiding base pairing. Assuming mutations arising independently at nucleotide sites at rate v, extension of a stem by one base pair via a single mutation from a nonmatching state has a probability of occurrence of u v u v n u −8 v n −6 N 6 The typical strength of selection operating on beneficial mutations may also have been unusually high in the RNA world. In today's organisms, the s associated with an adaptive mutation is seldom larger than 0.01 and more often probably less than 0.0001 ( Lynch 2024 Less can be said about effective population sizes in the RNA world, as most theoretical work devoted to the subject has focused on regimes in which mutation rates and selection coefficient are relatively low. With tiny individuals consisting of just a few molecules each, N e N e As noted above, there is the additional problem of early mutation rates being so high that well-adapted ribozymes are only transiently so, owing to almost immediately deleterious-mutation contamination. This problem might be mitigated in protective situations enabling single-stranded (parental) template molecules to replicate multiple times prior to loss from the environment, thereby helping ensure the appearance of rare mutation-free progeny molecules. In principle then, noting that a local population of molecules might contain Avogadro's number or more individuals, the further combination of large u s Finally, the above arguments ignore an additional mode of evolution that may have been dramatically stoked in the earliest stages of evolution. Situations exist in which a particular double mutant is advantageous over an ancestral type, with the first-step mutant being deleterious. A simple example is the switch between A:U and G:C bonds, which (in the absence of a double mutation) must go through an intermediate (single-mutation) state with disadvantage δ. Assuming the double-mutant does not arise in a single event, the rate of evolution is then proportional to the square of the mutation rate (because first-step mutations experience recurrent selection-mutation balance and remain at low frequency u/δ, whereas second-step mutations arise on such backgrounds at rate u). Under this process, known as stochastic tunneling (reviewed in Lynch 2024 For example, if u 6 12 There is, of course, the element of time to consider. The combination of phylogenetic and geological evidence suggests that the window within which first universal common ancestor emerged was on the order of 10 8 B. Origin of eukaryotic features By the time of the establishment of modern eukaryotes, mutation rates had settled down to levels like those of modern prokaryotes, likely as low as 10 −9 Lynch et al. 2023 There is an unfortunate tendency for most biologists to assume that every feature of cells is a product of adaptive evolution and that the goal of natural selection is the production of more and more robust and complex organisms. Based on an overly exuberant view of Darwin's early ideas, such uncritical thinking is based on a false caricature of evolutionary processes. On the contrary, a substantial amount of the evolution of cell biological features in eukaryotes is likely a product of nonadaptive processes and the limited bounds on the power of natural selection. More complex genomic and cellular features require elevated levels of energetic investment on the part of the host cell and are more sensitive to mutational breakdown (reviewed in Lynch 2007 Lynch 2024 Frank 2007 Frank 2023 Lynch 2012 Despite these reservations about their adaptive nature, there is no question that nearly all aspects of eukaryotic cell biology have become more complex than those in prokaryotes. For example, many protein complexes that are homomeric (all parts encoded in a single genetic locus) in prokaryotes are heteromeric in eukaryotes (with the units derived from different loci, typically products of gene duplication), although in no case is there evidence that such derived structures operate with enhanced speed or efficiency ( Lynch 2024 Lynch 2007 Notably, the mechanisms by which novel features arise need not be related to the current ways to which they have been put to use and maintained, and indeed the emergence of novel features by nonadaptive mechanisms can often alter the raw materials via which natural selection can mold future adaptations. For example, although large numbers of introns arose in the unicellular ancestor to the LECA, alternative splicing in today's multicellular eukaryotes provides a means for tissue-specific expression of alternative gene products. Thus, it should be kept in mind that the shift in the population-genetic environment in the eukaryotic domain likely resulted in indirect changes that altered the paths down which eukaryotic diversification was able to proceed. 1. Gene duplication Novel genes sometimes fortuitously arise in a de novo manner when previously noncoding DNA is transcribed into initially nondeleterious products ( Van Oss and Carvunis 2019 Zhao et al. 2024 Force et al. 1999 Lynch and Marinov 2015 The situation is different in eukaryotes, which have reduced effective population sizes, larger cells, and much more complex gene structures relative to prokaryotes. Owing to larger cell sizes (and overall larger total energy budgets per cell), the fractional energetic cost of a gene duplication is reduced relative to the case in prokaryotes even though the absolute cost is increased ( Lynch and Marinov 2015 Force et al. 2005 Although gene duplication plays a major role in the expansion of eukaryotic genome complexity, it is also a primary contributor to the other main engine of evolution in eukaryotes: the emergence of novel lineages by rapid speciation. Phylogenetic analysis suggests that very quickly after the emergence of the LECA, there was a rapid and explosive radiation of the major eukaryotic lineages ( Philippe et al. 2000 Cavalier-Smith 2002 Koonin 2007 A powerful mechanism for the passive emergence of reproductively isolated lineages in sexually reproducing eukaryotes involves the divergent resolution of pairs of duplicate genes by reciprocal silencing in sister lineages ( Lynch and Force, 2000 Lynch 2024 2. Constructive neutral evolution Many of the molecular features of cells superficially appear overdesigned. Examples already given are the heteromeric protein complexes of eukaryotes and the dramatic expansion of eukaryotic-gene complexity. Although it is commonly assumed that all such embellishments are products of adaptive processes, the evidence for enhanced function is generally lacking ( Lynch 2024 An alternative explanation is embodied in the concept of constructive neutral evolution (CNE), which postulates that cellular features can sometimes experience adventitious growth in an effectively neutral and ratchet-like manner ( Woese 1971 Zuckerkandl 1997 Stoltzfus 1999 Gray et al. 2010 Regrettably, although CNE is an attractive model for the nonadaptive emergence of gratuitous complexity, the model is currently entirely verbal in nature. For CNE processes to emerge as envisioned, specific combinations of mutation rates, effective population sizes, and selection coefficients are likely necessary. As noted above, there is at least some bioenergetic cost of relying on large complexes, and this presumably needs to be taken into consideration to explain the rarity of such structures in prokaryotes where the efficiency of selection is high. Future work will need to be done in a population-genetics framework to more firmly establish the conditions under which CNE is most likely to take place vs conditions under which it is likely to be nearly impossible. 3. Origin of the mitochondrion A signature and striking feature of eukaryotes is the presence of the mitochondrion, which like dozens of other unique features became established on the road from the first eukaryotic common ancestor to the LECA. Open questions remain about the order of events, and some have argued that by providing a superior platform for bioenergetics, the endosymbiotic establishment of the mitochondrion was the signature event in eukaryogenesis, generating a quantum leap in ATP production and paving the way for the emergence of all other eukaryotic structures and functions, including the growth in gene number, intracellular architecture, etc. ( Lane 2006 Lane and Martin 2010 Lynch and Marinov 2015 Lynch and Marinov 2017 Chiyomaru and Takemoto, 2020 Gabaldón 2018 This leaves unresolved the deeper question as to whether the primordial mitochondrion was a benign partner as opposed to some sort of parasite. Left out in most narratives about the mitochondrial origin is that species interactions are twoway streets. As both partners in a symbiosis will evolve in directions of self-interest, the key to understanding the mitochondrial-host cell interaction is resolving how and why each species could not only reap benefits from the other but do so in ways that are superior to free living. One possibility is that this was not initially the case, with the primordial mitochondrion being a self-proliferating parasite driven through the population that extracted more from the host than it provided in return. Although the precise phylogenetic location of the mitochondrion remains murky, many analyses position it within or close to clades of modern intracellular eukaryotic parasites ( Sassera et al. 2011 Wang and Wu 2014 Muñoz-Gómez et al. 2022 As noted above, parasites can eventually become permanent fixtures in two-species interactions under fortuitous circumstances in which each member of the pair loses a key function preventing a return to independent living. Although the initiating events are not known, this certainly became the case with respect the mitochondrion, which lost almost all of its genes (many of which were transferred to the nuclear genome of the host), and with respect to the primordial host cell, which lost the capacity for ATP production and relinquished pathways for the production of iron-sulfur clusters key to life. The primary point here is that, as with many central issues in the emergence of the LUCA, an understanding of the population genetics of coevolutionary interspecies interactions is central to clarifying the conditions under which the mitochondrion might have become established, whether this was in the best interest of the host cell, and whether the resultant energy consortium was even more than the sum of its parts. As this is another area of evolutionary theory that remains undeveloped, work in this area could have manifold effects beyond the issue of eukaryogenesis. 4. Systems drift and the evolution of intracellular languages With the emergence of individuality and multiple genes with different gene products came the reliance on mechanisms for gene expression. This in turn required the evolution of a variety of molecular “languages,” the genetic code being the first such instance. All of today's organisms rely on transcription factors and their binding sites and on sender and receptor molecules in signal-transduction systems. Eukaryotes developed further crosstalk involving elaborate vesicle transport systems, regulatory gates in nuclear-pore complexes, and protein-management and sorting systems. The evolutionary analysis of such systems again requires a coevolutionary perspective, as in each case there are at least two participating molecules. Like the languages in humans and communication systems in other animals, the mechanisms of intermolecular crosstalk are subject to divergence over time, which in many cases may accrue in a passive (nonadaptive) fashion. This can happen if there are multiple degrees of freedom by which two molecules can communicate. For example, the affinity between a transcription factor and its binding site generally depends on the number of nucleotide/amino-acid matches, with the precise motifs being of secondary importance provided a critical minimal matching criterion has been reached. This opens the opportunity for molecular languages to diverge over time as long as each step matches well enough, to a degree that depends upon the complexity of the networks involved. One-on-one interactions are particularly vulnerable to such drift as, like dance partners moving across a floor, all that is required is that each partner retains an overall affinity for the other. In contrast, one vs many systems experience greater constraints, as an increased affinity to one partner may decrease the affinity to others. In addition to molecular languages changing over time, the underlying agents of communication need not be stable. For example, comparative studies in evolutionary cell biology have revealed numerous examples in which key regulatory, biosynthesis, and post-translational modification pathways have retained their same topologies in different taxa while experiencing replacements in the underlying molecular participants ( Lynch 2024 McColgan and DiFrisco 2024 Again, there is room for considerable theoretical work here. Although some attention has been given to language drift in pre-existing systems ( Lynch and Hagner 2015 C. Summary The origin of life and the emergence of eukaryotes have generally been studied from the perspective of chemical, physical, and molecular challenges, with little consideration typically given to the evolutionary plausibility of the proposed scenarios. Given that the questions at hand are fundamentally evolutionary in nature and that there are specific rules by which evolution proceeds (just as such rules exist in physics and chemistry), this represents an unfortunate disconnect in these fields of study. Although the mathematical and computational infrastructure of population genetics provides the necessary tools for the evaluation of evolutionary hypotheses, almost all work in evolutionary genetics has focused on issues relating to contemporary species on short timescales, generally from a rather nonmechanistic perspective, i.e., without reference to the detailed molecular structure of individual traits. In contrast, the kinds of problems outlined above demand a more detailed molecular perspective, with the genotype constituting the phenotype in the most extreme case of an RNA world. In addition, the absolute strengths of the key components of population-genetic environments in the first billion or so years of the Earth's history were likely dramatically different than in today's species, which demands a substantial shift in the parameters typically explored in population-genetic analyses. A renewed focus on the population genetic features of the Earth's early biota provides a rich platform for investigation from the standpoint of evolutionary theory. Such investigation will provide an essential basis for ground-truthing alternative hypotheses derived from the perspective of the physical and chemical sciences. However, it likely will also lead to developments relevant to many issues concerning modern-day species, including the evolutionary features of viruses. VII. KNOWLEDGE GAPS AND FUTURE RESEARCH IN EARLY MICROBIAL LIFE AND EVOLUTION A. Introduction The study of early microbial life sits at the intersection of fundamental questions in biology: how did the first living systems emerge from abiotic chemistry, how did the complex cellular structures that define eukaryotes evolve from simpler prokaryotic precursors, and how did multicellular life repeatedly evolve from unicellular ancestors? These transitions represent profound innovations in the history of life on Earth, each transforming the biosphere and expanding the boundaries of biological complexity. Despite significant advances in our understanding of these transitions, critical knowledge gaps persist that limit our ability to fully reconstruct the events, mechanisms, and environmental conditions that shaped life's earliest chapters. These transitions occurred from hundreds of millions to billions of years ago, leaving limited and often indirect evidence of their occurrence. The temporal distance between these events and contemporary observation necessitates innovative approaches to inference and reconstruction. Moreover, these transitions involve complex interactions between biochemistry, genetics, cellular biology, and environmental conditions that demand interdisciplinary perspectives. This section integrates the input of the colloquia participants and provides an integrated exploration of the knowledge gaps and future research directions in understanding the major transitions in early microbial evolution, from abiogenesis to eukaryogenesis and the origins of multicellularity. B. Cross-cutting methodological and conceptual challenges 1. Temporal and evidential limitations A fundamental challenge in studying early microbial evolution is the vast distance separating these events from contemporary observation. The origin of life likely occurred at least 3.5-4 billion years ago, eukaryogenesis at least 1.7 billion years ago, and complex multicellularity multiple times over the past 600 million years (and perhaps earlier in prokaryotes). The fossil record, while invaluable, captures only fragmentary evidence of these transitions, particularly for microbial life forms and early transitional forms that often lack preservable hard structures. With direct evidence of these transitional forms being limited, indirect evidence and inference must contribute to our understanding as much as the gathering of more direct evidence. Abiotic geological forces have modified or erased much of the record of early life through tectonic processes, metamorphism, and weathering. The limited number of accessible rocks from these ancient periods constrains our ability to fully reconstruct the environmental conditions and biological diversity of early Earth, though more evidence is always being uncovered. Similarly, extinction events have eliminated entire lineages that might have provided crucial insights into transitional forms and evolutionary pathways. These evidential limitations necessitate innovative approaches to inference and reconstruction. Comparative genomics, molecular clock analyses, and phylogenetic methods provide important indirect tools for reconstructing evolutionary histories, but each comes with its own limitations and biases. For instance, long-branch attraction in phylogenetic analyses can obscure true evolutionary relationships, particularly when examining fast-evolving lineages such as viruses or when comparing very distantly related taxa. Developing more sophisticated models of sequence evolution and alternative methods for phylogenetic inference represents a critical priority for addressing knowledge gaps in early microbial evolution. 2. Conceptual frameworks Progress in understanding early microbial evolution is also hindered by conceptual challenges related to how we define and measure biological phenomena. A particularly significant issue is the lack of standardized approaches for measuring and comparing complexity across diverse life forms. For instance, in studies of multicellularity, the number of cell types has commonly been used as a proxy for complexity, but this approach has limitations: it relates only indirectly to morphological complexity and there is no consensus on how cell types should be defined. Similar definitional challenges pervade other areas of early microbial evolution. What precisely constitutes “life” in the context of prebiotic chemistry? How do we delineate the boundaries between cellular compartments in the absence of modern membrane structures? How do we define “individuals” in the context of symbiotic relationships that may have characterized early eukaryotes or in multicellular aggregates with genetic heterogeneity? These conceptual questions are not merely semantic; they shape how we frame research questions, design experiments, and interpret results. Developing more robust conceptual frameworks requires integration of diverse perspectives from biology, chemistry, physics, information theory, and philosophy. 3. Interdisciplinary integration The study of early microbial evolution is fundamentally interdisciplinary, with abiogenesis spanning the divide between chemistry, biochemistry, thermodynamics, and geology and understanding eukaryogenesis requiring expertise in phylogenetics, ecology, and cell biology. Despite the recognition of this interdisciplinary nature, research in these areas often remains siloed within specific disciplines or focused on particular model systems. Given that the questions being asked are evolutionary in nature, there is a particular need to integrate all of these areas with formal evolutionary theory, which has historically been primarily focused on recent events. This siloing is reinforced by academic structures, funding mechanisms, and the technical and conceptual barriers to cross-disciplinary communication. Overcoming these interdisciplinary barriers requires both structural changes in how research is organized and conducted and methodological innovations that allow integration of diverse data. Research networks connecting scientists working on different aspects of early microbial evolution could facilitate this. Similarly, dedicated data platforms could help synthesize evidence into more comprehensive reconstructions of early evolutionary events. C. Abiotic to biotic transition: origins of life 1. Main knowledge gaps in prebiotic chemistry and self-replication Our understanding of the origin of life remains incomplete, with fundamental knowledge gaps persisting despite decades of research. The complex interplay between environmental conditions and prebiotic evolution is poorly understood, with heterogenous early-Earth environments likely playing a crucial role in shaping prebiotic chemistry, be it from wet-dry cycles polymerizing organic matter ( Mamajanov et al. 2014 Vasiliadou et al. 2019 Lane and Martin 2012 These gaps exist not only because of the difficulty in experimentally recreating early Earth's geochemical conditions but also because of the lack of direct molecular or fossil evidence capturing critical transitions before the “phylogenetic event horizon” ( Lane 2015 De Duve 2005 A particularly critical gap exists in understanding the evolution of translation and information systems. How the various RNA, DNA, and polymerase systems coevolved to form the modern central dogma remains a major unresolved question. This is due in part to the lack of comprehensive experimental frameworks for studying these systems in their primordial forms as opposed to modifying modern forms. Questions surrounding the origins of replication, fidelity, and catalytic efficiency remain unanswered, as do the relative roles of early peptides and ribozymes in these processes. Similarly, the population genetics and genetic exchange in pre-LUCA systems has hardly been explored, as modern genomic frameworks mostly focus on the replication of large, high-fidelity genomes ( Doolittle and Brown 1994 2. Productive technological approaches for origin of life research Addressing these complex knowledge gaps requires a range of advanced technologies and interdisciplinary approaches. Synthetic biology stands out as a particularly transformative tool for studying a variety of phenomena. By designing and constructing minimal synthetic compartments and cells, researchers can replicate early metabolic and replication systems in controlled environments, testing hypotheses about the abiotic-to-biotic transition. For example, synthetic vesicles with encapsulated ribozymes ( Lai et al. 2021 Phylogenetics, when combined with new breakthroughs in structural biology, offers a powerful framework for reconstructing ancient protein structures and metabolic pathways. By tracing evolutionary trajectories through conserved protein structure, which is much more strongly conserved than mere sequence ( Illergård et al. 2009 Microfluidic technologies studying prebiotic chemistry directly provide another promising avenue. These systems allow researchers to precisely control chemical and physical conditions, simulating the dynamic environments of early Earth. Microfluidic platforms can be used to study processes such as abiotic carbon fixation, membrane formation, and redox metabolism using carefully controlled gradients of temperature, pH, and salinity ( Jordan et al. 2024 Novel computational models may also be helpful in this research. By studying the intersection of biochemistry, phylogenetics, and geochemical environments, such models might help predict plausible evolutionary pathways and guide experimental efforts. For example, simulations of autocatalytic networks under different geochemical conditions could identify the most promising scenarios for experimental validation ( Xavier et al. 2020 3. Future research directions in origin of life Future research on the origin of life should focus on targeted experiments and integrative approaches to address the most critical questions. A top priority is the development of experimental platforms that replicate key aspects of early-Earth environments. Synthetic-biology tools are becoming increasingly sophisticated, enabling the construction of complex systems that mimic early-life processes, especially via high-througput platforms allowing the examination of a wide variety of sequence possibilities, but these must be focused less on closely recapitulating features of modern-day biology and more on exploring the space of possible molecules. Experiments replicating the chemistry of so-called Lost City-style hydrothermal-vent environments could explore the role of redox chemistries in fostering abiotic synthesis and energy pathways ( Vasiliadou et al. 2019 Hudson et al. 2020 Reconstructing ancestral biochemical pathways, such as the apparently ancient Wood-Ljungdahl pathway, should also be a focus. By studying extant microbes with ancient metabolic traits, researchers can infer details about early shared biochemical processes and their evolutionary contexts. Computational approaches and metabolic modeling can guide these experimental efforts, integrating data from molecular evolution and geochemistry to refine hypotheses and experimental designs. These models should account for feedback loops between evolving metabolisms and their environments, providing a holistic view of life's early stages. Synthetic ecosystems or “minimal life” constructs could bridge theoretical models with empirical data, offering a tangible pathway to test hypotheses about not only the particular forces driving life's origin on Earth, but how it could plausibly originate anywhere. However, integrating these diverse approaches remains a significant challenge. Effective collaboration between disciplines requires shared methodologies, open data frameworks, and robust communication channels. Training programs to develop interdisciplinary expertise are also essential to ensure the success of these efforts. While initial costs may be high, the long-term benefits, both scientific and societal, justify these expenditures. Funding strategies should prioritize collaborative grants, public-private partnerships, and international cooperation to maximize resource efficiency. D. Emergence of cellular complexity: eukaryogenesis and diversification 1. Knowledge gaps in eukaryogenesis Despite increasing knowledge about microbial diversity across the tree of life, including the discovery of Asgard archaea together with progress in metagenomics and phylogenomics, many long-standing questions on eukaryogenesis remain open. Our understanding is advancing, but challenges remain due to the thorny nature of this question. Eukaryotes emerged at the very least 1.7 billion years ago ( Porter and Riedman 2023 Moody et al. 2024 Given the apparent extinction of any divergent stem groups during the extensive evolutionary remodeling that occurred after the first eukaryotic common ancestor (FECA) but before the last eukaryotic common ancestor (LECA), the complex process of eukaryogenesis is poorly constrained by phylogenetics of modern organisms. This means that we must rely on models of the ecological and evolutionary forces that drove eukaryotes into existence during this period. These models have converged on the idea that eukaryotes arose through merger(s) or symbioses between archaeal and bacterial partners; hence, extensive chimerism during the origin of eukaryotes adds to the challenge of inferring early events using models meant for more vertical transmission of information. Extant studies of the eukaryotic tree of life (eToL) have relied on studies of tractable model lineages to the exclusion of a considerable portion of the vast diversity of microbial species. This approach has limited our understanding of the origins and diversification of eukaryotes, which biases notions of early eukaryotes. While much can be gained by carefully making inferences about ancestors from extant organisms, such work requires careful sampling to capture the diversity of clades. Much of the biodiversity of eukaryotes, particularly microbial eukaryotes (also known as protists), has been neglected, and we run the risk of inferring rules of biology that are not universally applicable. 2. Expanding and interpreting the eukaryotic tree of life The eukaryotic tree of life is relatively poorly understood, with prokaryotic genome databases far exceeding their eukaryotic counterparts in scale. A concerted effort is needed to apply a natural history approach to capture biodiversity, expand knowledge of unculturable microbial eukaryotes, and prioritize sequencing of underrepresented eukaryotic lineages. Most microbial species are currently uncultivable, likely due to a combination of their ideosyncratic metabolic requirements and the fact that many species may require cohabitation with other partners, e.g., bacteria, archaea, and other microbial eukaryotes. Knowledge of these underrepresented lineages of eukaryotes is crucial for understanding deep evolutionary relationships, the origins of cellular complexity, and eukaryogenesis. Sample collection and sequencing of uncultivatable lineages from the widest possible variety of environments (particularly aquifers, deep oceans, exotic chemical environments, and all extremes of aerobic to anaerobic conditions) will pave the way for progress in this direction. A subset of molecular and microscopy tools can still be deployed to study uncultivable lineages ( Avcı et al. 2022 3. Focuses for technology and data in eukaryogenesis research Several technologies have been identified for their potential high impact on future research in eukaryogenesis. Effective study of the uncultivable majority of eukaryotic and archaeal lineages depends on single-cell analysis, especially via diverse microscopy and labeling approaches. Confocal microscopy advances including confocal scanning laser microscopy or related techniques to visualize natural fluorescently labeled cells via florescent in situ hybridization and immunohistochemistry have already allowed tagging and visualization of cytoskeletal proteins in archaea and bacteria and have already helped identify primitive versions of eukaryotic structures ( Rodrigues-Oliveira et al. 2023 Avcı et al. 2022 High-resolution methods to study subcellular prokaryotic structures such as cryo-transmission electron microscopy (cryoTEM) have been and continue to be critical for progress in the field, allowing study of subcellular structures from macromolecular complexes to membrane topologies in near-native states and their comparison to well-studied eukaryotic counterparts. Access to facilities for conducting this work is needed. The structural insights that can be gained are integral to the study of eukaryogenesis, revealing spatial organization of transcription and translation, membrane and cell-wall architecture, visualization of cytoskeletal features relevant to cell shape and division, and potential compartmentalization. The application of machine learning to structural biology represents another promising direction, allowing the identification of molecular homologies that are obscured in sequence comparisons by long phylogenetic branches, allowing comparative biochemistry on the molecular machines characteristic of eukaryotic life. Resolution of complexes with ligands is becoming very advanced due to improved cryoTEM image interpretation methods ( Callaway 2015 4. Key future directions in the study of eukaryogenesis With the above technologies and approaches, the field of eukaryogenesis research may be put on firmer footing. Perhaps more than any other major evolutionary transition examined in this colloquium, eukaryogenesis and eukaryotic diversification present a plethora of unique opportunities for expanded research given the wide variety of modern lineages to examine, the genetic evidence of the eukaryogenesis process burned into the LECA and everything descended from it, and the variety of new experimental and computational techniques that can be brought to bear on this information. Topics for further study include, but are not limited to, the following. LECA reconstruction. Eukaryote-specific genomic evolution. Viral coevolution. Reconstruction of prokaryotic lineages closest to eukaryotes. Identification of the bacterial lineage sourcing mitochondria. Analysis of the timing of the FECA and LECA. Protein structure and biochemistry. Origin of eukaryotic membranes and endomembrane systems. Further evaluation of prokaryotic contributions and innovations in the LECA is essential. Reconstructing the LECA implies a top-down approach involving comparative eukaryotic biology, genomics, and phylogenomics across the eToL, systematically sampling diversity to better understand deep relationships and evolutionary events. Eukaryotic genomes are larger and more complex than prokaryotic ones, with spiceosomal introns, longer intergenic regions, and extensive repeats, creating opportunities for de novo Viruses, particularly giant viruses, have been proposed to play a key role in eukaryogenesis, contributing genes, structures, and enhanced horizontal gene transfer and genome restructuring. Rapid viral evolution may have also generated divergent gene copies with novel functions, contributing to innovation. However, these interpretations may be affected by convergence and long-branch attraction (LBA) artifacts, which can misplace viral genes towards the base of phylogenetic trees. Even advanced evolutionary models struggle to correct for LBA necessitating alternative methods and modeling approaches to identify and mitigate these errors. This is crucial for determining whether LECA genes with apparent viral origins are actually fast-evolving cellular genes. Enhanced host and viral taxon sampling, refined phylogenetic methods, and modeling approaches will be essential to clarify viral contributions. Existing data suggest a mixed prokaryotic contribution to the LECA's gene complement. While Asgard archaea dominate the archaeal component, bacterial-derived genes are diverse, with alphaproteobacteria representing only a modest fraction. Recent analyses confirm this mixed heritage but differ on the relative archaeal and bacterial contributions. A refined phylogenetic and functional classification of the LECA's prokaryotic-like genes is needed to determine how many partners were involved in eukaryogenesis. Identifying and characterizing the closest prokaryotic lineages at the origin of eukaryotes is crucial, but may be difficult if contributing partners are members of extinct lineages without living representatives. Exploring microbial ecosystems and lineages without cultured representatives has already led to the discovery of Asgard archaea and a leap forward in understanding eukaryogenesis. Therefore, continuing this exploratory effort may allow the discovery of lineages that are even more closely related to eukaryotes than those already identified. Mitochondria do not seem closely related to any major alphaproteobacterial group sampled so far. Identifying the alphaproteobacterial lineage from which the mitochondrion evolved via structural alignments should help clarify its ancestral metabolism and whether the mitochondrial ancestor was more likely to be a parasite or a mutualistic syntrophic partner. Likewise, it will be important to further explore the diversity, genomics, biology, and ecology of other groups having potentially contributed many genes to eukaryotes in order to refine ideas about the FECA. Establishing a temporal and ecological framework for the origin of eukaryotes is another important direction. Determining when the FECA and LECA thrived would clarify whether eukaryogenesis was a long process or occurred rather rapidly once a particular type of symbiosis was established. It would also help clarify the type of ecosystem where eukaryotes likely arose. Prediction of protein-protein interactions on the scale of whole proteomes may soon be possible. Given good enough genomes and gene models, it will probably very soon be possible to mostly do away with the difficult business of directly characterizing large protein complexes (a signature trait of eukaryotes) across different organisms experimentally, although this is still a long distance from knowing gene functions with certainty. Coupled with cryo-electron tomography, this could nonetheless allow a deep systematic description of how proteomes have varied in structure and function across eukaryogenesis and since the diversification of eukaryotes. If an Asgard archaeon was the host of the mitochondrial ancestor, there must have been some sort of transition of membrane phospholipids from archaeal-to-bacterial type ( Summons et al. 2022 For a more detailed discussion of the spread of research opportunities that the study of eukaryogenesis presents, see the Appendix. E. Transition to multicellularity 1. Knowledge gaps in multicellular evolution After decades of research examining the origins and diversity of multicellular life, we now have unprecedented opportunities to address fundamental questions about how independent lineages have repeatedly evolved complex multicellular forms from unicellular ancestors. Advances in genomics, developmental biology, biophysics, and computational approaches have provided powerful new tools for investigating multicellularity. Yet despite these advances, significant gaps limit our understanding of this transformative evolutionary transition. The path forward requires not only continued technological innovation but strategic investments in areas where focused research could yield substantial conceptual breakthroughs, such as new model systems and synthetic biology. By addressing these challenges through interdisciplinary collaboration and sustained investigation, we can develop a more comprehensive framework for understanding one of life's most profound evolutionary innovations. A primary barrier to progress in understanding multicellular evolution is the lack of standardized approaches for measuring and comparing complexity across diverse multicellular forms. The most common approach has been to use the number of cell types produced by an organism as a proxy for complexity, but this approach has two major limitations: the number of cell types an organism produces is only indirectly related to morphological complexity and, like many definitions in biology (i.e., species, individual, or organisms), biologists do not currently agree on how cell types should even be defined. Recent work applying computational entropy measures to morphological data offers a promising new approach, but require careful standardization to enable meaningful comparisons across different scales and imaging modalities ( Gonzalez et al. 2009 Shannon 1948 One of the most important aspects of multicellular evolution is the approximately billion-year gap between the origin of eukaryotes and the widespread emergence of many modern large, complex multicellular forms. This pattern demands explanation, as it suggests that either specific enabling conditions or key innovations were required before complex multicellularity could evolve. Several hypotheses have been proposed and argued for and against, including the role of increased atmospheric oxygen, the constraints of the unicellular genomic toolkit, Snowball Earth events, and altered tectonic or oceanic cycles. Distinguishing among these numerous possibilities requires careful integration of geological, comparative, and experimental approaches. Testing alternative hypotheses will require the integration of diverse methods, including the development of first-principles biophysical and evolutionary theory, experimental tests with modern organisms, and corroboration with the geological record. Importantly, we should resist the temptation to seek a single causal explanation for all evolution of macroscopic multicellularity. Multicellularity has evolved dozens of times across the tree of life, for distinct reasons in distinct clades. In the past several decades, comparative genomics has shown that multicellular organisms invent surprisingly little cell biology, evolving relatively few new genes despite considerable expansions in developmental regulatory space. Co-option, rather than invention, has the most influence. However, we still lack a clear understanding of how these genes were co-opted for new functions in multicellular contexts. This gap is particularly relevant for understanding why some lineages have repeatedly evolved complex multicellularity (e.g., fungi) while others remain relatively simple (e.g., cyanobacteria) and others make this transition precisely once (e.g., animals), despite billions of years of evolution. 2. Expanding our understanding of prokaryotic multicellularity While bacteria and archaea have long been known to form simple aggregates and biofilms, emerging evidence suggests that prokaryotic multicellularity can be far more sophisticated than previously recognized. New imaging technologies and molecular approaches have revealed intricate cellular organization, differentiation, and even metabolic division of labor in bacterial and archaeal systems. These findings demand a reconsideration of how we conceptualize and study multicellular evolution across all domains of life. The diversity of prokaryotic multicellularity is particularly striking. From the tissue-like organization recently discovered in certain archaeal species ( Rados et al. 2025 The discovery of genetically heterogeneous but functionally integrated bacterial consortia, such as in multicellular magnetotactic bacteria, suggests that prokaryotic multicellularity may follow different evolutionary rules than those typically observed in eukaryotes. These systems challenge our traditional models of multicellular evolution, which often emphasize the importance of genetic uniformity in preventing internal evolutionary conflict. 3. Origin of multicellular development A central challenge in understanding multicellular evolution lies in explaining how novel developmental processes arise from unicellular ancestors. While we have made significant progress in identifying the genetic toolkit used by complex multicellular organisms, we know surprisingly little about how these tools were initially deployed to create new developmental programs in the earliest stages of multicellular evolution, where developmental processes must be created de novo. The role of physical forces in shaping multicellular development has emerged as a crucial area of investigation. Recent work suggests that many aspects of multicellular organization may arise as emergent properties of cell-cell interactions, requiring minimal genetic regulation. Understanding how these physical constraints both limit and enable evolutionary innovation is essential for explaining the repeated evolution of similar multicellular forms across diverse lineages. Future research must better integrate biophysical approaches with evolutionary and developmental biology. The co-option of existing cellular traits for new functions in multicellular contexts represents another critical area for investigation. While we know that many genes involved in animal development were present in unicellular ancestors, the evolutionary processes by which these genes were repurposed for multicellular development remain poorly understood. New experimental approaches, including the study of transitions between unicellular and multicellular states in modern organisms and the development of novel model systems via synthetic biology, could help illuminate these processes. 4. Leveraging the power of synthetic biology for future research The emergence of synthetic biology as a tool for studying multicellular evolution represents a powerful new approach to testing evolutionary hypotheses. By engineering artificial genetic circuits and cellular systems, researchers can directly test ideas about the minimal requirements for multicellular development and the constraints that shape evolutionary trajectories. These approaches offer the unique advantage of allowing precise control over initial conditions and evolutionary parameters, enabling rigorous tests of theoretical predictions. Recent work has demonstrated the feasibility of engineering synthetic developmental programs that recapitulate key aspects of multicellular evolution. These efforts have revealed both the surprising ease with which some multicellular traits can emerge and the challenges involved in creating stable, integrated multicellular systems. Future work should focus on creating more sophisticated synthetic systems that can test specific hypotheses about the role of genetic regulation, physical emergence, and environmental factors in shaping multicellular evolution. The development of synthetic multicellular life cycles offers particularly promising opportunities for understanding how different modes of multicellular organization evolve and persist. By engineering systems with different patterns of cell adhesion, division, and dispersal, researchers can explore how life cycle features influence the evolution of multicellular complexity. Such work could help explain why certain evolutionary trajectories appear more common than others in nature and what constraints might limit the evolution of complex multicellularity in some lineages. F. Integrated implementation strategy 1. Technical and methodological needs Advancing our understanding across these three transitions in early microbial evolution requires targeted technological and methodological innovations. While each transition presents unique challenges, several common needs emerge that could catalyze progress across multiple areas of research. One critical need is the development of improved imaging technologies that can capture subcellular structures and processes at high resolution across diverse scales. Advances in cryo-electron microscopy and tomography have already transformed our ability to visualize cellular structures, but further improvements in resolution, sample preparation, and data analysis would enable more detailed characterization of subcellular organization in both extant microbes and fossil specimens. Similarly, innovations in fluorescence microscopy and labeling techniques would facilitate in situ observation of cellular processes in living cells, particularly in non-model organisms. Another shared need is the development of computational tools for integrating and analyzing diverse data types. Machine learning approaches, particularly those leveraging artificial intelligence for pattern recognition and prediction, show tremendous promise for extracting meaningful patterns from complex biological data. For instance, AI-driven prediction of protein structure and function at the scale of whole proteomes could accelerate our understanding of how ancient proteins functioned and interacted, offering insights into early metabolic and signaling networks. Similarly, computational approaches for quantifying morphological complexity based on information theory principles could provide standardized metrics applicable across diverse life forms and even fossil specimens. Single-cell analytical technologies represent another high-priority area for development. Advances in single-cell genomics, transcriptomics, and metabolomics would enable characterization of unculturable microbes that may hold clues to evolutionary transitions. Additionally, technologies for isolating and analyzing individual cells within multicellular contexts could help elucidate how cellular differentiation and specialization emerge during the evolution of multicellularity. Developing new model systems represents perhaps the most pressing need. There is no singular model system of multicellularity; this process occurs in different lineages at different times for different reasons. To understand the evolution of multicellularity in general, we need diverse model systems. Expanding our repertoire of experimental systems, particularly to include representatives from understudied lineages such as holozoan relatives of metazoans, brown algae, and diverse prokaryotic groups, would provide crucial comparative insights into convergent and divergent evolutionary pathways to multicellularity. The taming of new unicellular eukaryote model systems will also greatly aid the study of eukaryotic diversity and the nature of the LECA, as it will provide detailed information on cell architecture outside those cells which have already been well studied. Synthetic biology approaches offer a powerful means of testing hypotheses about evolutionary transitions through experimental reconstruction. Engineering minimal cells, artificial symbioses, or synthetic multicellular systems allows researchers to explore the constraints and possibilities of different evolutionary pathways in controlled laboratory settings. Expanding the toolkit for precise genetic manipulation across diverse microbial lineages would enable more sophisticated experiments probing the genetic basis of key transitions. Current work in this area includes gene replacement experiments (such as testing the function of transcription factors from unicellular ancestors in multicellular contexts), engineering of artificial cell-cell communication systems, and the creation of synthetic developmental programs. These approaches have already yielded insights into the role of genetic co-option in multicellular evolution, but much more could be done. For instance, engineering synthetic morphogen gradients, artificial cell adhesion systems with defined properties, or minimal developmental circuits could help test specific hypotheses about the minimal requirements for multicellular innovation. Similarly, creating chimeric regulatory systems from different multicellular lineages could help identify universal versus lineage-specific components of the multicellular toolkit. 2. Building collaborative research communities Successfully addressing knowledge gaps in early microbial evolution requires building and maintaining robust interdisciplinary research communities, spanning evolutionary biology, biochemistry, cell biology, geochemistry, biophysics, computational science, and paleontology. Progress will depend on creating structured opportunities for sustained interaction among these diverse fields. Research networks facilitating this cross-pollination could be organized around specific questions or themes that cut across evolutionary transitions, such as the role of compartmentalization in biological organization or the emergence of information processing systems and could focus on utilization of common approaches, such as synthetic biology. Efforts like the Early Microbial Life (EML) project and similar meetings are essential toward meeting this goal. Colloquium participants recommend the following. Strengthening the EML community at the American Society for Microbiology Microbe Meeting. Establishing ongoing multidisciplinary EML colloquia. Forming small (~2-4) initial investigator clusters to develop and test new ideas, supported by short-term funding (2436 months). Providing a research presentation forum for these clusters. Inviting additional scientists to contribute fresh insights. Dedicated data integration and synthesis centers focused on early microbial evolution would provide crucial infrastructure for collaborative research. These centers could serve as repositories for diverse data types, i.e., genomic, structural, geological, paleontological, and develop tools for integrated analysis across disciplines. They would also provide physical and virtual spaces for collaborative work, hosting workshops, working groups, and visiting researchers focused on specific questions in early evolution. 3. Data curation for accessibility and progress A major practical bottleneck in EML research is the fragmentation of genomic, transcriptomic, and proteomic datasets, particularly among non-model eukaryotes. Efforts should be made to do the following. Convert transcriptome data into protein annotations for broader usability. Standardize and update genomic databases. Improve accessibility for non-expert researchers. A eukaryogenesis “data synthesis center” could take the model of NSF centers on a smaller scale for funding community gatherings and activities that will utilize currently available public data. In this case, the effort could focus on the coupled topics of eukaryogenesis, understudied eukaryote diversity, and the structure of their deep evolution. Resources should include funding for exploratory and experimental research, but also the maintenance or the establishment of open curated databases of genomes/transcriptomes/proteomes/lipidomes of microbial eukaryotes across the eToL and prokaryotes. It will be extremely useful to have a Genome Taxonomy Database ( https://gtdb.ecogenomic.org/ This also requires the assembly and maintenance of genomic data sets, updating of transcriptomes, and capabilities for organelle genome analysis. The group recognized that there are individual investigators developing software for this purpose (for example, Phylatol by Dr. Katz) and that there is a developing community of databases (Figshare, for example), but it was emphasized that there needs to be a more comprehensive effort in this area. However, an even more glaring gap is the absence of a community effort in the area of comparative protein structure, which ultimately will be necessary for understanding the causes and consequences of the great transitions in life. These data may provide a multitude of practical applications immediately (for example, drug discovery, food supply applications, and cataloging responses to environmental perturbations) aiding in the practicality of its establishment Training programs that explicitly cultivate interdisciplinary expertise are essential for developing the next generation of researchers equipped to tackle complex questions in early evolution. Funding structures must also evolve to support these interdisciplinary endeavors. Traditional grant mechanisms often favor incremental advances within established disciplines over the riskier, longer-term, and more integrative approaches needed to transform our understanding of multicellular evolution. Developing dedicated funding streams that explicitly value cross-disciplinary collaboration and longer time horizons would provide the foundation for sustained progress and must provide for the maintenance of critical research infrastructure and the continuation of successful collaborative networks. One such set of infrastructure that would benefit from long-time-horizon support would be laboratory evolution experiments, especially as pertaining to the evolution of multicellularity in model organisms. These experiments continue to emit surprises decades after their inception ( Blount et al. 2008 Stroud and Ratcliff 2025 4. Funding and resource allocation Effectively addressing knowledge gaps in early microbial evolution requires funding structures adapted to the interdisciplinary, long-term nature of the research. Traditional grant mechanisms often favor incremental advances within established disciplines over the riskier, integrative approaches needed to transform our understanding of evolutionary transitions. Dedicated funding streams that explicitly value cross-disciplinary collaboration and longer time horizons would provide the foundation for sustained progress. These could include center grants supporting collaborative teams working on specific questions in early evolution, technology development grants focused on tools with applications across multiple evolutionary transitions, and training grants for interdisciplinary education and career development. The economic feasibility of these initiatives is high, given the recent great advances in relevant technologies and the potential for leveraging existing research infrastructure. Initial investments should prioritize the establishment of dedicated experimental facilities, the development of interdisciplinary training programs, and the promotion of international collaboration. These actions will lay the foundation for sustained progress in understanding the key transitions in early microbial evolution. G. Broader impacts and societal outcomes Research on the key transitions in early microbial evolution has profound implications that extend far beyond the scientific community. By elucidating the processes that gave rise to life and shaped its increasing complexity, this research addresses fundamental questions about our origins and place in the universe. It also informs our search for life beyond Earth and our understanding of life's potential diversity in the cosmos. The conceptual frameworks developed through this research ultimately contribute to our broader capacity to comprehend complex, emergent systems in general across scales and contexts and provides critical insight into the evolutionary mechanisms driving current human-microbe interactions (from pathogens to biogeochemical cycles). The practical applications of this research are substantial and diverse. Understanding the biochemical principles underlying the origin of life stands to inspire the development of novel catalysts, materials, and energy systems based on biological principles. The general process by which matter organizes into stable, active forms is of fundamental interest for understanding the stability of all complex dynamic systems, living and otherwise, and their capacity to attain homeostasis in the face of a fluctuating world. The spontaneous origin of structure from previously unstructured matter is ultimately the source of all complexity of any sort. The study of eukaryogenesis will shed light on numerous practical matters. Microbial eukaryotes, particularly photosynthetic protists, play crucial roles in global carbon cycling and ecosystem function. Many understudied lineages, particularly protists and fungi, serve as keystone species in nutrient cycling, carbon fixation, and food webs. Understanding their evolutionary history and diversity is essential for modeling and managing Earth's biogeochemical cycles in the face of environmental change. Insights into eukaryotic evolution also enhance our understanding of basic cellular processes, critical for studying diseases such as cancer, neurodegeneration, and aging. Many disease-related mechanisms stem from ancient eukaryotic features like the cytoskeleton, mitochondrial conflict, and cell cycle regulation. Mitochondrial evolution research in particular aids in understanding mitochondrial diseases and age-related disorders. In drug discovery and parasite control, pathogenic protists (e.g., Plasmodium, Trypanosoma, Leishmania) The mechanisms through which multicellular organisms suppress within-lineage genetic conflict have direct applications in synthetic biology and biotechnology. For example, researchers like Tong and Khalil are applying principles derived from studying cancer suppression in multicellular organisms to enhance the stability and productivity of engineered microbes for biotechnological production. Similarly, the regulatory networks that coordinate cell differentiation and spatial organization in natural systems provide blueprints for designing artificial multicellular consortia with enhanced metabolic capabilities for bioremediation, biofuel production, biomaterial synthesis, and synthetic biology. The study of the origins of biological complexity and transitions across scales addresses one of humanity's most profound questions: how did we get here, and more broadly, how does complex life arise in the universe? By identifying the key triggers, bottlenecks, and facilitating conditions for the evolution of complex biology, we gain insight into whether similar transitions might occur on other worlds. This knowledge directly informs astrobiology and the search for extraterrestrial life by helping distinguish between conditions that permit simple life versus those necessary for complex multicellular organisms. The existential implications of this work extend to our understanding of humanity's place in the cosmos: are we the product of highly contingent evolutionary pathways, or the predictable outcome of universal biological principles? All research on early microbial evolution offers a powerful framework for science education and public engagement. The story of life's origin and early evolution captures the imagination and provides an accessible entry point to fundamental concepts in biology, chemistry, and Earth science. By communicating the excitement and significance of this research to diverse audiences, scientists can foster a greater appreciation for the scientific process and the interconnectedness of life on Earth. Eukaryotic organelle evolution has applications in biotechnology, metabolic engineering, and biomedicine. Artificial organelles could revolutionize drug delivery (vesicles), regenerative medicine (synthetic mitochondria producing therapeutic factors), synthetic immunology (pathogen-destroying organelles), and detoxification (ROS-scavenging organelles). Industrial applications include isolating toxic intermediates in chemical production and protecting host cells from metabolic byproducts in biofuel synthesis. Artificial enzyme cascades could enhance drug synthesis or biopolymer production, while artificial chloroplasts could improve photosynthesis in engineered algae for biofuel production. H. Conclusion The study of major transitions in evolution, from the origin of life through eukaryogenesis to the evolution of multicellularity, stands at an exciting juncture. Technological advances, conceptual breakthroughs, and the integration of diverse disciplinary perspectives provide unprecedented opportunities to address long-standing questions about life's early history. By taking an integrated approach to these evolutionary transitions and recognizing their interconnectedness while respecting their unique features, we can develop a more comprehensive understanding of how life has repeatedly achieved greater complexity. Addressing the gaps identified in this section will require sustained investment in interdisciplinary research, technological innovation, and collaborative infrastructure. It will demand the development of new conceptual frameworks and methodological approaches that can bridge traditional disciplinary boundaries. Further, it will necessitate a willingness to embrace the complexity and contingency of evolutionary processes while searching for general principles that might govern biological organization across scales and contexts. The potential rewards of this endeavor are substantial. By understanding how life originated and evolved increasing complexity, we gain insight into profound questions: how did we get here and are we alone in the universe? By elucidating the mechanisms underlying key evolutionary transitions, we develop tools for addressing contemporary challenges in health, environment, and technology. In addition, by telling the story of life's early chapters, we contribute to a deeper understanding of our connection to the broader web of life that has evolved on our planet over billions of years. APPENDIX: ADDITIONAL FUTURE RESEARCH DIRECTIONS FOR THE STUDY OF EUKARYOGENESIS AND EUKARYOTIC DIVERSITY Introduction Perhaps more so than any other major evolutionary transition examined in this colloquium, eukaryogenesis and eukaryotic evolution have an enormous landscape of potential near-future productive research. This is because of novel data analysis and machine-learning techniques, an expanding set of model organisms including both diverse modern eukaryotes and the new understanding of the Asgard archaea sister to the eukaryotic nuclear genome, and a broadened perspective on the possibilities of early eukaryotic evolution revealing possibilities that would have previously been rejected. Significantly more information survives to be productively examined as compared to attempts to study the origin of life itself, and the utter transformation of cell biology that occurred at the base of the eukarya makes the largest genetic changes of multicellularization seem small. In the interest of assisting future researchers formulate research questions and attack them via appropriate techniques, we here provide an expanded and detailed account of particularly pressing open questions related to the study of eukaryogenesis and the techniques we believe could be productively applied to them now or in the near future. Sequencing of underrepresented eukaryotic lineages for LECA reconstruction The following are key lineages needing more genomic and transcriptomic data. Deep-branching and uncultured eukaryotic lineages such as Collodictyonids, Rigifilids, Mantamonads, Hemimastigophora Metamonads Carpediemonas-like Marine and unicellular eukaryotes with unique genomic features (Ancyromonads, Telonemids Centrohelids, Heliomonads Plastid-bearing lineages with cryptic evolutionary histories, i.e., Glaucophytes Apicomplexan Chromera, Vitrella, Perkinsids Early-diverging fungi and opisthokonts, e.g., Rozellids Microsporidia Fonticulids Nuclearids Asgard-related eukaryotes, i.e., hypothetical and uncultured groups (evidence of recent lateral gene transfer from Asgard archaea and unknown eukaryotes may be uncovered in deepsea or hydrothermal-vent ecosystems). Eukaryotic genome evolution Understanding the origin of other typical eukaryotic processes or structures, such as the spliceosome, an RNA interference system, mitosis and meiosis will also require a combination of comparative prokaryotic and eukaryotic genomics and molecular phylogeny across a representative diversity of prokaryotic and eukaryotic taxa. Gathering genomic, phylogenetic, as well as cell and molecular biology data from the bacterial and archaeal taxa that seem to have contributed the most genes to the LECA and developing model systems for them will offer a solid starting point to test for evolutionary relatedness (via phylogenomics) and potential mechanisms (via observation and experimental approaches). The origin of mitosis and crucially meiosis as inextricably related to sex is intriguing. Beyond identifying prokaryotic homologs from which part of the underlying mechanisms may derive, getting data at the level of populations should inform about the selective pressures (e.g., purging deleterious mutations) or stochastic phenomena at play. Very little is known about mutation rates and effective population sizes in microbial eukaryotes across the eToL, or many prokaryotes in natural environments. How these vary with growth rate and environmental conditions, including the establishment of facultative and obligatory symbioses, remains to be established. Developing approaches to derive these types of data or equivalent proxies from metagenomes or whole natural communities would be extremely helpful. Also, experimental evolution approaches testing how different population parameters vary as symbioses form and how they influence fitness will provide interesting information. These types of studies could be done through inducing artificial symbioses [e.g., cyanobacteria in yeast or, better, prokaryote-in-prokaryote symbioses] or analyzing, ideally, the behavior of (Asgard) archaea-bacteria symbioses under different evolutionary constraints. Studying genome evolution and gene transfer along these processes should reveal potential convergent patterns of gene gain and loss. In parallel, studying the role of stochasticity and constructive neutral evolution during emerging obligate prokaryotic symbioses might also provide clues about how novelty and complexity can arise in the context of eukaryogenesis. Viral coevolution Viruses, particularly giant viruses, have been proposed to play a key role in eukaryogenesis, contributing genes and structures. However, these interpretations may be affected by convergence and long-branch attraction artifacts, which can readily misplace viral genes at the base of phylogenetic trees. Even advanced evolutionary models struggle to correct for LBA, necessitating alternative methods and modeling approaches to identify and mitigate these errors. This is crucial to determine whether LECA genes with apparent viral origins are actually fast-evolving cellular genes. Improved sampling of eukaryotic host genomes in phylogenetic trees could help reposition viral branches more accurately. Viruses likely influenced eukaryogenesis by facilitating horizontal gene transfer (HGT), accelerating gene evolution, and reshuffling genomes. Their rapid evolution may have generated divergent gene copies with novel functions, contributing to innovation. Enhanced host and viral taxon sampling, refined phylogenetic methods, and modeling approaches will be essential to clarify viral contributions. Additionally, improved viral sampling could help determine whether eukaryote-specific viruses originated mainly from bacterial or archaeal sources. Reconstructing prokaryotic lineages closest to eukaryotes Identifying and characterizing the closest prokaryotic lineages at the origin of eukaryotes is also crucial. Our knowledge of microbial diversity is continuously expanding. Exploring microbial ecosystems and lineages without cultured representatives led to the discovery of Asgard archaea and a leap forward in understanding eukaryogenesis. Therefore, continuing this exploratory effort may allow the discovery of lineages that are even more closely related to eukaryotes than those already identified. Existing data suggest a mixed prokaryotic contribution to the LECA's gene complement. While Asgard archaea dominate the archaeal component, bacterial-derived genes are diverse, with alphaproteobacteria representing only a modest fraction. Recent analyses confirm this mixed heritage but differ on the relative archaeal and bacterial contributions. A refined phylogenetic and functional classification of the LECA's prokaryotic-like genes is needed to determine whether two or more partners were involved in eukaryogenesis. If Asgard archaea contributed most genes, models favoring a complex archaeal host for the mitochondrial ancestor gain support. Conversely, significant bacterial contributions could imply permanent or transient symbiotic interactions. Many bacterial genes may have resulted from HGT within the early eukaryotic ecosystem, complicating their distinction from symbiotic gene transfer. Advanced phylogenetics, dating methods, and phylostratigraphy will be key to resolving these uncertainties. Provenance of the mitochondrial genome Mitochondria do not seem closely related to any major modern alphaproteobacterial group sampled so far. Identifying the alphaproteobacterial lineage from which the mitochondrion evolved should help clarify its ancestral metabolism and whether the mitochondrial ancestor was more likely to be a parasite or a mutualistic syntroph. Likewise, it will be important to further explore the diversity, genomics, biology, and ecology of other groups having potentially contributed many genes to eukaryotes. Many of those lineages, such as Myxococcota or Planctomycetota, are widely diverse and poorly studied in the anoxic ecosystems where Asgard archaea preferentially thrive. This might lead to pinpoint major contributors to eukaryogenesis and perhaps decipher if they were symbionts or simple gene donors. Collectively, this information should help to refine ideas about the first eukaryotic common ancestor (FECA), strictly speaking the oldest obligatory symbiotic consortium which evolved into the first eukaryotic cell. Establishing a time and ecological framework for the origin of eukaryotes Determining when the FECA and LECA thrived would allow us to know if eukaryogenesis was a long slow process or, on the contrary, occurred rather rapidly once a particular type of symbiosis was fixed. It would also help in constraining the type of ecosystem where eukaryotes likely arose. The ancestor of mitochondria likely was a facultative aerobe, suggesting that the oxygenation of the atmosphere (~2.4 Ga) and the evolution of oxygen respiration had already occurred. At that time, the deep ocean was still anoxic, which suggests that the microbial ecosystems where eukaryotes evolved might have been either shallow oceanic or freshwater sediments or microbial mats. Asgard archaea mostly thrive in anoxic settings, but some Asgard archaea might be tolerant to oxygen or microaerophilic. These observations support redox transition environments as favorable for the development of the proto-eukaryotic symbiosis. Microbial ecology studies of environments hosting Asgard archaea consortia will be useful to unravel the diversity and specificity of bacterial partners and propose a plausible context for eukaryogenesis. The fossil record is highly fragmentary, especially for microbes and for so ancient dates. Therefore, expanding the eukaryotic microfossil record, developing criteria to differentiate members of current taxa from potentially ancestral ones, and combining this knowledge with phylogenetic dating approaches will be required. Improving molecular phylogenetic dating and having reliable calibration points is needed, although challenging. Establishing relative timing events, for instance, whether mitochondria (or other bacterial contributions) were incorporated early or late during eukaryogenesis with respect to the Asgard partner, seems to be achievable by considering average branch lengths from eukaryotes to their closest prokaryotic ancestors in phylogenetic trees. However, these approaches might be subject to different biases, notably due to differences in evolutionary rate across lineages, such that improving these methods or finding alternative ways to validate inferred times (e.g., via reconciliation approaches) will be also needed. Biochemistry of eukaryogenesis Complementary to the described phylogeny and organelle research, the participants discussed evolution of protein structure and function, addressing mechanisms governing biochemical evolution in eukaryogenesis. At the moment, it remains challenging to make detailed inferences about when pre-existing components gain their eukaryote-specific functions or indeed if any one function we think originated in the LECA based on limited sampling really is that old. The issue remains that protein-protein interactions and functions are not trivial to predict from gene content alone. How can we make progress? One significant technical development is the advent of cryo-electron tomography, with which low-resolution structures of complexes can be solved directly inside frozen cells. This obviates the need to purify large molecular machines from difficult non-model organisms and could help resolve the diversity of structures across the eukaryotic and Asgard trees. It has been used, for example, to visualize the structure of actin filaments directly inside Asgard archaeal cells. This technique, however, still has its limits and works best for large and abundant assemblies (like ribosomes or the proteasome) and for very thin cells. In many cases, it also cannot tell us how these assemblies function. Returning to the exosome, an in situ structure of the plant exosome would not have revealed that its ring is an active RNAase. This still required isolation and biochemical characterization, because the overall topology of the plant exosome is very similar to that of animals. Another promising technology is machine-learning-driven prediction of protein-protein interactions on the scale of whole proteomes. Given good enough genomes and gene models, it will possibly very soon be possible to mostly do away with the difficult business of characterizing complexes across different organisms experimentally. Instead, effort would be focused on species where structural or functional changes are suspected based on such predictions. Together with cryo-electron tomography, this is perhaps the most exciting technical development for our ability to decipher the biochemistry of eukaryogenesis. Ascertaining the origin of bacterial-like eukaryotic membranes and the endomembrane system If an Asgard archaeon was the host of the mitochondrial ancestor, there must have been a transition of membrane phospholipids from archaeal-to-bacterial type. Although there is a wide phospholipid diversity in prokaryotes, such a transition has never been observed in nature, likely due to the highly specific properties of archaeal versus bacterial phospholipids, with archaeal membranes generally more adapted to low-energy, often extreme, ecosystems. Here experimental approaches could help test the feasibility of such a transition. Vesicles with mixed archaeal and bacterial lipids are stable. Also attempts of expressing and incorporating archaeal phospholipids in Escherichia coli Having a developed cytoskeleton is a prerequisite for membrane remodeling. In eukaryotes, membrane remodeling can happen externally, e.g., to reshape morphology (in wall-less cells) or internally, for the dynamics of most membranous compartments (e.g., Golgi apparatus, the endoplasmic reticulum, or, most notably, the nucleus). It is also required for phagocytosis, a property thought to be exclusive to eukaryotes for a long time and, in some models of eukaryogenesis, a strict requirement for the acquisition of the mitochondrial ancestor by an archaeal host. However, if it seems clear that the eukaryotic cytoskeleton has an archaeal origin, it is also notable that phagocytosis relying on different cytoskeletal proteins occurs in some planctomycete bacteria. Likewise, many bacteria have relatively complex endomembrane systems, some of them exhibiting nuclear-like compartments, such as some planctomycetes and Atribacteria. Although less frequently described, some archaea also seem to display endomembranes. Studying the origin and mechanisms underlying the formation of endomembrane systems in prokaryotes and their dynamics could shed new light on the evolution of endomembranous systems in eukaryotes. Studying cell biology from a strong evolutionary perspective is highly needed in eukaryotes and, most importantly, prokaryotes. Unfortunately, cell size and difficulties in culturing most prokaryotic species make this task difficult. High-resolution microscopy techniques, notably cryoTEM and related techniques, and developing fluorescent labeling techniques for specific intracellular components in model and non-model organisms will be crucial to make progress. The evolutionary origin of the eukaryotic nucleus and the nuclear pore remains particularly open, from both a mechanistic and a phylogenetic point of view, but also from the perspective of the causes (the selective forces) that led to the evolution of this defining eukaryotic structure. An integrative view that considers how (and why) intracellular membrane compartments formed, the evolution of the eukaryotic genome, the origin and spread of introns, as well as gene editing and trafficking will be most likely needed to propose a realistic model for the evolution of the nucleus and the eukaryotic cell. Experimental studies of LECA biochemistry A different strategy would be to skip the intermediary and assay the properties of the LECA's biochemical machinery directly. This is in principle possible using ancestral sequence reconstruction, with which probabilistic estimates of the sequences of LECA proteins can be madeClick or tap here to enter text.. If these sequences can then be produced recombinantly and reconstituted into functional complexes, their biochemical properties can be measured directly instead of inferred from the properties of their living descendants. However, the hurdles to this approach for LECA proteins are significant. Many of our most pressing questions about the LECA and its archaeal predecessors are about the structure and function of multi-subunit complexes. Resurrecting such entities and then reconstituting them in vitro More importantly, it comes with many phylogenetic challenges. First, ancestral sequence reconstruction invariably introduces mistakes into its reconstructed sequences. The larger the number of components that need to be resurrected, the larger the chance that one of them contains a non-functionalizing mistake that could doom the entire enterprise. Whether this is likely depends on the complex in question and how fast it evolves. As a reference point, the research group of Hochberg has in unpublished work successfully resurrected complexes that existed in the last common ancestor of fungi and animals and contain 10 separately reconstructed components, which form a functional complex. This indicates that in principle even relatively complex machines are accessible to this approach. Root of the eukaryotic tree of life and eukaryogenesis protein structure studies A much bigger issue is the phylogenetic uncertainty around the root of the eukaryotic tree. There is currently no universally accepted root position for this tree, but resurrecting LECA protein complexes via ancestral sequence reconstruction absolutely requires choosing one. One way to circumvent this problem would be to resurrect proteins for a number of plausible roots and test directly what impact this has on the properties of LECA proteins. However, multi-protein resurrections are very time consuming and expensive, and the number of plausible root positions is currently still large enough to make this impractical. It may seem as if another solution would be to leave unresolved relationships at the base of eukaryotes as polytomies to reflect our ignorance and use this topology for ancestral sequence reconstruction. However, this artificially inflates the statistical confidence of reconstructed sequences because it treats taxa as independent that in reality share common ancestors. Forcing hard polytomies is therefore not an appropriate solution for the problem of an unknown eukaryotic root position. The last problem is that there may not even be a single root of the eukaryotic tree that applies to all proteins. Horizontal gene transfer and incomplete lineage sorting can both lead to deviations of individual gene histories from the “real” species phylogeny, if such a thing exists for the earliest eukaryotes. Both processes are very plausible at the base of the eukaryotes: single-cell organisms tend to engage in HGT and speciation intervals for the deepest branches in the eukaryotic tree may have been very short. Distinguishing these processes from a simple lack of phylogenetic signal (in which case the real root could be enforced through a constraint) is near impossible for the deepest splits in the eukaryotic tree. These are all reasons to be cautious, but for some problems with the LECA's biochemistry the exact topology near the root of eukaryotes may matter little. This would, for example, be the case if there were no extreme variation in a particular protein's function between eukaryotic supergroups (as is the case for actin). In such cases, topological uncertainty about the root and relationships among supergroups would probably not influence the reconstructed sequence at functionally crucial sites. We do have to ask ourselves, however, what value there is in resurrecting LECA proteins whose functions in the LECA are very obvious from the properties of all extant descendants of those proteins. The LECA's actin, for example, will almost certainly behave like any other eukaryotic actin. Resurrecting ancestral proteins only makes sense if it solves a problem without an obvious solution about the function of extinct proteins; in that case, we may have to worry about the uncertainties in deep relationships of eukaryotes. Can these phylogenetic problems be solved? One promising avenue is more complex and flexible amino-acid substitution models, which may help settle on one root for the eukaryotic tree of life. Another development is the advent of structural phylogenetics. This technique uses a translation of protein structures into strings of letters that can be used directly by phylogenetic software as if they were amino acids. The advantage of this technique is that structure evolves much slower than sequence such that branches that are pathologically long on protein phylogenies shorten significantly. This can help solve some deep phylogenetic problems, but its impact on eu-karyogenesis seems limited to very specific problems for now. It will not, for example, help us resolve the root of the eukaryotic tree or the branching order of the eukaryotic supergroups, even though these problems involve some troublesome long branches. The reason is that the deepest branches in the eukaryotic tree of life are already very short on protein trees and would shrink to near zero on structure trees, resulting in an unresolved polytomy at the base of eukaryotes. Structural phylogenetics could however help resolve the order of some very deep gene-duplication events that occurred over the LECA branch, such as all the duplications that lead to eukaryotic exosomes and TRiC chaperones comprising many more paralogs than their archaeal counterparts. However, the order of such duplications is not currently a burning question about how eukaryogenesis proceeded, so for the moment the impact of structural phylogenetics on this topic seems limited. How will we explain the biochemistry of conversion from prokaryotes to eukaryotes? We must continue the hard work of doing and funding LECA-relevant biochemistry in non-model eukaryotes and archaea for some years. Developments in AI prediction of protein structure and function are all but certain to significantly accelerate this endeavor in the near future. There will however likely be no single discovery that “solves” the biochemistry of eukaryogenesis, unless lineages much closer to eukaryotes than even the Asgard archaea are discovered. Instead, it seems probable that the biochemistry of eukaryotes will seem less and less singular, as we understand better how it relates to the biochemistry of their prokaryotic ancestors. Alas, the perceived impact of proving that yet another apparently eukaryote-specific protein or function can be traced back to prokaryotes will probably decrease with every such discovery. However, perhaps this is as it should be: concluding that eukaryogensis was biochemically unremarkable, if indeed it was, would be an immense leap in our understanding of this event. BIBLIOGRAPHY Bibliography for Executive Summary and Sec. I Brocks JJ, Nettersheim BJ, Adam P, Schaeffer P, Jarrett AJM, Güneli N, Liyanage T, Van Maldegem LM, Hallmann C, Hope JM. 2023. 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Proc R Soc B 287:20192377. [ PMC free article : PMC7126077 PubMed : 32156207 The American Academy of Microbiology (Academy) is the honorific branch and scientific think tank of the American Society for Microbiology (ASM), a nonprofit scientific society with more than 30,000 members. Fellows of the Academy have been elected by their peers in recognition of their outstanding contributions to the microbial sciences. Through its colloquium program, the Academy draws on the expertise of these Fellows and other experts to address critical issues in the microbial sciences. This report is based on the deliberations of experts who gathered to discuss a series of questions developed by the steering committee. All participants had the opportunity to provide feedback, and every effort has been made to ensure that the information is accurate and complete. The contents reflect the views of the participants and are not intended to reflect official positions of the Academy or of the American Society for Microbiology. Footnotes 1 1 Doolittle and Brunet 2016 © 2025 American Society for Microbiology. All rights reserved. This work is being shared with NCBI under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License Bookshelf ID: NBK621464 PMID: 41955394 10.1128/AAMCol.Jun.2025 Share Views PubReader Print View Cite this Page Early Microbial Life: Our Past, Present, and Future Project Report [Internet]. Washington, DC: American Society for Microbiology; 2025. PDF version of this title In this Page Governors, American Academy of Microbiology ACKNOWLEDGEMENTS EXECUTIVE SUMMARY I. INTRODUCTION: EVOLUTION OF MICROBES, A JOURNEY FROM PRE-LIFE TO MULTICELLULARITY II. PLANETARY, GEOCHEMICAL CONDITIONS, AND FOSSIL RECORD OF EARLY MICROBIAL LIFE III. EARLY ORIGINS FROM ABIOTIC PROCESS TO CELLULAR ORGANIZATION AND HIGHER-ORDER MECHANISMS OF EVOLUTION IV. DIVERSIFICATION AND ORIGIN OF EUKARYOTES V. EVOLUTION AND MECHANISMS GOVERNING MULTICELLULARITY VI. EVOLUTIONARY THEORY AS A GUIDE TO UNDERSTANDING THE PLAUSIBILITY OF EARLY-LIFE SCENARIOS VII. KNOWLEDGE GAPS AND FUTURE RESEARCH IN EARLY MICROBIAL LIFE AND EVOLUTION APPENDIX: ADDITIONAL FUTURE RESEARCH DIRECTIONS FOR THE STUDY OF EUKARYOGENESIS AND EUKARYOTIC DIVERSITY BIBLIOGRAPHY Other titles in this collection American Academy of Microbiology Colloquia Reports Related information NLM Catalog Related NLM Catalog Entries PMC PubMed Central citations PubMed Links to PubMed Recent Activity Clear Turn Off Turn On Early Microbial Life: Our Past, Present, and Future Project Report Early Microbial Life: Our Past, Present, and Future Project Report Your browsing activity is empty. Activity recording is turned off. Turn recording back on See more... 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