APPENDIX: ADDITIONAL FUTURE RESEARCH DIRECTIONS FOR THE STUDY OF EUKARYOGENESIS AND EUKARYOTIC DIVERSITY

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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 University of Wisconsin-Madison Karen C. Carroll, M.D. Johns Hopkins University School of Medicine Sean Crosson, Ph.D. Michigan State University Suzanne Fleiszig, O.D., Ph.D. University of California Berkeley Susan Golden, Ph.D. University of California San Diego Jay T. Lennon, Ph.D. Indiana University Melissa B. Miller, Ph.D. University of North Carolina (UNC) at Chapel Hill Susan E. Sharp, Ph.D. Copan Diagnostics Alfredo Torres, Ph.D. University of Texas Medical Branch Paul E. Turner, Ph.D. Yale University Susan Weiss, Ph.D. University of Pennsylvania Henry Neal Williams, Ph.D. Florida A&M University This project was supported by a grant from the Gordon and Betty Moore Foundation. PROJECT STEERING COMMITTEE Michael Lynch, Ph.D. (Co-Chair) Arizona State University Vaughn Cooper, Ph.D. (Co-Chair) University of Pittsburgh Susana Coelho, Ph.D. Max Planck Institute Betül Kaçar, Ph.D. University of Wisconsin-Madison William Ratcliff, Ph.D. Georgia Institute of Technology Paul E. Turner, Ph.D. Yale University PROJECT PARTICIPANTS Frank Aylward, Ph.D. Virginia Tech Devaki Bhaya, Ph.D. Carnegie Science Thibaut Brunet, Ph.D. Institut Pasteur, Université Paris Cité, CNRS Shelley Copley, Ph.D. University of Colorado Boulder Andrew Ellington, Ph.D. The University of Texas at Austin Laura Eme, Ph.D. University of Rhode Island Toni Gabaldón, Ph.D. Barcelona Supercomputing Center and Institute for Research in Biomedicine Peter Gogarten, Ph.D. University of Connecticut Holly Goodson, Ph.D. University of Notre Dame Georg Graf von Hochberg, Ph.D. Max Planck Institute Galen Halverson, Ph.D. McGill University Katrin Hammerschmidt, Ph.D. Christian-Albrechts-Universität zu Kiel Matthew D. Herron, Ph.D. National Science Foundation Julie Huber, Ph.D. Woods Hole Oceanographic Institution Laura A. Katz, Ph.D. Smith College Eugene Koonin, Ph.D. National Center for Biotechnology Information Eric Libby, Ph.D. Umeå University Purificación López García, Ph.D. Centre National de la Recherche Scientifique, Université Paris-Saclay Timothy W. Lyons, Ph.D. University of California, Riverside John McCutcheon, Ph.D. Arizona State University Elizabeth Ostrowski, Ph.D. University of Washington Bothell Eva Pillai, Ph.D. European Molecular Biology Laboratory, Institut Pasteur Susannah Porter, Ph.D. University of California, Santa Barbara Thomas Richards, Ph.D. University of Oxford Patricia Sanchez-Baracaldo, Ph.D. University of Bristol Julia Schwartzman, Ph.D. University of Southern California Arnau Sebé-Pedrós, Ph.D. Centre for Genomic Regulation Carl Simpson, Ph.D. University of Colorado Boulder Michael Travisano, Ph.D. University of Minnesota Arvind Varsani, Ph.D. Arizona State University Paula V. Welander, Ph.D. Stanford University Tom A. Williams, Ph.D. University of Bristol Peter Yunker, Ph.D. Georgia Institute of Technology 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 ). Such ideas were used as a springboard in Colloquium 1 to consider a wide variety of compelling questions. Where on Earth did life originate in order to acquire both the simple monomeric building blocks essential for biochemistry and the production of information-containing polymers? In what sequence did key fundamental biological systems evolve to sustain protocells and inheritance across generations of cell division, including metabolic function, preservation of genetic information, and membranes that protect against environmental perturbations? How did random genetic variation, population dynamics of competing variants, and symbiotic interactions (parasitism and mutualism) within biological communities contribute to the emergence of complexities associated with protocells? What features characterized the LUCA, and how can we infer the genotypic and phenotypic characteristics of the LUCA from extant microbial genomes? 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 ). These findings imply that by the time the LUCA emerged, cellular life had already undergone significant evolutionary refinement, although it remains unclear whether the LUCA had made a full transition to the DNA world as we currently know it. Importantly, the colloquium addressed ecology, especially uncertainties over conditions on Earth at key times in its history that would have challenged early microbial life to deal with various environmental contexts that could have presented serious constraints. For example, life likely emerged during a period on Earth without an atmospheric ozone layer, exposing nascent life forms to harmful UV radiation. However, the abundant uncertainties and debates over these environmental conditions necessitated that the participants grappled with examining alternative geochemical niches as cradles of early life, ranging from deep-sea hydrothermal vents, mineral surfaces, and subsurface aquifers. In the search for how microbial life shaped and was shaped by geochemical conditions, a clear challenge is to determine how different environments influenced mutagenesis, chemical stability, and the feasibility of the evolution of larger genome sizes. 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 ) proposed how models of “Earth-system evolution” could help align molecular evolutionary patterns with the geological and geochemical record, creating a more holistic picture of life's emergence. Computational approaches and increasing efforts in synthetic biology could present theoretical and empirical platforms capable of reconstructing ancient biochemical systems (Kacar 2024). These and other exciting tools could test hypotheses about metabolic evolution under controlled conditions and simulate evolutionary scenarios not observable in nature. 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 date back over 1.5 billion years ago, the lipid biomarkers typically associated with modern eukaryotes (e.g., sterols) only appear ~1 billion years ago ( Brocks et al. 2023 ). This 500-million-year discrepancy hints at a cryptic phase in which proto-eukaryotic biochemistry evolved before it became recognizable in the geochemical and fossil records. Clearly, reconstructing early eukaryotic evolution requires cross-referencing genomic evidence with structural and chemical data, presenting a challenge for elucidating the evolution of early microbial life. Many early innovations, such as phagocytosis and endosymbiosis, are expected to leave limited traces in the fossil record, making molecular fossils and ancestral genome reconstruction indispensable tools as we move forward. A similarly challenging exercise concerns the evolution of bioenergetics and genetic architecture, to better understand the role of energy demands and metabolism in shaping biological complexity. Although mitochondria derived from an ancient endosymbiotic event serve as the platform for energy production in today's eukaryotes, considerable evidence suggests that eukaryotes are not energetically superior to prokaryotes, and it remains unclear whether the emergence of mitochondria occurred prior to the establishment of many of the other embellishments of the eukaryotic cell. 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 showcase cell fusion and aggregation, often without cell proliferation. These organisms demonstrate that multicellularity can emerge through various mechanisms, including aggregation, clonal expansion, and cell fusion. The multiple instances of yeasts within the fungi provide compelling examples of independent reversions to unicellularity. Colloquium 3 addressed the role of genomics to uncover how genetic changes enable the evolution of multicellularity. These discussions focused on identifying core regulatory networks and genetic toolkits shared across multicellular lineages. Many genes used in multicellular development existed prior to multicellularity, suggesting they were co-opted for new roles. Understanding how this co-option occurred is central to uncovering the origin of multicellular complexity. Interestingly, while bacteria exhibit biochemical diversity, their morphological complexity remains similar across their evolved time on Earth. This paradox was explored in Colloquium 3 in the context of evolutionary constraints and tradeoffs. For example, bacterial cell walls and division mechanisms may limit morphological elaboration, despite genomic innovation. Participants asserted that a need exists to better quantify complexity; to move beyond simplistic metrics like cell-type number, participants discussed new ways to quantify complexity, such as regulatory network density, modularity, and the diversity of developmental pathways. The colloquium centered on timing and environmental triggers as well. Why did complex multicellular life emerge only begin to emerge around a billion years ago, despite the presence of eukaryotes long before then? Ideas were discussed centered on possible environmental triggers, particularly oxygenation events in the history of Earth, and whether these were necessary to support metabolically expensive multicellular organisms. 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 ). This means that just as we can rely on principles of chemistry, physics, and geology to guide our understanding of early microbial life scenarios, we can use evolutionary theory to refine the focus on situations that are likely to be possible from the standpoint of evolutionary processes, remaining rigorous. 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 ). Even more ancient minerals, e.g., zircon grains from the Jack Hills region in Western Australia, crystallized from magma during the Hadean, with the oldest dating to approximately 4.4 Ga ( Wilde et al. 2001 ; Cavosie et al. 2007 ). These tiny but robust crystals provide evidence for magmatic differentiation and perhaps early steps toward the formation of rock like that in modern continental crust within 200 million years of planetary accretion. 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 ; Gamaleldian et al. 2024). This evidence for an early hydrosphere is critical, as liquid water is considered an essential prerequisite for life as we know it. A recent study based on divergence-A time analysis of gene duplication events prior to the last universal common ancestor (LUCA) estimated the origin of life to date to about this time (4.33-4.09 Ga) ( Moody et al. 2024 ). However, the precise timing of the LUCA's establishment remains unresolved, with several prior studies proposing a somewhat later emergence, closer to ~3.8 Ga, based on molecular clock analyses and geological evidence (e.g., Doolittle 2000 ; Glansdorff et al. 2008 ; Weiss et al. 2016 ). A potential source of these discrepancies is the existence of higher rates of evolution in the earliest stages of evolution, owing to high mutation rates prior to the establishment of accurate polymerases. 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 ) has significant implications for atmospheric composition. 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 ). Further, the segregation of iron into the core also left the residual mantle too oxidizing to release voluminous reduced gases like hydrogen and methane, instead favoring the more oxidized CO 2 and water and challenging the classic Miller-Urey model of prebiotic synthesis. 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 ). These events could have supported prebiotic organic chemistry crucial for many origin-of-life scenarios ( Wogan et al. 2023 ). Nitrogen cycling plays a particularly important role in these models, as nitriles such as hydrogen cyanide formed in a reducing atmosphere ( Wogan et al. 2023 ) could have contributed to prebiotic synthesis of nitrogen-containing nucleobases and ultimately RNA ( Benner et al. 2020 ) in a process more difficult in a more oxidized environment. The emerging model depicts an early atmosphere with high levels of CO 2 released from Earth's interior through volcanic processes, providing sufficient warming to sustain oceans or at least vast bodies of liquid surface water. A potential side effect of high atmospheric CO 2 would have been ocean pH values well below 7 ( Halevy and Bachan 2017 ), which is inconsistent with some prebiotic synthesis models that require borate rather than boric acid ( Kim and Benner 2021 ) but consistent with others that rely on pH gradients as an engine of organic synthesis. 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 ). Several transition metals are essential for core biosynthetic and energy-related processes, and the evolution of the metallome was likely strongly influenced by their abundance in ancient environments ( Anbar and Knoll 2002 ; Rico 2024). 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 ). This finding challenges the idea that metal use strictly followed environmental abundance and raises the possibility that Mo's chemical properties made it uniquely valuable, even in trace amounts (Rucker and Kacar, 2024). However, early ocean environments could have provided sufficient metal availability to support the early evolution of life (Evans 2024). 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 ) or highly reactive forms of reduced P produced by lightning strikes or delivered with meteorites ( 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 ). Cycles of drying and rewetting, such as in hot springs, could have driven polymerization reactions essential for life ( Song et al. 2024 ). Though early land was likely limited due to small quantities of buoyant continental crust, wet-dry cycles may have occurred on volcanic islands ( Bada and Korenaga 2018 ) or within the topography of impact craters ( Korenaga 2021 ). Frequent large impacts may have played diverse roles in the origin of life, including (i) delivery of organic precursor molecules, light elements, and water; (ii) formation of crater rims and central highlands rising above the ocean surface; (iii) creation of transient reducing atmospheres; (iv) triggering of prebiotic reactions and organic synthesis through impact energy; (v) stimulation of hydrothermal activity; and (6) delivery of reactive phosphorus and bioessential trace metals. 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 ). The Pilbara's “dome-and-keel” geology, i.e., rising granitoid domes and sinking greenstone keels, likely reflects early crustal processes unlike those of today and predating modern-style plate tectonics, which may have begun around 3.0 Ga ( Windley et al. 2021 ). Together, these features offer a window into the biosphere and geodynamics of early Earth. 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 ). Therefore, the Pilbara stromatolites provide a minimum age for the earliest bacteria. Additional indirect evidence for microbial life from the North Pole Dome comes from very light carbon isotope (δ 13 C) signatures from methane preserved in fluid inclusions in barite interbedded with a seafloor basalt unit in the Dresser Formation ( Ueno et al. 2006 ). Only biological methanogenesis is known to create 13 C depletions of this magnitude, and known methanogens are restricted to modern Archaea ( Garcia et al. 2021 ); hence, these highly negative δ 13 C signals are interpreted as a minimum age constraint for the appearance of Archaea. Importantly, these same rocks also bear sulfur isotope (δ 34 S) signatures suggestive of active bacterial sulfate reduction, hence yielding a possible minimum age for the advent of this biogeochemically important metabolism ( Shen et al. 2001 ), though not providing supporting evidence for the divergence of Archaea or Bacteria since this metabolism is found in both prokaryotic groups. Barite and signs of bacterial sulfate reduction in these rocks suggest local sulfate buildup, likely from abiotic sulfide oxidation processes such as UV-driven radiolysis in an O 2 -free atmosphere ( Westall and Xiao 2024 ). The well-preserved North Pole Dome succession and stable barite make these rocks ideal for further isotopic studies to explore early marine metabolisms and microbial life around 3.5 Ga. 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 ). However, their biological affinity was rigorously challenged based on their occurrence within hydrothermal veins cutting oceanic basalt and possible abiotic processes capable of generating similar structures through Fischer-Tropsch reactions ( Brasier et al. 2002 ). Nevertheless, given the multiple independent lines of evidence for microbial populations in these rocks and supporting evidence for the biogenicity of the Apex chert fossils ( Schopf et al. 2007 ), a biological origin remains plausible. 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 ). The most straightforward explanation for these seemingly contradictory lines of evidence is that these oases record the production of O 2 by oxygenic photosynthesizers in quantities insufficient to overcome massive aqueous and atmospheric sinks and consumption by decomposition, but adequate to drive local redox transformations and generate the geochemical signatures preserved in shallow seafloor sediments. Several recent molecular estimates independently indicate a middle to late Archean origin (~3.4-2.8 Ga) origin for oxygenic photosynthesis by cyanobacteria ( Fig. 1 ) (e.g., 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 quickly overwhelmed the O 2 sinks and led to the permanent presence of O 2 in the atmosphere, i.e., the start of the Great Oxidation Event (GOE) ( Fischer et al. 2016 ). There is something closer to a consensus, however, that the rise of O 2 at the GOE may have triggered global glaciation due to rapid drawdown of atmospheric methane. While a late, GOE-synchronous emergence of oxygenic photosynthesis handily dispenses with the long delay between the origin of oxygenic photosynthesis and the onset of the GOE suggested by the other model, an alternative hypothesis more compatible with both geological and molecular-clock data is that early oxygenic photosynthesizers were limited by habitat and possibly nutrient supply. The predicted result is that limited early O 2 production was mostly swamped by O 2 sinks. Early cyanobacteria were strictly benthic, small, and unicellular and formed thin mats at best ( Sánchez-Baracaldo et al. 2022 ). A world with minimal shallow illuminated margins, low bioavailable P, and abundant alternative electron donors (namely, Fe II) could have favored anoxygenic photosynthesis, with these factors diminishing as the time of the GOE approached. Thus, ecological expansion of cyanobacteria across the Archean-Proterozoic boundary could have been a major trigger for the GOE, linked to a combination of increased continental breadth and height ( Bindeman et al. 2018 ), the advent of thicker mats, and limitation of alternative electron donors ( Blank and Sánchez-Baracaldo 2010 ; Sánchez-Baracaldo et al. 2022 ). However, there are many other models focused on decreasing O 2 sinks at the GOE that must have also been important, and the isotopic data on the carbon cycle at this time are open to debate. 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 above a critical threshold (~10 −5 -10 −7 present atmospheric level) and the formation of an ozone layer ( Farquhar et al. 2000 ; Pavlov and Kasting 2002 ). The subsequent Lomagundi-Jatuli Event (~2.3-2.1 Ga), a prolonged interval of elevated δ 13 C values, reflects temporary high burial rates of organic carbon and possibly a transient “oxygen overshoot” ( Lyons et al. 2014 ). Geochemical proxies from this time, such as bedded sulfate evaporites and redox-sensitive isotopes, point to increased oxygenation of the atmosphere and oceans. 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 and the resulting reduction of greenhouse warming. However, this may have been a dynamic, oscillatory process, associated with multiple additional glaciations during the Paleoproterozoic (Bekker 2021) (see Fig. 1 below). Intriguingly, a recent study proposes that the Lomagundi-Jatuli Event represents a major evolutionary shift from fermentation-based microbial ecosystems to those dominated by cyanobacteria performing oxygenic photosynthesis (Prave 2024). This microbial transition would have fundamentally altered surface redox conditions and paved the way for later biological complexity. However, following this oxygen overshoot, both primary productivity and pO 2 appear to have declined sharply around 2.05-1.85 Ga, as suggested by mass-independent oxygen isotopes and redox-sensitive proxy data ( Crockford et al. 2018 ; Hodgskiss et al. 2023 . This downturn coincided with the formation of the supercontinent Nuna and the onset of a billion-year interval of environmental and evolutionary stasis marked by low atmospheric oxygen, widespread anoxic oceans, and nutrient limitation ( Lyons et al. 2014 ; Lyons et al. 2021 ; Anbar and Knoll 2002 ). FIG 1 Estimates for the evolving oxygen (O 2 ) content of the early atmosphere from 4 billion years ago to the present reported in terms of partial pressure of O 2 (left y axis) and partial pressure relative to the present atmospheric level (PAL) (reproduced from (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 ). Environmental change remained a central driver of biological innovation, beginning with the biological production of O 2 and followed by its eventual rise around 2.3-2.4 Ga ( Lyons et al. 2024 ). This oxygenation opened new aerobic niches, first in surface waters and eventually in the deeper ocean. Early O 2 -utilizing life may have harnessed the high energy yield of aerobic respiration while mitigating oxidative stress using enzymes originally evolved to manage reactive oxygen species generated through abiotic processes ( 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 ) (see Fig. 2 below). This evolving redox landscape impacted the availability of essential nutrients, most notably P, N, and bioessential trace metals that are and were woven deeply into enzymatic function as cofactors such as the role of nickel in methanogenesis (Konhauser 2015) and vanadium and iron in nitrogen fixation (Cuevas et al. 2024). Moreover, the eventual rise of biological methane production as a result of metal availability provided an opportunity for additional pathways to climatic warming, as well as cooling as methane concentrations waned under the later rise of atmospheric oxygen. Beyond the first steps of microbial innovation, researchers increasingly think in terms of how and when particular metabolisms flourished to the point of shaping Earth surface environments on a planetary scale ( 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 ). Well-known examples include Valeria lophostriata, Tappania plana, and Satka favosa ( Porter and Riedman 2023 ), though their exact placement within the eukaryotic tree remains uncertain, let alone whether they represent stem or crown group eukaryotes ( Adam et al. 2017 ). While fossils dating to ~1.65 Ga were long considered the earliest unambiguous evidence for eukaryotes, new reports push this back to at least ~1.75 Ga, with fossil diversity observed comparable to that seen later in the Proterozoic ( Porter and Riedman 2023 ). These findings suggest that total group eukaryotes originated well before 1.75 Ga. 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 ). Recent studies question the idea that rising oxygen alone enabled complexity, as modern O 2 levels postdate the molecular dating of the appearance of major multicellular groups ( Sperling et al. 2013 ). New ideas, like Simpson's hypothesis that increased ocean viscosity during Snowball Earth events promoted multicellularity, are being explored ( Simpson 2012 ). Exploring these models opens a new frontier for microbiologists, one that demands the integration of evolutionary theory, laboratory experiments, and geological data to uncover how complex life emerged. It is relatively easy to concoct hypotheses based on singular geological events, but these need ground truthing in terms of plausibility with respect to basic principles of evolutionary theory. For example, catastrophic events causing dramatic reductions in population size will dramatically alter the ability of natural selection to promote adaptive changes vs passively accumulated nonadaptive alterations. 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 ). More complex fossils appear in the Paleo-proterozoic, including the coiled carbonaceous compression Trypanin spiralis (~1.9 Ga) ( Walter et al. 1976 ; Han and Runnegar 1992 ), septate filaments like Siphonoseptum bombycinum (~1.6 Ga) ( Riedman et al. 2023 ), and bead-like chains of Horodyskia ( Horodyski 1982 ). While cellular detail is often lacking, their size and organization suggest multicellularity, possibly of eukaryotic origin. Well-preserved filamentous and lobate fossils like Rafatazmia chitrakootensis and Ramathallas lobatus may represent early red algae, though their age (~1.6 Ga) and phylogenetic placement remain under debate ( Bengtson et al. 2017 ). Some other reported macroscopic fossils might instead be abiotic structures or fortuitously shaped microbial mats ( Zhu et al. 2016 ; Albani et al. 2010 ). Additional forms, some with differentiated cells, appear by the end of the Mesoproterozoic. Bangiomorpha pubescens (~1,050 Ma) shows differentiated holdfasts and gametangia, resembling basal red algae ( Butterfield 2000 ). Aimonema (~1,030 Ma) consists of non-septate, anastomosing filaments similar to fungal mycelia ( Butterfield 2015 ). Proterocladus (~1,000 Ma) possessed branched, septate filaments interpreted as siphonocladalian green algae ( Tang et al. 2020 ), while Bicellum brasieri (~1,000 Ma) consists of tightly packed isodiametric cells inside a layer of sausage shaped cells and have been compared to multicellular stages in holozoans ( Strother et al. 2021 ). Later fossils like Archaeochaeta guncho (~900 Ma) ( Maloney et al. 2021 ) and Tappania (~800 Ma) ( Butterfield 2005 ) further expand the known diversity of pre-Ediacaran multicellular eukaryotes. 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 , and the diverse, widespread Ediacara Biota appear, some reaching meter-scale sizes. Though initially thought to be part of a single clade, the Ediacara Biota is now recognized as a polyphyletic grouping of taxa ( Xiao and Laflamme 2009 ); many are interpreted as stem animals, but some have been interpreted as giant protists ( Seilacher et al. 2003 ) or even colonies of bacteria ( Grazhdankin and Gerdes 2007 ). The earliest biomineralizing animals emerged by ~545 Ma, with dozens more animal lineages acquiring mineralized skeletons in the ensuing 20 million years. Thus, the Ediacaran witnessed a major flourishing of macroscopic life. 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 , evolved rarely, and once evolved was never lost, and it is these organisms that transformed the post-Cryogenian world. If such multicellularity arises as a consequence of an increase in size of simple multicellular organisms ( Bonner 2004 ), then it seems that the question is not what favored the evolution of multicellularity per se in the Ediacaran, but rather what favored the evolution of large size in lineages that had already evolved simple multicellularity. 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 may have limited complexity. However, recent evidence shows no major stepwise oxygen increase during the Ediacaran, but rather a dynamic redox landscape stabilizing only in the mid-Paleozoic ( Krause et al. 2022 ; Dahl et al. 2010 ). More recent hypotheses propose that large size evolved because low oxygen thresholds were finally crossed and that pulsed oxygenation events supported intermittent radiation of macroscopic life ( Sperling et al. 2015 ). Recent work in the Ratcliff laboratory on model “snowflake yeast” further supports the idea that rising oxygen may explain the timing of the evolution of increased multicellular size, with low to intermediate oxygen levels selectively favoring very small multicellular groups while modern levels favor size expansion ( Bozdag et al. 2021 ). However, some evidence suggests earlier intervals may have experienced even higher O 2 levels (e.g., Krause et al. 2022 ; Karhu and Holland 1996 ), raising the question of why complex multicellularity did not evolve at those times. 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 ). Other ideas highlight the potential influence of Cryogenian glaciations, where cold, nutrient-limited conditions may have favored large motile eukaryotes ( Crockett et al. 2024 ). It is also worth considering possible “null models”: perhaps the rise of complexity simply reflects increasing diversity and evolutionary turnover after the Cryogenian glaciations ( Tang et al. 2024 ). Alternatively, this transition may reflect the transformational effects of a single unusual clade, the animals. Animals had a profound influence on their environment as mobile heterotrophs with major effects on both nutrient cycling and available ecological niches and could have helped drive the subsequent evolution of complex multicellularity in other clades. If true, we are left with the difficult task of explaining a singularity in the history of life: why a single clade originated and diversified when it did. 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 ). Incorporating principles from microbial ecology into evolutionary studies will also be essential for building a more complete picture of life's early development. 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 ). By the very nature of the origin-of-life problem, it cannot be restricted to the domain of biology but rather is inherently multidisciplinary. Given that the origin of life field focuses on the transition from inanimate matter to living organisms, identification of geophysical and geochemical conditions that could support the origin of life is of paramount importance. If the nature of the cradles of life is understood, this will be a gigantic step towards the solution of the origin-of-life problem as a whole. We know little about the events that led to the emergence of the first organisms, and yet our knowledge should not be underestimated either as we can be confident about a number of crucial constraints. Thus, from the side of geology, it is clear that at the origin of life, there was little or no continental crust, and in terms of geochemistry, there was no oxygen, and accordingly no ozone layer, so that any surface areas were subject to harsh UV light. This potentially limited the type of habitats where life could evolve. Further, the emergence of life was undoubtedly constrained by the availability of chemical elements, ions, and small molecules. Assuming that the chemical composition of living organisms is the heritage of the primordial geochemistry, the likely conditions and habitats for the origin of life can be further constrained ( Mulkidjanian et al. 2012 ). For example, life is based on phosphate, the concentration of which in both the primordial and modern ocean was and remains very low. Similarly, the intracellular concentration of potassium is much higher than the concentration of sodium, reversing the ratio of these ions in the ocean. All life is built from about 20 chemical elements and about 1,000 organic compounds, of which at least the most common ones must have been initially produced abiotically. Theoretical and experimental models of the origin of life should account for these biogeochemical constraints. 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 below). The semi-permeable phospholipid membranes surrounding cells enable the formation of ion gradients that are converted into the energy of chemical bonds and selective transport of chemicals. Such membrane-bound compartments appear to be essential for life. The origin-of-life problem is often cast in terms of “metabolism first vs replication first.” However, this dilemma appears to be moot beyond the very earliest steps of abiogenesis because nucleic acids required for replication could not become available at high concentration and purity without a proto-metabolic network supplying nucleotides. Such a network of reactions operating within compartments, likely lipid vesicles, appears to be an essential prerequisite for the origin of cells ( Babajanyan et al. 2023 ) as discussed in Sec. III F. 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 ). Such compartments are capable of both concentrating various molecules and supporting ion gradients “for free.” The framework of cellularity based upon compartmentalization, metabolism, and energy was a key consideration of the colloquium (Sec. III B). Another direction starts from the premise that cells were preceded in evolution by vesicles bounded by simple lipid membranes. Experiments with such vesicles performed, in particular, by Szostak and colleagues, have demonstrated impressive capability of such vesicles to support various chemical reactions including limited templated RNA synthesis ( Schrum et al. 2010 ; Chen et al. 2004 ; Mansy et al. 2008 ). These are by no means the only possibilities. For example, another proposed scenario involves terrestrial geothermal fields ( 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 ). However, evolutionary reconstructions confidently map many other genes to the last Universal common ancestor (LUCA) of all modern life forms, leading to the striking conclusion that, with about 3,000 genes, the LUCA was a fully fledged cell of about the same complexity as modern bacteria and archaea ( Moody et al. 2024 ). Furthermore, the LUCA has been inferred to have been the host to a broad diversity of viruses ( Krupovic et al. 2023 ). Indeed, the colloquium found understanding of the nature of coevolution of viruses and their host cells (for which the taxonomy of prokaryotic viruses is crucial) to be an essential aspect of the study of early microbial evolution (Secs. III D and III F). The LUCA with its accompanying viromes could not possibly demarcate the first cells in existence but rather represents an advanced stage of evolution that must have been preceded by a complicated history of molecular innovations on the path from simple chemicals to cellular complexity. The existence of the LUCA has been dated at least 4 billion years ago ( Mahendrarajah et al. 2023 ), indicating that the origin of life and this crucial first stage of its evolution all occurred within several hundred million years, at best, focusing work on the origin-of-life problem to a relatively rapid process compared to some previous hypotheses. 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 ). Analysis of families of paralogous proteins shows that all the major protein folds had already evolved before the time of the LUCA. Furthermore, substantial evolutionary diversification of proteins apparently occurred before the formation of the modern translation system in which the specificity of amino-acid incorporation into polypeptides is determined by dedicated proteins, aminoacyl-tRNA synthetases ( Aravind et al. 2002 ). Thus, an advanced, high-fidelity translation system based primarily on RNA must have existed at the pre-LUCA stage of evolution ( Aravind et al. 2002 ). This inference supports the concept of a primordial RNA world in which RNA molecules performed both informational and catalytic functions ( Pressman et al. 2015 ; Szilagyi et al. 2020), or at least an RNA-peptide world in which abiogenic peptides but not modern-type nucleic acid-encoded proteins might have been important. The RNA world scenario is further supported by the central role of RNA catalysis in the peptide-bond formation in modern ribosomes ( Nissen et al. 2000 ), the reconstruction of the primordial peptidyltransferase ribozyme (Agmon 2017; Bose et al. 2022 ), and by increasing experimental demonstrations of the catalytic versatility of ribozymes ( Wilson and Lilley 2021 ). Crucially, the RNA world can resolve the inherent chicken and egg paradox of the origin of life: all reactions occurring in modern cells require versatile and efficient protein enzymes, but the formation of these proteins depends on the translation machinery, which itself includes many essential proteins ( Wolf and Koonin 2007 ). The evolution of translation and the genetic system for coding and heritability were topics addressed in depth during the colloquium (Secs. III E and III F). 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 ). The idea of compartmentalization or membrane formation envisions that an enclosed structure allowed the localization of prebiotic self-replicating RNAs to carry catalytic reactions and would have allowed for the ability to generate energy gradients that then evolved into to more complex and higher energy producing metabolisms ( Lane and Martin 2012 ). While these first compartments may have been within mineral or rock structures, there eventually was the emergence of primitive lipid membranes that evolved into the membrane structures we observe today ( Jordan et al. 2019 ). Thus, the formation of the cell membrane is one of the key evolutionary landmarks in our attempts to understand the origins of life and the biology of the LUCA. However, the characteristics of that initial membrane structure and how it evolved into the modern-day membranes are still unclear. It has even been suggested that alternative modes of compartmentalization, such as coacervates or polyester microdroplets, could be relevant at some intermediate point before modern membranes evolved ( 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 ). The majority of cellular membranes observed in nature are maintained as lipid bilayers with similar polar headgroups. Membrane fluidity and structure is often regulated through modifications such as unsaturations and through the incorporation of accessory lipids such as cardiolipins, sterols, and hopanoids (Saenz et al. 2015; Harayama and Riezman 2018 ; Subczynski et al. 2017 ). Although the basic functions and structures of cell membranes are well conserved, it is well known that microbial life today exhibits two distinct membrane chemical compositions ( Fig. 4 ) ( Koga and Morii 2007 ). Bacteria, like eukaryotes, harbor fatty-acid-based alkyl chains ester linked to glycerol-3-phosphate (G-3-P). 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 ) (Pearson 2014; 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 ) and the occurrence of these synthesis pathways varies between domains. Fatty acid and G-3-P synthesis, for example, are not observed in many archaea, but isoprenoid biosynthesis is required in all domains of life ( Lombard and Moreira 2011 ). This pattern reflects the significance of isoprenoids as building blocks for other important cellular lipids such as quinones and sterols in addition to forming the chemical backbone of archaeal membranes. From a functional standpoint, the occurrence of isoprenoid-based ether-linked lipids in archaeal membranes has been attributed to the harsh environments that extremophilic archaea inhabit. In particular, ether linkages render these lipids resistant to cleavage under conditions in which ester linkages are more readily degraded such as high temperatures in combination with alkaline or acidic pH ( Koga 2012 ; Tourte et al. 2022 ). However, all mesophilic archaea also possess isoprenoid-based ether-linked membranes while extremophilic bacteria harbor fatty-acid-based ester-linked membranes indicating that the divergence of membrane chemical composition between archaea and bacteria is not solely a response to environmental factors but also a fundamental separation between these two domains of life, potentially the result of historical contingency along their evolutionary trajectories. 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 ). Over the past decade, genomic approaches have been undertaken to better understand the evolutionary history of membrane lipid synthesis ( Sahonero-Canavesi et al. 2022 ; Villanueva et al. 2017 ; Hoshino and Gaucher 2018 ). Attempts to identify ancestral pathways in lipid metabolism have suggested that minimal isoprenoid synthesis was most likely present in the LUCA ( Moody et al. 2024 ). In addition, extensive lipid analyses of archaeal and bacterial species in cultures as well as lipidomic studies of environmental samples have revealed the existence of different membrane lipid components in both bacteria and archaea that bridge the lipid divide ( Weijers et al. 2006 ; Villanueva et al. 2021 ). For example, in bacteria fatty-acid-based membrane lipids that are ether rather than ester linked have been identified in thermophilic bacteria such as Thermotoga species (Damste et al. 2007) and soil dwelling Acidobacteria ( Halamka et al. 2023 ). A handful of studies have also identified a small amount of free fatty-acid production in different archaea, although the functional role of these lipids remains unclear ( Hamerly et al. 2015 ). Taken together, these data have led to the idea that the pre-LUCA and/or the LUCA possessed mixed membranes, i.e., a mixture of isoprenoid and fatty-acid-based lipids that can be ester linked or ether linked to either isomer of glycerol phosphate ( Lombard et al. 2012 ). It was then in the bifurcation of bacteria and archaea that the split between membrane structure that we observe today may have occurred ( 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 ) and possibly even 4.1 billion years ago ( Bell et al. 2015 ). Recent phylogenetic analyses suggest that life may have arisen even earlier, perhaps by 4.2 billion years ago ( Moody et al. 2024 ). This indicates a successful transition from prebiotic chemistry to metabolism as we know it by this time, as well as the emergence of cellular organization and genetic inheritance. 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 ) 1.8-2.7 billion years ago ( Vosseberg et al. 2024 ). In theory, we should be able to predict the characteristics of the last universal common ancestor using phylogenetics; genes found in both bacteria and archaea likely trace back to the LUCA. However, gene duplications, horizontal gene transfer, and gene losses complicate the picture. Strikingly different predictions of the proteome of the LUCA have been obtained by groups working with different datasets and using different algorithms ( Crapitto et al. 2022 ). Despite this difficulty, it is clear that the LUCA was a complex organism, with a genome encoding on the order of 2,500 proteins ( Moody et al. 2024 ). A lipid bilayer membrane enclosed a nucleic acid genome, ribosomes, and a metabolic network capable of converting environmental resources to cellular components. The LUCA most likely fixed carbon using the reductive acetyl-CoA pathway (the Wood-Ljungdahl pathway) present in both modern bacteria and archaea and harnessed energy from proton transport across the membrane using ATP synthase ( Moody et al. 2024 ; Sousa et al. 2013 ; Mahendrarajah et al. 2023 ). Given the complexity of the LUCA, it is clear that it was preceded by simpler cells. Thus, the origin of cellularity occurred earlier than phylogenetic analyses can easily reach. 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 ; Zhaxy-bayeva and Gogarten 2004). Vestiges of other life forms that used a genetic system similar to that in extant life may linger in modern genomes due to horizontal gene transfer before the LUCA; indeed, it has been suggested that signs of this horizontal gene transfer linger in the structure of the genetic code itself ( Vetsigian et al. 2006 ). However, other genetic systems are conceivable; chemists have explored the possibility of different types of genetic systems that used different backbones and different nucleotides ( Hud et al. 2013 ; Hoshika et al. 2019 ). Any traces of life forms that evolved a different, entirely incompatible type of genetic system have been forever unknown, but we should not assume that they never existed. 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 ) or in deep-sea or surface hydrothermal systems driven by redox gradients ( Baross and Hoffman 1985 ; Martin et al. 2008 ) (roughly corresponding to replication-first and metabolism-first approaches, respectively). The warm little pond scenario dates back to Darwin, Oparin, and Haldane and gained traction with the famous Miller-Urey experiment ( Miller 1953 ), which showed that amino acids could be produced when electricity was passed through an atmosphere (incorrectly) believed to be similar to that on the early Earth. The discovery of deep-sea hydrothermal vents in 1977 raised a different possibility ( Baross and Hoffman 1985 ; Martin et al. 2008 ). Hydrothermal systems offer the possibility of early compartmentalization in the pores of vent walls, chemical disequilibrium between H 2 -containing vent fluids and the CO 2 -rich ancient ocean driving both the production of organics and energy availability ( Catling and Zahnle 2020 ), a steady supply of small inorganic molecules for abiotic synthesis of small organic molecules, catalytic minerals in vent walls, temperature gradients between hydrothermal fluids and the surrounding ocean water, and, in some locations, proton gradients between alkaline vent fluids and the more acidic ocean. The debate over whether life emerged in warm little ponds or hydrothermal vents is critical because the sources of organic compounds, catalysts, amphiphiles, and energy differ entirely between these two sites. The warm little pond scenario has had considerable momentum because it was the only conceivable hypothesis before 1977, but the hydrothermal vent scenario has become more widely accepted in recent years ( Weiss et al. 2016 ; Martin et al. 2014 ). Decades of microbiological studies have produced information relevant to this debate. The reductive acetyl CoA pathway, possibly the oldest carbon fixation pathway on Earth and present in both bacteria and archaea, relies on transition-metal catalysts to reduce CO 2 to acetyl CoA using electrons donated from H 2 . H 2 is abundant in hydrothermal-vent systems and CO 2 would have been available in seawater. In contrast, straightforward connections between chemical processes at the surface of the Earth and biology are lacking. 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 ). The vast majority of these amino acids were not incorporated into genetically encoded proteins. Further, in a complex mixture of organic compounds, most components will be present at low concentrations, limiting the rates of second-order reactions that might produce more complex compounds. In contrast, the simple inorganic feedstocks at hydrothermal vents could have enabled buildup of a sparse matrix of metabolites directly downstream of catalyzed reactions present at higher concentrations ( 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 ). With caveats in mind, the following discussion will be cast in the context of a hydrothermal-vent origin of life. 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 ), including some components of ATP synthase and electron transport chains that allow organisms to capture energy from electrochemical gradients across membranes. The nature of the LUCA's membrane is unclear, however, because bacteria and archaea synthesize phospholipids via evolutionarily unrelated pathways. Bacteria synthesize phospholipids by attaching fatty-acid side chains to glycerol 3-phosphate through ester linkages, while Archaea attach isoprenoid chains to glycerol 1-phosphate through ether linkages. This “lipid divide” poses a difficult problem regarding the nature of phospholipid membranes in the LUCA. It is possible that the earliest cells made both types of phospholipids. If this idea is correct, enzymes for synthesizing ester-based phospholipids were lost in the archaeal lineage, while enzymes for synthesizing ether-based phospholipids were lost in the bacterial lineage. Alternatively, the LUCA and, by extension, pre-LUCA cells, might have had a different type of membrane and the two solutions for synthesizing phospholipids might have arisen after the split into the bacterial and archaeal lineages ( 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 and H 2 (generated in situ by decomposition of formate) after incubation for 2-3 days under simulated hydrothermal conditions (175 °C in stainless steel vessels using the mineral montmorillonite as a catalyst). Yields of longer-chain amphiphiles are low however ( McCollom et al. 1999 ). Possibly sustained production over long periods of time allowed amphiphiles to accumulate to levels sufficient to form vesicles inside pores in vent walls. 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 ). Above a critical concentration, amphiphiles spontaneously assemble into vesicles bound by lipid bilayers. Mixtures of C 10 -C 15 fatty acids and alkanols form vesicles in seawater; vesicle formation is optimal at 70 °C and at alkaline pH, conditions available within hydrothermal vents ( Jordan et al. 2019 ). Together, these studies suggest the possibility that amphiphiles produced abiotically within hydrothermal vents could have formed vesicles that encapsulated the first cells under known conditions. The invention of enzymatic machinery for synthesizing ester and ether lipids could have allowed the first bacteria and archaea, respectively, to later “escape” from the confines of these environments ( 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 ) or whether the first genetically encoded enzymes “invented” reactions and pathways that bear little resemblance to primordial geochemistry ( Lazcano and Miller 1999 ; Pross 2004 ; Orgel 2004 ). A major challenge for the genetics-first hypothesis is to explain how the ribonucleotide building blocks for macromolecular RNAs could have been generated in the absence of a proto-metabolic network. Synthetic chemists have endeavored to meet that challenge by identifying mechanisms for synthesis of ribose and nucleobases ( Yadav et al. 2020 ). However, the conditions required are often prebiotically implausible, requiring high concentrations of reactants and/or addition of reagents in specific orders along with wet-dry cycles that could occur at the surface of the Earth but not in hydrothermal vents. Further, conversion of ribose and nucleobases to nucleotides has been elusive. 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 ). This argument is less than ironclad though, as there may be many ways of synthesizing biologically relevant compounds that are not relevant to either life today or the first forms of life. The metabolism-first hypothesis is appealing because it allows for the possibility of gradual changes that conserve the underlying structure of prebiotic pathways while improving efficiency. An analogy would be the widening of a footpath to a dirt road and finally to a superhighway, improving flux along a pre-existing route. Nevertheless, intellectual appeal is not a reason to accept a hypothesis. The challenge for the metabolism-first hypothesis is to explain how the simple molecules available on the early Earth were elaborated into a network comprising thousands of metabolites. E. coli has more than 3,700 metabolites ( Sajed et al. 2016 ). The metabolic networks of the earliest cells were undoubtedly simpler, but likely still contained hundreds of metabolites in order to perform the tasks we now see as core biochemistry. A recent computational study provides an interesting perspective on the metabolism-first hypothesis ( Goldford et al. 2017 ). The stoichiometries of biochemical reactions in the KEGG database were used to guide a network expansion algorithm that built up organic compounds from prebiotically plausible seed compounds (CO 2, H 2 S, NH 3 , N 2 , H 2 , formate, and acetate). The products of each iteration were added to the set of reactants for the next iteration. Fourteen iterations led to a network of 260 compounds. Critically, though, many reactions in the predicted network are thermodynamically uphill and therefore unlikely to have occurred at hydrothermal vents without sophisticated energy metabolism. Adding pantetheine, a simpler version of coenzyme A, to the seed set and allowing it to substitute for CoA in reactions that involve thioesters and also allowing primitive electron donors/acceptors rather than NAD(P) and FAD to catalyze redox reactions resulted in a broader network of thermodynamically accessible reactions reaching 814 compounds. Notably, addition of polyphosphate as a substitute for ATP instead of pantetheine did not support expansion of a thermodynamically accessible network, suggesting the possibility of a phosphate-independent primordial core of metabolism in which thioesters provided the driving force for endergonic reactions, an idea that harks back to de Duve's proposal of a thioester world (de Duve 1991). A subsequent computational study that began with a more complex seed set (19 small organic molecules, NH 4 +, N 2 , H 2 S, H 2 , CO 2 , phosphate, and metal ions) plus polyphosphate as a primordial phosphorylating agent further expanded the network to more than 4,000 compounds ( Goldford et al. 2024 ). Although many questions remain, particularly about the sources of primordial phosphorylating agents and critical organic cofactors, this work supports the plausibility of continuity between ancient geochemistry and extant biochemistry and the notion that simple building blocks present at hydrothermal vents could have been elaborated into the thousands of compounds in extant metabolic networks. e. Critical role of catalysts Conversion of CO 2 and H 2 to organic compounds is thermodynamically favorable under hydrothermal-vent conditions, with biomass actually downhill of these feedstocks ( Amend and Shock 1998 ; Shock and Schulte 1998 ). However, the availability of suitable reactants and thermodynamically favorable sequences of reactions are necessary but not sufficient for supporting a primordial proto-metabolic network. The rates of chemical reactions are determined by the activation energy barrier rather than the overall free-energy change. Many prebiotic chemical reactions would have been too slow to be useful in the absence of catalysts. For example, at pH 7 and 25°C the t 1/2 for hydration of fumarate is 730,000 years and the t 1/2 for decarboxylation of orotidine monophosphate is 78 million years ( Wolfenden and Snider 2001 ). Thus, the rate accelerations provided by catalysts were likely essential for the emergence of life. 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 ). Such pruning allows accumulation of higher concentrations of fewer compounds, simplifying reaction networks and enhancing the rates of second-order reactions that are proportional to the concentrations of reactants. Extant protein enzymes accelerate chemical reactions by up to 26 orders of magnitude ( Edwards et al. 2012 ). Such potent catalysis was surely not available on the early Earth. The earliest catalysts must have been minerals. Minerals such as greigite, magnetite, and awurite catalyze formation of formate, acetate, pyruvate, and methanol from CO 2 and H 2 at 100 °C ( Preiner et al. 2020 ). Fe 2+ catalyzes reactions in glycolysis under neutral and mildly acidic conditions and reactions in the pentose phosphate pathway under alkaline conditions ( Keller et al. 2016 ). Reactions in the reverse TCA cycle are catalyzed by Fe 0 , Zn 2+ , and Cr 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 ). Ever-better catalysts for individual steps could have been swapped into existing pathways or network without disturbing the function of the whole. Thus, the structure of an ancient proto-metabolic network might have been maintained over billions of years as flux was gradually improved by the discovery of more efficient catalysts. 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 ). The field of organocatalysis has been driven by synthetic organic chemists interested in practical applications rather than prebiotic chemistry and as such has primarily focused on reactions in organic solvents. However, numerous examples of organocatalysis in aqueous conditions (van der Helm et al. 2019) from recent efforts exploring more environmentally friendly methods provide proof of principle for small-molecule catalysis in prebiotic geochemical networks. Perhaps most relevant to prebiotic chemistry, acetate, and other small organic acids are effective general acid catalysts for a variety of reactions and amino acids can catalyze aldol condensation reactions ( 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 ). Molecules of this size would have enhanced catalytic abilities even in the absence of genetic coding due to the availability of more functional groups that could act as general acids, general bases, or nucleophiles and a greater ability to bind metal ions, cofactors, and reactants. 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 of 0.12 min-1 ( Fig. 8 ). Catalysis is achieved by binding of the GCCU substrate via three base pairs to the tiny catalyst in an orientation that positions the 2’ hydroxyl of the substrate in close proximity to the carbonyl of the Phe-AMP ( 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 ), phosphoryl transfer ( Saran et al. 2005 ), peptide-bond formation ( Zhang and Cech 1997 ), aldol condensation ( Fusz et al. 2005 ), and methyl transfer ( Deng et al. 2022 ), but in most cases they are not actually particularly impressive catalysts. For example, a ribozyme that catalyzes an aldol reaction has a k cat of 2.2 × 10 −7 s −1 and a k cat / K M of 2.3 × 10 −4 M −1 s −1 ( Fusz et al. 2005 ). (For comparison, E. coli fructose bisphosphate aldolase has a kcat of 10 s −1 and a k cat / K M of 59,000 M −1 s −1 ( Zgiby et al. 2000 ). However, there is no reason to suppose that early RNA catalysts acted alone and every reason to suspect they had cofactors. Macromolecular RNAs are adept at binding small molecules. Naturally occurring riboswitches bind small molecules such as amino acids, S-adenosylmethionine, and B12 to regulate gene expression ( Kavita and Breaker 2023 ). Selective enhancement of ligands by exponential enrichment experiments have generated an enormous number of aptamers that bind amino acids, peptides, ions, and cofactors. RNA aptamers as small as 30-40 nucleotides can bind small-molecule ligands. An idea that deserves further investigation is the possibility that the catalytic efficiencies of early RNAs might have been enhanced by binding catalytic auxiliaries such as metal ions, amino acids, and peptides that coexisted in the prebiotic milieu in which life arose. This notion is supported by the in vitro evolution of a ribozyme that catalyzes a reduction reaction using NAD+ and Zn 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 ). Irrespective of whether we are considering obvious nucleotide or dinucleotide derivatives (NAD, FAD, and ATP) or less obvious relatives (folate, derived in a few steps from guanosine), the nature of the cofactors provides a key insight into the nature of early metabolism. Thus, it is likely that there were a variety of reactions (energy transfer, redox, 1-carbon additions, and radical transformations) that arose in an RNA-dominated world, which in turn suggests that metabolism was already quite complex in the RNA world. In contrast, there are few catalytic auxiliaries built from amino acids or peptides that are permeant in metabolism, and even when amino acids are involved in cofactor formation (such as PLP synthesis), there are a variety of pathways for its biosynthesis, suggesting a relatively late evolutionary origin ( Tanaka et al. 2005 ). This conversely suggests that uncoded peptides originally played only a small role in early evolution, and only upon the advent of the ribosome (its own sheer size further suggesting a complex RNA world) was takeover of catalysis by protein possible. 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 ). Additional enzymes arose over the ensuing billions of years by both gene duplication and divergence and de novo gene emergence from non-coding sequences. The expansion of metabolic capabilities allowed organisms to colonize a vast range of habitats on Earth. Extant organisms have complex and well-regulated metabolic networks that allow them to efficiently utilize the resources available within their environments. Remarkably, the core of extant metabolic networks was already well established by the LUCA ( 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 ). Evolutionary trees tracing modern life back to a common ancestor were introduced in the 19th century immediately upon the understanding of descent with modification, and in the late 20th century molecular phylogenetic analyses of universally conserved genes made the inference of the universal tree a practical, and then a statistical, project for the first time (Zuckerland and Pauling 1965; Woese and Fox 1977 ; Lake et al. 1984 ; Cox et al. 2008 ; Hug et al. 2016 Spang et al. 2022 ). A vast network of unicellular and multicellular organisms has evolved on Earth based on the foundation of compartmentalization, metabolism, and catalysis embodied in the LUCA (Secs. III A and III B). Throughout recorded history, humans have sought to understand how this natural world is structured and our place within it. The structure of the relationships between these diverse organisms encodes information about the earliest life and the nature of the process of its diversification. 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 ). Instead, microbial evolution has both vertical and horizontal components, with microbial lineages being remodeled by gene transfer, gene birth, and gene loss as they proceed through evolutionary time (Dolittle 2004; Williams et al. 2024 ). Genes involved in translation and some other core cellular processes are transferred over long evolutionary distances only rarely, and these have been used as a starting point to infer lineage trees that trace back into deep time ( 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 ). As a result, studies of prokaryotic phylogeny and metabolic evolution bear directly on ideas about the nature of the last universal common ancestor and the course of early evolution. The eukaryotes, our domain of life, evolved later, perhaps 2-2.5 billion years ago ( Betts et al. 2018 ; Mahendrarajah et al. 2023 ), long after the emergence of the main extant lineages of prokaryotes. The origin of eukaryotic cells involved endosymbiosis between an Asgard archaeon and at least an Alphaproteobacterium, the latter of which evolved to become mitochondria ( Martin et al. 2015 ; Roger et al. 2017 ; Eme et al. 2017 ). All of the deep branches and most of the modern branches of the tree are microbial, emphasizing the centrality of microbiology to studies of biodiversity and life's evolutionary history. 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 ). Methano-genesis is a hallmark metabolism of the Euryarchaeota and is also found in other archaeal lineages ( Garcia et al. 2022 ), but a key unanswered question is whether the common ancestor of the Archaea was already a methanogen. This is due to debate about the position of the DPANN Archaea, a diverse group of small-genome organisms that includes symbionts, parasites, and some free-living forms, within the archaeal tree. An archaeal root within, or near, the Euryarchaeota would provide stronger support for the antiquity of methanogenesis, but whether Euryarchaeota or DPANN are the deepest-branching archaeal lineage has proven difficult to resolve, due to the challenges of phylogenetic analysis in deep time (discussed in more detail below). On the bacterial side of the tree, some analyses have suggested that acetogenesis might indeed be the ancestral form of carbon fixation ( Xavier et al. 2021 ; Coleman et al. 2021 ), but there is also debate about whether phototrophy, or even oxygenic photosynthesis, might already have been underway at the time of the last bacterial common ancestor (Oliver 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 ), although the magnitude of the difference depends on the genes used to infer the tree. There is no strong reason to think that current metagenomic approaches might be biased towards the recovery of bacterial rather than archaeal genomes, and so it seems either that the Archaea have experienced a lower diversification rate than the Bacteria since the time of the LUCA, which would then represent the most striking case of the differing success of sister lineages yet described, or alternatively that major new lineages of Archaea await discovery, perhaps in the subsurface or other environments not yet comprehensively sampled by metagenomics. Another potential explanation is that the root of the universal tree might lie not between Archaea and Bacteria, but within the bacterial domain with an extremely long fast-evolving branch leading to the Archaeal domain ( Cavalier-Smith and Chao 2020 ; Lake et al. 2009 ; Guoy et al. 2015), although there is currently no compelling evidence for this possibility. Answering these questions will be important for understanding the Earth's biogeochemical evolution, for interpreting the geochemical record in terms of microbial metabolism, and for understanding how microbes have shaped, and coevolved with, the Earth system through geological time. 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 ; Koonin 2024) and the inference of lineage (species?) trees for some groups, such as the large DNA viruses ( Aylward et al. 2021 ). The consensus view is that viruses are likely at least as old as cells as indicated by the reconstruction of the virome of the LUCA based on the spread of different groups of viruses across the tree of life ( Koonin et al. 2020 ). This view is supported by analyses suggesting the LUCA and other early cells already encoded Cas proteins and potentially other anti-viral defense mechanisms ( Moody et al. 2024 ). However, direct evidence of the antiquity of viruses is lacking, and their evolutionary history in deep time is very poorly constrained. This is a key area that is ripe for future progress, and recently developed phylogenetic methods that use structural information (for example, that model evolution in a way that accounts for protein structure which evolves much slower than sequence) may be a promising avenue ( Garg and Hochberg 2024 ). The precise details of the emergence of various viral clades remain unclear, and their potential role in shaping cellular evolution also remains debated and was an active topic of discussion at the colloquium (see Secs. III D and III F below). At a minimum, viruses likely imposed an important selective force on cellular life throughout its history ( Koonin 2016 ), but there is also evidence of an important role for viruses as agents of evolutionary innovation, providing a vector for gene exchange between distantly related organisms (Irwin 2021) as exemplified by the exaptation of the retrovirus enveloped protein for the syncytin function in mammalian placenta ( Mi et al. 2000 ) and contributing genes (or even the entire nuclear compartment) alongside Archaea and Bacteria to the origin of eukaryotic cells ( Bell 2020 ; Takemura 2020 ). Evidence for the first two of these hypotheses is compelling, while a direct role in eukaryogenesis remains speculative at this time. However, direct evidence of the antiquity of viruses is lacking, and their evolutionary history in deep time is very poorly constrained. This is a key area that is ripe for future progress, and recently developed phylogenetic methods that use structural information (for example, that model evolution in a way that accounts for protein structure which evolves much slower than sequence) may be a promising avenue ( Garg and Hochberg 2024 ). The precise details of the emergence of various viral clades remain unclear, and their potential role in shaping cellular evolution also remains debated and was an active topic of discussion at the colloquium (see Secs. III D and III F below). At a minimum, viruses likely imposed an important selective force on cellular life throughout its history, but there is also evidence of an important role for viruses as agents of evolutionary innovation, providing a vector for gene exchange between distantly related organisms (Irwin 2021) as exemplified by the exaptation of the retrovirus enveloped protein for the syncytin function in mammalian placenta ( Mi et al. 2000 ) and contributing genes (or even the entire nuclear compartment) alongside Archaea and Bacteria to the origin of eukaryotic cells ( Bell 2020 ; Takemura 2020 ). Evidence for the first two of these hypotheses is compelling, while a direct role in eukaryogenesis remains speculative at this time. 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 ). However, there are grounds for caution: perhaps the most important lesson from the history of phylogenetics is that we will get things wrong, sometimes badly wrong, and we will have to update, revise, and sometimes entirely discard our previous hypotheses as new data and new analyses come to light. The reason is that to infer the tree of life, the method we use to draw the tree based on the data is critically important. The main tools are substitution models, statistical descriptions of how we think the process of evolution works, in terms of the rate at which genes and genomes change, and the relative frequency of different kinds of evolutionary events. The model also includes a representation of the tree, along which our imagined process(es) of evolution occur. If our model of evolution is too simple, then our analysis is at risk of favoring the wrong tree. This is what now appears to have happened in the case of the classic “three domain” tree, in which Archaea, Bacteria, and eukaryotes were each an independently branching, monophyletic domain of life: at the risk of too greatly simplifying a rather technical topic, simple models of evolution mistook convergent evolution in the common ancestors of Bacteria and eukaryotes as evidence that these two groups were closely related, artifactually pulling the eukaryotes outside the Archaea and giving the appearance of a distinct third domain of life (Tourasse and Guoy 1999; Williams et al. 2020 ; Spang et al. 2022 ). That is why future progress will depend on progress both in microbiology and in computational evolutionary biology; we will need new techniques to bring interesting new organisms into culture and render them amenable to experimentation, and we will also need better phylogenetic methods with which to draw more accurate evolutionary inferences from the data generated by sequencing and cultivation efforts. 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 ), is arguably too complex to have been assembled by chance alone, and so the earliest stages of life's history might have involved a process of assembly, accumulation, or enrichment of simpler components that were not yet capable of inheritance as we know it today. Several contributors drew on the more avant-garde end of current evolutionary theory in proposing new ways to think about these earliest systems, suggesting, for example, that symbioses between early genetic elements and metabolic compartments might have preceded the emergence of organisms as we understand them today ( Babajanyan et al. 2023 ) (see Sec. III F for further discussion of this scenario). Once genetics was underway, there was some discussion of how the antecedents of viruses might have interacted with early organisms and promoted the evolution of the first cells, and how multi-level selection ( Okasha 2006 ), on individual genes, emergent genomes, and cells, would have constrained early evolution. 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 ) or perhaps the concept of constructive neutral evolution ( Stoltzfus 1999 ; Brunet and Doolittle 2018 ) might help us understand the course of early evolution prior to and during the origin of cells. For example, the concatenation of individual replicating molecules into multi-gene genomes would have introduced clonal interference for the first time and reduced the efficacy of selection on individual genes. Were these negative gene-level effects overcome by selective benefits at the level of the whole genome (or organism) or were they fixed as slightly deleterious mutations in a marginal population genetic environment? Fully working out the theoretical basis of some of these ideas and designing ways to test them with experiments, perhaps using the tools of synthetic biology and ancestral sequence reconstruction ( Kacar et al. 2017 ), are clear priorities for the next stage of applying evolutionary biology to the origin of life. D. Coevolution of infectious/parasitic relationships in early life 1. Viral classification After considering the status of prokaryotic phylogenetic research defining the relationships between microbial species, the colloquium's focus turned toward analysis of the coevolution of infectious/parasitic relationships. Viruses are ubiquitous molecular parasites that infect all domains of life. They comprise a diverse assortment of lineages with distinct genome architectures and nucleic acid composition, i.e., DNA or RNA, and as such appear to have multiple independent evolutionary origins. Since the early 1970s, viruses have been classified using the Baltimore classification system, which organizes viral diversity according to genome architecture and the manner in which viral mRNAs are produced during infection ( Baltimore 1971 ). In recent years, however, progress has been made to produce a so-called megataxonomy of viruses based on analysis of hallmark proteins that are present in large subsets of viral taxa. This has led to major changes in viral classification that reflect our evolving knowledge of viral evolution ( Koonin et al. 2020 ). As one example, while Baltimore group I contains all double-stranded DNA viruses, it is now recognized that many of these viruses have distinct capsids and processing enzymes with independent evolutionary origins ( Koonin et al. 2020 ) stemming from separate cellular “escapes” or replacement of viral genes with derived cellular genes. As a consequence, double-stranded DNA viruses are now divided into multiple distinct higher-order groups, referred to as realms in the new megataxonomy, which is reflected in current classifications in the International Committee on the Taxonomy of Viruses, while all RNA viruses are subsumed under a single realm with presumed common ancestry. 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 ), leading to a model in which viruses emerge through co-option of host machinery ( 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 ). In the early stages of viral emergence after capsid recruitment, replication modules could be potentially acquired multiple times independently and/or swapped with existing viral groups, leading to a chimeric re-assortment of morphogenetic and replication modules and leading to the adaptive radiations of new lineages. Even in extant viral lineages, swapping of morphogenetic or replication modules remains commonplace. The result is a deep, difficult to read palimpsest of modular genome evolution that has gone on for billions of years. 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 (also known as giant viruses), where genomes can reach up to 2.7 Mbp, but a variety of other viral lineages also contain lineages with genomes more than 100 kbp, including herpesviruses, baculoviruses, tailed phages, and mirusviruses. Over the course of their evolution, members of these viral lineages have often acquired genes from their hosts, in some cases leading to an unexpected complement of “cell-like” genes in viral genomes. For example, tailed bacteriophages (class Caudoviricetes) often encode a wide range of auxiliary metabolic genes (AMGs) that manipulate the physiology of their hosts during infection ( Warwick-Dugdale et al. 2019 ; Tian et al. 2024 ). Some of the best-studied examples of AMGs are photosynthesis genes encoded by phages that infect marine cyanobacteria; in these cases, it has been shown that these viral genes can markedly influence host physiology during infection ( Thompson et al. 2011 ). A wide range of other AMGs involved in carbon, nitrogen, and sulfur metabolism has also been identified, revealing a vast array of viral-encoded physiological capabilities ( Tian et al. 2024 ). In animal viruses, several lineages been shown to convergently acquire components of the vertebrate immune system (so-called virokines and viroceptors) that they use to subvert the host defenses during infection (Schonrich et al. 2017; Johnston and McFadden 2003 ). The diversity of viral-encoded cellular functions is most impressive in giant viruses of the phylum Nucleocytoviricota. In genomes of this phylum, a wide range of functions has been identified, including TCA cycle and glycolysis components, cytoskeleton proteins (actin, myosin, and kinesin), nutrient transporters, histones, and various components of the translation system (Moniruzzman et al. 2023; Moniruzzaman et al. 2020 ). Conceivably, the broadly varying functional repertoires of host-derived genes in viruses reflect different pressures experienced by viruses inside infected cells ( 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 ). For the largest viral genomes in the Nucleocytoviricota, most evolutionary reconstructions support the hypothesis of genome accretion in which viruses in this lineage acquired these genes from cellular lineages over a stepwise process over time (Moniruzzman et al. 2023; Iyer et al. 2006 ). According to this view, even the largest giant viruses emerged from smaller ancestral viruses ( Yutin et al. 2014 ). Robust phylogenetic assessments remain difficult due to the long timescales involved, and in some cases an ancient viral origin of some eukaryotic genes has been proposed ( Da Cunha et al. 2022 ; Guglielmini et al. 2019 ). Regardless of the initial origin, the presence of viral genes predicted to be involved in diverse cellular functions demonstrates that viruses are capable of evolving to a startling level of complexity. 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 ). Traditionally, most endogenous viruses in eukaryotes were thought to derive from RNA viruses, but recent work has highlighted a broad diversity of DNA viruses that can be found in the genomes of most eukaryotic lineages. Even endogenous giant viruses greater than 1 Mbp in length have been found in a wide range of eukaryotic lineages, including green and brown algae, fungi, amoeba, and several protist lineages ( Moniruzzaman et al. 2020 ; Zhao et al. 2023 ; Sarre et al. 2024 ; Delaroque and Boland 2008 ). This is an emerging field of research, and more work is needed to assess the impact of endogenous DNA viruses on host evolution throughout the diversification of cellular life. 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 ). These virophages (phylum Preplasmaviricota) can integrate into eukaryotic genomes, re-activate upon infection of a giant virus, and subsequently parasitize the replication machinery of the giant virus. This often leads to a reduction in the burst size of the giant virus, which may lead to decreased incidence at the population level (Koslova et al. 2024). This parasitic interaction has only been demonstrated in a handful of preplasmaviruses, but their high prevalence in diverse eukaryotic genomes hints at a much broader occurrence of tripartite host-virus-virophage interactions in the biosphere ( Bellas et al. 2023 ). Indeed, putative virophages associated with entomopoxviruses have recently been found, broadening this phenomenon to insects ( Barth et al. 2024 ). These groundbreaking findings have highlighted the importance of understanding viral diversity for advancing our knowledge of eukaryotic genome evolution, the evolution of antiviral strategies, and even the evolution of complexity. 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 ) and the long-term consequences of this, as well as its potential involvement in the evolution of complexity, will be an important future direction. 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 ). This uniformity is also reflected in the central dogma that says that the sequence-encoded information flows from nucleic acids (usually form DNA to RNA) to proteins ( Crick 1958 ). Here we consider how the study of molecular evolution can shed light on the early evolution of the translation machinery, even though it is a feature shared between all cellular organisms alive today. 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 or last universal common ancestor. However, another possibility for traits to be found in Bacteria, Archaea, and Eukaryotes is genetic transfer between the domains that occurred sometime after the organismal LUCA. At first sight, the latter might appear far-fetched. However, components of the translation machinery have been identified as having been transferred between bacterial phyla ( Brochier et al. 2000 ) and even between archaea and bacteria ( Wolf et al. 1999 ; Ibba et al. 1997 ). This observed transfer of ribosomal proteins and aminoacyl tRNA synthases (aaRSs) might be related to transfer of resistance against naturally occurring antibiotics ( Brochier et al. 2000 ; Andam and Gogarten 2011 ). While genetic exchange between archaea and bacteria has undoubtedly occurred frequently, phylogenetic reconstruction of aaRS histories does reliably separate the archaeal and bacterial homologs into separate groups, except for particular aaRSs transferred between the domains. These transfers are revealed as an archaeal aaRS grouping within bacterial homologs or bacterial sequences grouping within archaeal homologs ( 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 ). This observation gives some confidence that molecular phylogenies reflect organismal evolution ( Pace et al. 2012 ); however, the flip side of the observation that gene transfer mainly occurs between close relatives is that organisms not participating in genetic exchange appear more divergent ( Gogarten et al. 2002 ). In addition, highways of gene sharing connect unrelated organisms ( Beiko et al. 2005 ) and individual gene trees, especially in the presence of faster evolving lineages, cannot reliably resolve evolutionary events that occurred during the early evolution of life ( Lapierre et al. 2014 ). The transfer of genetic information between lineages and the fusion of different lineages (as in eukaryogenesis and endosymbiosis) results in a reticulated organismal phylogeny that complicates the reconstruction of early evolution; however, it also provides information on relative timing (the donor had to exist before the recipient) and the presence of a transferred gene can create taxonomically useful markers (see, e.g., 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 ). Alas, components of complex machineries were found to be transferred at least occasionally ( Brochier et al. 2000 ; Yap et al. 1999 ), and some important components, such as the ribosomal RNAs, are so conserved they allow for within-gene recombination following transfer ( Gogarten et al. 2002 ; Yap et al. 1999 ). Furthermore, complete complex molecular machines are found to have been transferred between divergent organisms on at least rare occasions. For example, the photosynthetic machinery was transferred between divergent bacteria on several occasions ( Igarashi et al. 2001 ) and the multi-subunit archaeal rotary ATPase/ATPsynthase was transferred from an archaeal donor to the ancestor of the Deinococcaceae ( 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 ). The hope is that this tree of cells, provided that gene transfer and fast evolving lineages are taken into consideration, is approximated by the dominant phylogenetic signal that can be extracted from multiple gene phylogenies (see Puigbo et al. 2013 and Puigbo et al. 2009, in particular the sections on the LUCA and citations therein for examples). 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 contrast, the ribosome that is the most recent common ancestor of all extant ribosomes (the cenancestral ribosome) already had incorporated many ribosomal proteins, revealing a long pre-LUCA evolutionary history connecting the ancestral ribozyme to the cenancestral ribosome. 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 ) followed a pathway of constructive neutral evolution ( Brunet and Doolittle 2018 ; Lukes et al. 2011) and not a takeover by ribosomal proteins. 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 ). The same argument applies to ancient duplicated genes such as the catalytic and non-catalytic subunits of ATPsynthases and the class I and II aaRSs that evolved through gene duplications ( Fournier and Gogarten 2007 ). Ancient proteins that were established under a genetic code with fewer genetically encoded amino acids should have fewer of the later added amino acids in conserved positions than proteins that evolved after the genetic code had been expanded to 20 genetically encoded amino acids. Using this reasoning, Fournier and Gogarten ( Fournier and Gogarten 2010 ) inferred that the LUCA, placed using ancient gene duplications 1 (Zhaxybayeva et al. 2005; Gogarten et al. 1989 ; Iwabe et al. 1989 ; Brown and Doolittle 1995 ; Gribaldo and Cammarano 1998 ), had an inferred amino-acid composition of its ribosomal proteins that does not correspond to the amino-acid usage observed in hyperthermophiles, an observation in agreement with the reconstruction of ancestral ribosomal RNAs ( Boussau et al. 2008 ; Galtier et al. 1999 ) and the evolution of reverse gyrase ( 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 ) that diverged before the organismal LUCA. The analysis of individual aaRS phylogenies reveals that at the time of the LUCA other lineages existed that harbored deeper branching and more divergent aaRSs and that these divergent aaRSs were later acquired by lineages with extant representatives through horizontal gene transfer (Fournier et al. 2015; Fournier et al. 2009 ). These genes are vestiges of organismal lineages present at the time of the LUCA that have since gone extinct. 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 ), with subsequent amendments ( 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 ). In contrast, the ancestor of valRS and ileRS had both isoleucine and valine in distinct conserved positions, revealing that at the time valRSs and ileRSs diverged, both amino acids were already part of the genetic code ( Fournier et al. 2011 ), implying that another tRNA charging system was in place at this time that has since been entirely replaced. Today's aaRS proteins represent a surprisingly late takeover of a more ancient, likely ribozyme-based, system. 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 ) is divided into phases beginning with the peptidyl transfer center core to which other units were added over time. This accretion of additional RNA stem loops is illustrated in the more recent evolution of evolution of the eukaryotic cytoplasmic ribosome from the archaeal ancestor ( Petrov et al. 2015 ) and in some archaeal lineages ( Bowman et al. 2020 ). The peptidyl transferase center, exclusively formed by RNA ( Noller et al. 1992 ) is considered a vestige of an ancient pre-LUCA ribozyme that synthesized peptides that were not genetically encoded. The peptidyl transferase center shows rotational symmetry, suggesting a dimer or heterodimer as the first peptide bond forming ribozyme ( Agmon et al. 2005 ). Peptide-bond formation by reconstructed protoribosomes using activated amino acids as substrates was shown experimentally ( Kawabata et al. 2022 ; Bose et al. 2022 ; Xu and Wang 2021 ) and reviewed in Agmon 2024 . However, the early evolution of the activation of amino acids remains shrouded in mystery. As discussed in the previous section, present-day aaRSs represent a takeover by sophisticated “modern” proteins. While attempts to reconstruct ancestral aaRSs proteins are ongoing (see, e.g., Patra et al. 2024 ; Douglas et al. 2023 ), the pre-protein precursors of today's aaRS have not left clear traces in modern biology; thus the specifics of these pre-protein world aaRSs remains difficult to explore. 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 ). Viruses and other mobile genetic elements (MGEs), by contrast, realize virtually all possible interconversions of nucleic acids in their replication-expression cycles, with single-stranded (ss) or double-stranded (ds) RNA or DNA molecules serving as genomes (that is, the form of nucleic acids incorporated into transmissible virions) ( Koonin et al. 2020 ) ( Fig. 11 ). This diversity of the replication-expression strategies among viruses might provide important clues to the evolution of the genetic system along the path from the first cells to the LUCA. 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 ). A central role of RNA, and in particular, ribozyme catalysis (likely with inorganic and organic cofactors), in the early cellular evolution appears to be a logical necessity, the only realistic solution to the inherent chicken and egg paradox of the origin of cells ( Wolf and Koonin 2007 ). Indeed, for the genetic system to function, ensuring replication and expression of genetic information with sufficient fidelity, sophisticated molecular machinery consisting of numerous proteins is essential, but the production and, in the first place, emergence and diversification of these proteins required an efficient, accurate genetic system. The RNA world concept offers a plausible potential solution whereby in primordial protocells, RNA molecules functioned both as genetic material and as catalysts. The RNA world must have started with abiogenic, spontaneously formed oligoribonucleotides (potentially in the context of some form of proto-metabolism), some of which might possess ribozyme activity that could enhance reactions occurring within protocells and promote their growth and division. Amplification and evolutionary fixation of such ribozymes in protocell populations would give those populations a selective advantage ( Babajanyan et al. 2023 ; Joyce and Szostak 2018 ). FIG 12 Coevolution of primordial replicators and reproducers (Babajanyan et al. 2023 ) . In this model of two-level selection, protocells containing genetic elements (blue) compete for common resources (black shapes) with protocells lacking genetic elements (yellow). (more...) 3. Replication of genetic material in an early system A broad diversity of ribozyme activities has been reported ( Wilson and Lilley 2021 ), but a hard and still unresolved problem is the emergence of an efficient, processive ribozyme RNA replicase. Nevertheless, experimental evolution approaches are gradually progressing towards this goal, yielding increasingly efficient and compact replicase ribozymes ( Papastavrou et al. 2024 ; Tjhung et al. 2020 ). Interestingly, ribozymes are far more efficient as catalysts of ligation of oligonucleotides than as polymerases of mononucleotides ( Dhar et al. 2017 ; Briones et al. 2009 ). It therefore appears likely that complex ribozyme replicases initially evolved via non-templated and possibly also templated ligation of untemplated short oligonucleotides. 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 ). Although all RNA viruses and viroid-like agents depend, for their replication, on protein enzymes, encoded in either the viral or the host genome, their persistence and evolutionary success demonstrate the efficacy of very small RNAs as genetic material, and their replication and expression strategies might recapitulate those that operated in the RNA world and during the transition to modern cellular systems. 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 ). Protocells carrying replicating genetic elements (initially, in all likelihood, RNA molecules) can be usefully considered as symbiotic systems in which the reproducer is the host and the replicators are symbionts ( Babajanyan et al. 2023 ). From the onset of evolution, some of the symbiotic replicators would be mutualists benefiting the protocells, in particular, through ribozyme activities, whereas others would develop into parasites. The two-level selection would benefit efficiently replicating mutualist collectives, but the continuous emergence and presence of parasitic replicators in evolving protocells appear inevitable. 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 ) of the emergence of translation that was a prerequisite for the transition from the RNA world protocells to modern-type, primarily protein-based cells. Ribozymes with non-templated, probably non-specific peptide synthetase activity could have initially been selected for, due to the utility of the produced peptides. Such a ribozyme still functions in the peptidyl-transferase center of the large ribosome subunit in all modern cells ( Nissen et al. 2000 ; Hiller et al. 2011 ) and the ability of the minimal form of this ribozyme to catalyze peptide bond formation has been demonstrated experimentally ( Bose et al. 2022 ). The evolution of rRNA from this primordial peptide synthase appears to be well described by the “onion” model, which details accretion of RNA modules Bokov and Steinberg 2009 ; Petrov et al. 2014 ). Aminoacylation of oligonucleotides, a key reaction in translation, is readily catalyzed by small, simple ribozymes ( Turk et al. 2010 ), further supporting an RNA-based, perhaps, RNA-only translation as a stage in early evolution (Hlouchova 2024). Evolution of the modern translational machinery. There are additional indications that RNA played key roles in the early translation system, particularly in defining the specificity of amino-acid incorporation into proteins. The enzymes that are responsible for charging tRNAs with the cognate amino acids in the modern translation system, aminoacyl-tRNA synthetases, are universal across cellular life, are products of extensive evolution of protein families, and the same holds for translation factors ( Aravind et al. 2002 ; Leipe et al. 2002 ; Fer et al. 2025 ). Thus, considerable diversity of proteins must have evolved at the stage of evolution when the translation systems consisted (almost) exclusively of RNA, which may be considered the transitional stage from the RNA world to modern-type cell biology. For protein evolution to occur, the primitive, RNA-based translation system would need to have efficiency and fidelity comparable to those of the modern translation machinery with its large set of essential proteins. 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 ). One potentially attractive possibility is that RNA-based templated translation emerged concomitantly with ribozyme-catalyzed replication where the replicases initially polymerized trinucleotides rather than mononucleotides, that functioned similarly to experimentally studied trinucleotide ligase ribozymes ( 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 ). The synthesis of DNA on RNA templates catalyzed by RT that is an essential stage in the replication of such MGEs likely shares an origin with that of DNA genomes. Given the broad spread of MGEs with all types of genomes across the diversity of modern life forms, it appears likely that the primordial pool of replicators that likely inhabited protocells already contained many types of replicators ( Krupovic et al. 2019 ). One of these, the dsDNA genome, was selected to become the universal genetic material of cellular life forms. This selection was conceivably driven by the advantage of large genomes which, for the reason of chemical stability, could consist only of dsDNA and enabled coordinated replication and segregation of multiple genes, as well as the separation of the genetic material of the host from that of some of their RNA parasites ( 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 ). Three distinct, unrelated varieties of DNA polymerases (DNAPs) are responsible for the elongation of the genome replication in bacteria, archaea, and eukaryotes. In bacteria, the replicative polymerase (PolC) core has the polymerase-beta fold shared with a variety of non-replicative DNAPs. In most of the archaea, the key role in replication belongs to PolD, with the core double-psi beta barrel (DPBB), but in several groups of archaea, PolD is replaced by PolB, a DNAP with a core RNA recognition motif (RRM) domain. In eukaryotes, several DNAPs are involved in replication, all of them variants of PolB. Finally, in most of the viruses with large dsDNA genomes that encode their own DNAP, this role belongs to PolB. A potential clue to the earliest stages in the evolution of the replication apparatus is the homology between the core domains of PolD and those of the large subunits of DNA-directed RNA polymerases (RNAPs) that are universal in cellular life forms and encoded by many viruses as well ( Raia et al. 2019 ; Sauguet 2019 ). This homology suggests concomitant evolution of transcription and replication whereby RNAP and PolD evolved from a common ancestor that might have functioned as an RNA-dependent RNA polymerase (RdRP) and subsequently diversified into RNAP and DNAP ( 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 ) and the demonstration that mitochondria and plastids derive from ancient endosymbiotic bacteria, respectively related to Alphaproteobacteria (purple bacteria) and Cyanobacteria (“blue-green algae”) ( Schwartz and Dayhoff 1978 ; Gray and Doolittle 1982 ). Accordingly, a long-prevailing model arose in which eukaryotes were thought to derive from an unidentified proto-eukaryotic lineage sister to archaea that developed complex features, including endomembranes and the nucleus, prior to the acquisition of the mitochondrial ancestor by phagocytosis (Lopez-Garcia and Moreira 2023; Embley and Martin 2006 ; Gabaldon 2021; Cavalier-Smith 2002 ). Although some models evoked a symbiotic origin of eukaryotes directly from bacterial and archaeal ancestors, they were far from mainstream ( Embley and Martin 2006 ; Lopez-Garcia and Moreira 2015; Lopez-Garcia et al. 2017), even when phylogenomic analyses with more data and adapted sequence evolution models started to suggest that archaea and bacteria were the only two primary domains derived from the last universal common ancestor ( Williams et al. 2013 ). However, the relatively recent discovery, by metagenomic and enrichment culture approaches, of the Asgard archaea, which harbor many genes previously thought to be present only in eukaryotes and are their closest relatives in phylogenomic trees based on highly conserved genes, strongly supports scenarios that place the symbiosis of one Asgard-like archaeon and one or more bacteria at the origin of the eukaryotic cell (Lopez-Garcia and Moreira 2023; Gabaldon 2021; Lopez-Garcia and Moreira 2015; Eme et al. 2017 ; Lopez-Garcia and Moreira 2020; Imachi et al. 2020 ; Rodrigues-Oliveira et al. 2023 ; Zaremba-Niedzwiedzka et al. 2017 ) ( Fig. 14 ). In this view, based on the subset of the total genome content present now and at the origins of the domains of life, archaea and bacteria are primary domains (Williams et al. 2019) whereas eukaryotes form a third, composite domain of life, with the origin for the nuclear genome nested within the archaea. It is prudent to recognize that all such analyses are based on relatively small subsets of the total genome content present now and at the origins of this domain. 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 ). Asgard archaea were first identified from metagenome-assembled genomes (MAGs) in marine sediments, but are also present in microbial mats and other, mostly anoxic, environments ( Zaremba-Niedzwiedzka et al. 2017 ; Spang et al. 2015 ; Valentin-Alvarado et al. 2024 ). Asgard archaea encode more so-called eukaryotic signature proteins, i.e., proteins formerly thought to be exclusively eukaryotic, than all other sampled archaea ( Zaremba-Niedzwiedzka et al. 2017 ; Eme et al. 2023 ). Many of these proteins are putatively annotated as involved in membrane remodeling and trafficking, including GTPases, endosomal sorting complexes (ESCRT), and cytoskeletal proteins ( Hatano et al. 2022 ; Tran et al. 2024 ; Akil et al. 2021). The interplay of ESCRT and ubiquitin systems seems to have already been present in Asgard archaea ( Lu et al. 2024 ). Likewise, the presence of actin and related regulatory proteins (profilin and gelsolin) in Asgard archaea, some of which can complement the function of their homologs in eukaryotic systems, point to an Asgard origin of the eukaryotic cytoskeleton and its dynamics including manipulation of the membrane shape (Akil et al. 2021; Stairs and Ettema 2020 ). Intriguingly, a close Asgard archaeal homolog of tubulin has only been identified in Odinarchaeota (Akil et al. 2022). Based on their inferred metabolic potential, most Asgard archaea seem to be H + /electron donors or scavengers, which suggests that they live in syntrophy (metabolic symbiosis) with other members of the complex microbial communities they thrive in ( Liu et al. 2021 ; Spang et al. 2019 ; Lopez-Garcia and Moreira 2019). In agreement with this prediction, the first two cultured Asgard archaea, Candidatus Prometheoarchaeum syntrophicum and C a . Lokiarchaeum ossiferum, are anaerobic organisms growing in syntrophic co-culture with sulfate-reducing deltaproteobacteria, methanogenic archaea, or both and can manipulate membrane architecture in an astonishing way ( 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 ), it became progressively clear that mitochondria were already present in the last eukaryotic common ancestor (LECA) ( Embley and Hirt 1998 ; Gray 2012 ; Roger et al. 2017 ). The LECA is a cornerstone in the study of life's complexity, bridging the gap between prokaryotic simplicity and the vast diversity of eukaryotic organisms today. Mitochondria and some of their more highly derived forms (e.g., hydrogenosomes) are directly involved in modern eukaryotic cell bioenergetics. Accordingly, energy metabolism functions in eukaryotes mostly have a bacterial origin. In addition, eukaryotic membranes are bacteria-like; their phospholipids are primarily composed of a glycerol-3-phosphate moiety ester linked to fatty-acid lateral chains (Pereto et al. 2004; Lombard et al. 2012 ). By contrast, archaeal lipids use glycerol-1-phosphate ether linked to isoprenoid lateral chains. 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 ) This clade may be directly relevant in the context of eukaryogenesis as some Asgard archaea, including those that have been cultured, establish syntrophic interactions with representatives of the these taxa ( Imachi et al. 2020 ; Rodrigues-Oliveira et al. 2023 ). Several other eukaryotic metabolic pathways, such as the beta oxidation of fatty acids in mitochondria (Schluter et al. 2011) and the synthesis of sterols ( Hoshino and Gaucher 2021 ), also apparently have myxobacterial origin. 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 ). Symbiogenesis can account for the chimeric nature of eukaryotes ( Koonin 2015 ; McInerney et al. 2014), which overall have archaeal-like “informational” processes (e.g., replication, transcription, and translation) but bacterial-like bioenergetics membranes ( Jain et al. 1999 ) and “housekeeping” ( Lombard et al. 2012 ). Most current models of eukaryogenesis propose a two-partner symbiosis, mostly based on syntrophic interaction, involving an Asgard archaeon that hosts the alphaproteobacterial ancestor of mitochondria ( Fig. 15 ) (Lopez-Garcia and Moreira 2015; Spang et al. 2019 ; Imachi et al. 2020 ; Baum and Baum 2014 ; Sousa et al. 2016 ) . These models need to postulate an archaeal-to-bacterial-like membrane phospholipid transition. A few models propose more than one bacterial partner. Some of these propose transient symbioses of an Asgard archaeon with sulfate-reducing bacteria, such as the E3 model ( Imachi et al. 2020 ), or with diverse bacteria, such as the pre-mitochondrial symbiotic hypothesis, prior to the establishment of the mitochondrial symbiosis (Pittis and Gabaldon 2016; Gabaldon 2018). One model, the syntrophy hypothesis (Lopez-Garcia and Moreira 2020), proposes a tripartite metabolic symbiosis involving a sulfate-reducing deltaproteobacterial host, an endosymbiotic H 2 -producing Asgard archaeon (the future nucleus), and a facultatively aerobic, sulfide-oxidizing alphaproteobacterium coexisting in a realistic microbial ecology context. A bacterial host would easily explain the bacterial-like eukaryotic membrane. Alternatively, massive lateral gene flow from bacterial partners could have resulted in the replacement of the Asgard phospholipid biosynthesis pathways (Pittis and Gabaldon 2016; Bernabeu et al. 2025; Tobiasson et al. 2025). The ancestral eukaryotic virome, seemingly more related to bacterial than to archaeal viruses, would agree with the importance of an ancestral bacterial membrane during eukaryogenesis ( 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 ), mostly flagellated and heterotrophic. Much of this diversity remains undescribed, as suggested by environmental studies (Moreira and Lopez-Garcia 2002; Burki et al. 2021 ). Several eukaryotic lineages, such as animals, fungi, plants, and brown algae, developed complex multicellularity and/or acquired photosynthesis through the primary endosymbiosis of a cyanobacterium or secondary or tertiary endosymbiosis of their chloroplast-carrying protists ( Adl et al. 2019 ; Burki et al. 2020 ) (Sec. IV A, Fig. 14 ). Heterotrophic protists are generally phagotrophic, preying on bacteria or other protists ( Adl et al. 2019 ). Other heterotrophic eukaryotes such as fungi and oomycetes became osmotrophic, feeding from the absorption of extracellularly digested organic molecules; several of them further evolved into parasites ( Adl et al. 2019 ; Torruella et al. 2018 ). With their diversity of trophic modes, protists play crucial roles in ecosystem's networks and the carbon cycle ( Worden et al. 2015 ). Eukaryotic lineages are currently known to form several large supergroups, but their deep phylogenetic relationships remain unresolved ( Burki et al. 2020 ), as is the root of the eukaryotic tree ( Al Jewari and Baldauf 2023 ). These uncertainties can be explained by an inherent phylogenetic signal limitation due to a rapid diversification in a short time span, methodological artifacts linked, among others, to heterogeneous evolutionary rates, hidden paralogy, and horizontal gene transfer, as well as to patchy sampling across the eukaryotic diversity ( 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 ). The addition of this diversity has increasingly enlarged, reshaped, and consolidated eukaryotic supergroups. For instance, the long-supported Opisthokonta clade, comprising metazoans, fungi, and their unicellular relatives (e.g., choanoflagellates, ichthyosporeans, nucleariids, aphelids, and rozellids) ( Galindo et al. 2023 ; Ruiz-Trillo et al. 2023 ), was found to form a robust supergroup, the Amorphea, with classical amoeba (Amoebozoa) plus two groups of flagellated protists previously considered incertae sedis, the amoeboflagellated Breviatea and the biflagellated Apusomonadida ( Brown et al. 2013 ). Opisthokonta and Amoebozoa were previously thought to share a uniflagellated ancestor ( Cavalier-Smith 2002 ) and the root of the eukaryotic tree was inferred to lie between this Unikonta clade and the rest of the eukaryotes (Bikonta) ( Richards and Cavalier-Smith 2005 ). The Bikonta unified several lineages sharing a biflagellate ancestor, including Archaeplastida (glaucophytes, red algae, green algae, and plants) and SAR, composed of Stramenopiles (e.g., oomycetes, diatoms, and brown algae), Alveolata (e.g., dinoflagellates and ciliates), and Rhizaria (e.g., radiolarians and cercozoans) ( Richards and Cavalier-Smith 2005 ). However, the nested position of the apusomonads within unikonts rejected the idea of a uniflagellated ancestor of the (former) Unikonta clade, and the new names Opimoda and Diphoda were proposed for those clades ( Derelle et al. 2015 ; Torruella et al. 2025 ). Archaeplastida and SAR, together with other lineages of unicellular protists, many photosynthetic (cryptophytes and haptophytes), and their allies (e.g., telonemids, centrohelids, and Palpitomonas), often cluster in a large supergroup called Diaphoretickes ( Torruella et al. 2025 ; Heiss et al. 2018 ; Lax et al. 2018 ) whereas another group of poorly characterized protists called CRuMs (collodictyonids, rigifilids, and Mantamonas) appears to branch as a sister to the Amorphea ( Burki et al. 2020 ; Torruella et al. 2025 ; Brown et al. 2018 ). To complete the global picture, Excavata, once thought to be a major eukaryotic supergroup characterized by the shared phenotypic feature of a ventral feeding groove, lacks molecular phylogenetic support and is no longer considered monophyletic, being split into Discoba (e.g., euglenids and jakobids), Malawimonadida, and Metamonada (e.g., trichomonads, Trimastix, and 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. Some examples are ancyromonads ( Atkins et al. 2000 ) or malawimonads ( Heiss et al. 2018 ), so far not clearly related to any eukaryotic supergroup, although they might represent the deepest-branching clade of the Opimoda ( Torruella et al. 2025 ). Other newly described phylum-level lineages such as Provora ( Tikhonenkov et al. 2022 ) [including Ancoracysta (Janouskovec et al. 2017)] Hemimastigophora ( Lax et al. 2018 ), and Meteora ( Galindo et al. 2022 ; Eglit et al. 2024 ) also branch deeply in the eukaryotic tree. Recent studies suggest that these branches belong within the Diaphoretickes and might constitute a newly identified supergroup ( Torruella et al. 2025 ; Eglit et al. 2024 ). In summary, although new genetic information continues to clarify the relationships among the major eukaryotic groups, the precise relation of major supergroups remains controversial and the affinities of many groups remain uncertain. 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 ). Although these analyses were certainly limited by the biased taxon sampling of the time ( del Campo et al. 2014 ), that conclusion has been subsequently confirmed with the addition of more genomes and/or transcriptomes for an increasingly diverse set of lineages ( Vosseberg et al. 2021 ; Newman et al. 2019 ). Gene content comparisons together with the observed distribution of cellular traits across the eToL suggest that the LECA possessed essentially all typical traits of extant eukaryotes ( Richards et al. 2024 ; Koonin 2010 ). These include nuclear and chromosomal structure (the compartment itself, nuclear pores, nucleolus, lamina, histone-based nucleosomes, and linear chromosomes with telomeres) as well as an elaborate actin and tubulin-based cytoskeleton allowing for phagocytosis and cell shape remodeling, flagella, diverse membranous organelles in addition to the alphaproteobacteria-derived mitochondria (Golgi apparatus, lysosomes, and peroxisomes), intron-containing genes and the spliceosome, the ubiquitin system, and RNA interference. In addition, the ubiquity of mitosis and meiosis suggests that the LECA already had a complex life cycle potentially alternating sexual and asexual phases. The LECA likely was a facultative anaerobe ( Newman et al. 2019 ) bearing two flagella ( Derelle et al. 2015 ; Torruella et al. 2025 ; Suzuki-Tellier et al. 2024 ). Some authors suggest that the LECA had an excavate phenotype, with a ventral groove hosting the posterior flagellum ( Torruella et al. 2025 ; Suzuki-Tellier et al. 2024 ). This inference seems further supported by a recent study attempting to root the eukaryotic tree with alphaproteobacteria ( Williamson et al. 2025 ). However, an amoeboflagellate architecture cannot be ruled out given that some excavates (e.g., Naegleria) exhibit this phenotype. 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) and the deepest evolutionary relationships among known eukaryotic supergroups remain highly uncertain. This uncertainty hampers reliable reconstruction of ancestral states. In addition, the position of the root of the eukaryotic tree remains controversial, with the current most popular hypothesis placing it among the excavate taxa and/or between Opimoda and Diphoda ( Williamson et al. 2025 ) ( Al Jewari and Baldauf 2023 ). In the following, we highlight specific drawbacks and limitations to current research and suggest potential strategies and developments for improvement. Some of these points have been summarized in a recent consensus paper ( 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 ). Cultured species for some of them have become available only recently ( 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/ ) may provide an initial database of proteomes derived from genomes or transcriptomes to reach that goal. Unfortunately, however, eukaryotic genome data are very often contaminated with foreign genes of prokaryotic or eukaryotic origin, derived from prey, endosymbionts, or coexisting organisms in culture. Therefore, curating these data is essential to prevent artifactual phylogenetic reconstruction. Improved curation methods from massive datasets would be extremely useful. 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 ) can be helpful. However, improvement of these approaches is clearly needed, not only to detect contaminants but, importantly, paralogs. Novel machine-learning approaches should facilitate or even replace manual curation steps, ideally, with even better accuracy. In addition, eliminating contaminants and improving gene/protein clustering and the detection of orthologs are essential. Here AI-derived tools taking into account structural conservation which decays much slower than sequence conservation should be useful. However, eukaryotic proteins often result from the fusion of different modules. Developing bioinformatic tools to reliably detect these discrete modules and avoid artificial clustering of proteins with different module arrangements (or, conversely, take advantage of that information to study gene/protein evolution) would be helpful. 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 ) is also needed to accurately reconstruct the eToL. Several complex models of sequence evolution have been developed within the Bayesian and maximum likelihood frameworks, but many of these are computationally intensive and therefore not practically applicable to large datasets. The development of (bio)informatics tools for ameliorating this problem is needed. 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 ) but also the impact on hosts, which so far is much less explored. In this regard, a good case study can be that of the evolution of primary, secondary, and higher-order photosynthetic endosymbioses in eukaryotes, which are not yet well understood or described. They are possibly easier to follow at the scale of the eToL and could help unravel symbiosis-derived mechanisms and patterns of evolution across the eukaryotic tree. 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 ) or reconciliation approaches ( Williams et al. 2024 ). However, these attempts are still partial and not fully conclusive such that work in this area is needed to disentangle the different the different hypotheses on the deep relationships among eukaryotic lineages. 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 ). They allow inference of gene origination, duplication, transfer, and loss in a maximum likelihood framework. However, the underlying steps are not always easy to understand and evaluate. Improving reconciliation approaches for clarity and reliability would be useful. Also, linking function to genes and collections of functionally annotated genes to specific traits would be required to achieve the goal of inferring functions and traits from genome/transcriptome data. Machine learning approaches might be useful in this direction. Combining this with dating approaches should help anchor specific innovations in natural history. So far, with the available data, molecular dating analyses suggest a rather late LECA ( Chernikova et al. 2011 ; Eme et al. 2014 ), which might imply a lost world of complex forms ( Brocks et al. 2023 ). Although the fossil record is extremely patchy, the use of high-resolution techniques combining microfossil morphology, ultrastructure, and chemistry at particularly unaltered sites (if the signals are at all preserved) may help establish minimal dates of emergence of specific traits, which could then be useful as calibration points for dating eToL nodes. 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 (archaeal viruses with A-form dsDNA), Duplodnaviria (dsDNA viruses encoding an HK97 capsid protein), Monodnaviria (ssDNA viruses encoding an HUH endonuclease), Riboviria (RNA viruses and retroviruses), Ribozyviria (small satellite RNAs), Singelaviria (DNA viruses with single vertical jelly roll proteins), and Varidnaviria (DNA viruses with double vertical jelly roll proteins) ( https://ictv.global/vmr ). Nonetheless, significant differences exist among viruses infecting bacteria, archaea, and eukaryotes. It is important to note that we currently have a limited knowledge of archaeal viruses, especially Asgard viruses. Certainly, sampling efforts here will help better define the composition of the LECA virome. 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 ) (see Fig. 16 ). With insights from their previous work showing viruses in the Varidnaviria and Duplodnaviria, and possibly Riboviria and Monodnaviria realms evolved at early stages of life predating the last universal common ancestor (LUCA) ( Krupovic et al. (2020) provide the most compelling evidence on the LECA virome, suggesting that it originated from a limited variety of bacterial viruses, rather than archaeal viruses. This evolutionary scenario could be linked to the bacterial origin of eukaryotic membrane lipid (glycerol-3-phosphate ester linked to fatty acids). This could be due to a bacterial replacement of membrane biogenesis due to horizontal gene transfer or a variety of endosymbiotic models with multiple nested endosymbioses such as the syntrophic eukaryogenesis model (Lopez-Garcia and Moreira 2020). These events could have resulted in the exclusion of archaeal viruses due to a lack of necesssary receptors on bacterial style outer membranes and the exclusion of the archaeal partner of the symbiosis from exposure to viral attack. 3. Bacterial origins of the eukaryotic virome Bacterial origins for the eukaryote-infecting viruses from Riboviria, Monodnaviria, Duplodnaviria, and Varidnaviria are elegantly summarized by Krupovic et al. ( Krupovic et al. 2023 ) and a few highlights of this synthesis are listed below. The deepest branch of the kingdom Orthornavirae includes leviviruses (phylum Lenarviricota) with an ancestral levivirus losing its capsid protein genes giving rise to capsidless eukaryotic replicators (classes Amabiliviricetes and Howeltoviricetes), Amabiliviricetes subsequently giving rise bacterial or archaeal ancestors, and Howeltoviricetes replicating in mitochondria. 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, phylum Cressdnaviricota) evolved from non-viral bacterial MGEs, namely, plasmids replicating via the rolling-circle mechanism. These plasmids provided the endonuclease and superfamily 3 helicase modules for initiation of replication, whereas the capsid proteins were likely acquired through with complementary DNA copies of ribovirus single jelly-roll capsid protein genes. Viruses in the phylum Preplasmiviricota, specifically polintoviruses (realm Varidnaviria), are likely descendants of tailless bacteriophages. Ancestral members of the phylum Nucleocytoviricota likely originated via recombination between a mirusviruslike duplodnavirus providing the replication module and a polintovirus providing the structural module. 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 ). Although Irwin et al. ( Irwin et al. 2022 ) found eukaryote-to-virus transfers were roughly twice as frequent as virus-to-eukaryote transfers, they highlighted that virus-to-eukaryote transfers were enriched in functions related to glycosylation and nuclear processes. In particular, the acquisition of viral-derived glycosyltransferases was correlated with several morphological transitions in eukaryotes and their diversification, e.g., algal cell wall elaboration (lipopolysaccharide and cellulose synthesis enzymes). 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 ), and some prokaryotes possess intracellular organelles like magnetosomes or carboxysomes ( Murat et al. 2010 ), there is still a large gap separating the subcellular complexity across the prokaryote/eukaryote divide. Consequently, the emergence of the intricate subcellular organization in eukaryotes and the origin of each individual organelle remain central questions in the field of eukaryogenesis ( 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 ). Most available reconstructions depict the LECA as a highly sophisticated unicellular eukaryote, comparable to extant free-living unicellular protists. The LECA possessed a nucleus, an already complex endomembrane system, mitochondria, peroxisomes, a cytoskeleton, and most of the eukaryotic-specific cellular machinery, such as the spliceosome and nucleosomes (see Fig. 17 ). This notion of a complex LECA is widely accepted in the community. In contrast, there are many different views and some important controversies surrounding the difficult question of how this complexity originated from the simpler organizations of the prokaryotic predecessors of the LECA. 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 ). In the endogenous case, the organelle emerges from within the cell, by duplication and repurposing of previously existing components. In the exogenous case, the organelle derives from an external organism that becomes integrated within a host cell, such as in the process of endosymbiosis. The idea that some intracellular organelles can originate through endosymbiosis of bacteria, popularized by Margulis in the 1960s (Lazcano 2017; Sagan 1967 ), provides an attractive mechanism for increasing cellular complexity, as it portrays one pre-existing system that ends up being a specialized subsystem within another cell, which readily becomes a more complex organism. However, of the organelles inferred to be present in the LECA, only the mitochondrion has received sufficient compelling evidence to support an endosymbiotic origin ( Gabaldón 2021 ). In this case, an exogenous origin from an alphaproteobacterial-related endosymbiont is supported by overwhelming evidence based on (i) the presence of a remnant organellar genome and protein-synthesis machinery and (ii) the semi-autonomous division of these organelles, i.e., all mitochondria derive from division of pre-existing mitochondria and cannot be formed 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 ), the nucleus ( López-García and Moreira 2006 ), or the peroxisome ( de Duve 2007 ), most of these ideas have been abandoned in favor of endogenous scenarios in the light of available molecular data ( Baum and Spang 2023 ). However, even in the context of an endogenous origin, the involvement of genes acquired from bacteria or of selective forces imposed by symbionts are often considered ( Raval et al. 2022 ; López-García and Moreira 2023 ). In this regard, an open question is whether, in addition to the mitochondrial ancestor, other prokaryotic symbionts might have played relevant roles during eukaryogenesis, not necessarily as domesticated endosymbionts that originate an organelle, but perhaps as gene donors or as ecological partners imposing selective pressures that favored the origin of a particular organelle. Finally, beyond prokaryotic partners, the potential involvement of viral interactions in the origin of some organelles such as the nucleus has gained increased attention ( 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 ). This idea was abandoned after it was realized that the molecular phylogeny results were artifactual, i.e., the result of long branch attraction, and that all extant eukaryotes had or previously had a mitochondrion ( Koonin 2010 ). Alternative scenarios were then proposed, the most popular of which was the hydrogen hypothesis, which suggested the engulfment of an alpha-proteobacterium by an archaeal host in a metabolic mutualism as the detonating event of eukaryogenesis ( Martin and Müller 1998 ), and the syntrophy hypothesis, which placed the mitochondrial endosymbiosis somewhat later, acquired by a host resulting from a previous endosymbiosis of an archeon into a delta-proteobacterium ( 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 ). However, there is no conclusive evidence that this was the case for all organelles. First, near-neutral processes could have also resulted in an increasing cellular complexity, especially if the ancestral eukaryotic populations had small population sizes or went through strong population bottlenecks ( Force et al. 2005 ). There is also the strong possibility of an initial symbiosis involving more than two partners, only one of which gave rise to a relict organelle genome but others of which could have contributed genetic material and molecular capabilities. Very few studies have aimed to test these ideas empirically. Analysis of branch lengths separating eukaryotic proteins from their prokaryotic ancestor in gene phylogenies suggest that proto-mitochondrial derived proteins were acquired later than eukaryotic genes stemming from (at least some) alternative prokaryotic groups, suggesting a late acquisition of mitochondria by a proto-eukaryote having a chimeric genome ( Pittis and Gabaldón 2016 ; Gabaldón 2018 ). However, similar analyses also suggest that following mitochondrial acquisition, the eukaryotic protein repertoire increased its complexity through extensive duplications ( Vosseberg et al. 2021 ), hinting at additional postmitochondrial complexification. 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 ). However, the supposed bioenergetic advantages provided by mitochondria have been broadly questioned ( Lane and Martin 2015 ; Lynch and Marinov 2016 ). In addition, that free-living organisms with complex subcellular organization can lose ATP-producing mitochondrial respiration ( Roger et al. 2017 ), or even mitochondria altogether ( Novák et al. 2023 ), suggests that the bioenergetic function of mitochondria is not a requirement for the maintenance of subcellular complexity. Finally, the discovery of Asgard archaea ( Spang et al. 2015 ), which contain homologs of some components of the eukaryotic cytoskeleton, and the observation that some of these organisms form long cellular protrusions have inspired many eukaryogenesis scenarios in which an Asgard host would have developed a complex cytoskeleton before the engulfment of mitochondria ( Vosseberg et al. 2024 ). Of note, scenarios involving an archaeal host implicitly involve a transition from archaeal-like cellular membrane to a bacterial-like one. Similarly, membrane transitions or a complete loss of one or more membrane systems is needed in any exogenous scenario involving the origin of a cellular organelle from an archaeal endosymbiont. How plausible these transitions are is an area of active research that could have implications beyond the field of eukaryogenesis, as the origin of this fundamental difference between archaeal and bacterial membranes is also relevant in questions relative to the origin and early evolution of life as considered in above in Sec. III B. 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 ), the chromatophore in Paulinella ( Gabr et al. 2020 ), the many endosymbionts in insects and unicellular eukaryotes ( McCutcheon 2016 ; Husnik et al. 2021 ), and the recently described nitroplast ( Coale et al. 2024 ) can be useful sources of information. Similarly, systems biology and manipulation of extant organisms to artificially create and study new symbioses can be useful ( Giger et al. 2024 ). The study of other endosymbioses can serve to assess potential paths for endosymbiont-to-organelle transitions and reveal common constraints and tendencies in these processes. In all these cases, however, we must keep in mind that the hosts of all these symbioses are modern, fully developed, eukaryotes and thus very different from the ancestors of the LECA. In this regard, deeper exploration of prokaryotic symbioses, particularly those involving Asgard archaea and bacteria, could provide us with groundbreaking discoveries of symbiotic systems more similar to those existing during eukaryogenesis. 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 ), but rather cases of organellar diversification or of emergence of a new organelle by co-opting or duplication of existing elements. 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 ) could be important processes to study to have a better understanding of what endogenous paths to new organelles could have existed. During the colloquium it was proposed that exhaustive examination of the subcellular structures of diverse eukaryotic lineages would reveal examples that are more recent, allowing study of organelle evolutionary pathways in greater detail. In this regard, advances in microscopy and imaging, and techniques such as subcellular fractionation or, more recently, localization of organelle proteins by isotope tagging ( Dunkley et al. 2004 ) offer promising avenues to characterize the subcellular localization of proteins. 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 ). It is plausible that more eukaryotic signature proteins will be discovered in the future, using new techniques that can detect very remote homology beyond the long-branch threshold such as Foldeek (van Kempen 2023). 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 ). In some cases, these non-animal proteins can even carry out what seem like extremely animal-specific functions when transferred into animal cells. We were recently involved in the discovery of the pluripotency-inducing transcription factors Sox and Pou in choanoflagellates ( Gao et al. 2024 ). Pluripotency is an animal-specific trait, so finding homologs of these proteins in single-cell relatives of animals was surprising enough. The Sox protein from choanoflagellates could even be used to generate stem cells in animals, meaning this protein already possessed all molecular features necessary to carry out its animal-specific function, long before animals ever evolved or gained the ability to produce pluripotent stem-cells. 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 ). This kind of “entrenchment” of useless additional components via constructive neutral evolution is expected to occur more often in eukaryotes, whose smaller effective population sizes make them less sensitive to the cost of otherwise useless complexity. This makes it difficult to tell if particular features of eukaryotic complexes are strictly necessary for their specific functions or whether they only appear so because of entrenchment. One example of this is the TRiC chaperone complex. This is a multimeric chaperone which exists in Archaea but in Eukaryotes consists of many more paralogs ( Archibald et al. 2001 ; Zhang et al. 2010 ). It has been theorized that the more complex eukaryotic architecture is necessary to fold eukaryotic actin, which the archaeal version cannot fold. However, this theory is difficult to test, because deletion of any one eukaryote-specific paralog now stops the complex from assembling altogether ( Liou and Willison 1997 ). Each eukaryotic paralog has “forgotten” how to assemble properly without its other paralogs through entrenchment, making it very difficult to test whether structural features of particular paralogs are what allow the eukaryotic complex to fold eukaryotic actin. 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 ). In archaea, this ring directly degrades RNA phosphorolytically. In humans and yeast the situation is much more complex. Here the core ring is made from six different paralogs and no longer has any RNAase activity. This activity is now outsourced to two peripheral RNAses, which bind to exosome and degrade RNA hydrolytically ( Kilchert et al. 2016 ). This arrangement is key for certain RNA cleavage reactions that are important for ribosome maturation in humans and yeast ( Fromm et al. 2017 ), and one might reasonably hypothesize that it has some connection to the complex maturation pathway of eukaryotic ribosomes as compared to that of Archaea and therefore likely evolved along the branch to the LECA. However, broader phylogenetic sampling reveals that this is false: plants have an exosome which is comprised of as many paralogs as that of humans, but its ring can still degrade RNA phosphorolytically without the aid of peripheral RNAs ( Sikorska et al. 2017 ). The peripheral RNAs are present in plant genomes, but apparently do not interact strongly with the exosome. Importantly, this only became apparent when this complex was purified directly from plants. 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 ). If the composition (and therefore plausibly function) of protein complexes varies rapidly across the eukaryotic tree of life, parsimonious inference about these qualities in the LECA becomes woefully difficult, unless sampling of extant diversity is much denser than it currently is. 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 ). However, for very many protein complexes, we simply have insufficient biochemical data to make good inferences about their exact assemblies and functions in the LECA or in the LECA's archaeal predecessors. 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 (through both mutations and deletions) is shown for a range of initial repeat densities ρ and LGT rates λ. For comparison, (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 ). To evaluate these hypotheses, it will be crucial to broaden the taxonomic scope of research model systems to encompass a greater diversity of organisms. Until now, much of the focus has been on a limited group of model species, which may not fully represent the diversity of sexual strategies. This narrow focus has likely hindered our ability to accurately infer the characteristics of the ancestral sexual eukaryote. 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 ). Such sequences may have been related to the spread of retrotransposons and group II introns in early eukaryotes, which are profoundly diverse and ancient in eukaryotes but rare in bacteria and archaea. 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 ). First, we can gain insights by identifying which mitotic processes were co-opted for use in meiosis, such as DNA repair through homologous recombination, which plays a central role in both processes. Second, we must investigate the selective forces that shaped the stepwise assembly of the full meiotic machinery, from its early origins to its current complexity. Understanding which modifications were advantageous at each stage of this evolution could shed light on how meiosis became a central feature of sexual reproduction. Finally, it is crucial to consider why certain forms of meiosis, or particular evolutionary pathways leading to meiosis, may have been less successful or even abandoned in some lineages ( Lenormand et al. 2016 ). By addressing these questions, we can begin to piece together the selective pressures and evolutionary innovations that led to the development of meiosis and the rise of eukaryotic life. 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 ). Traditionally mainly attributed to complex eukaryotic organisms ( Grosberg and Strathmann 2007 ), recent definitions of multicellularity diverge, encompassing terms such as groups of cells in physical contact, groups with cell-cell signaling, groups with cellular differentiation, groups with tissue-level organization, and groups with germ-soma differentiation. These discrepancies stem in part from recent discoveries in prokaryotic multicellularity, which challenge and expand classical definitions. To advance the study of multicellularity, the participants of the colloquium began with the recognition that it is essential to understand its distinguishing features: what exactly constitutes multicellularity? 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 ). “Multicellular groups” refer to collectives of cells that exhibit some degree of cooperation, while “multicellular individuals” are defined as multicellular entities within a population that can acquire adaptations as a unified group through natural selection. This requires reproduction of multicellular individuals and heritable variation in traits that affect fitness. Over evolutionary timescales, this can further lead to “multicellular organisms,” here defined as physiologically integrated entities with high within-group cooperation and limited within-group conflict that have gained novel traits and have become functionally and physiologically integrated. Of note, the concept of organismality (Queller and Strassmann) is still under debate; see Wilmsen and Kost 2025 for a recent account. The emergence of novel traits in multicellular organisms fundamentally depends on their evolution as multicellular individuals, which in turn requires the formation of multicellular groups. 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 ). Further complications are the quality of microscopic fossils and the difficulty in distinguishing multicellular prokaryotes from early multicellular eukaryotes. However, there is increasing evidence that multicellularity has evolved across domains, including archaea ( Rados et al. 2025 ; Tang et al. 2023 ) and bacteria ( Schwartzman et al. 2022 ; Tang et al. 2023 ). While simple bacterial multicellularity in the form of stromatolites dates back at least 3.4 billion years, eukaryotic multicellularity includes the more complex multicellular lineages, such as animals, plants, fungi, and algae. Complexity is defined here as the number of clonally derived cell types, but note that quantifying morphological complexity is challenging and that better metrics beyond simply counting cell types are needed ( 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 ), gene regulation ( Bingham and Ratcliff 2024 ; Olivetta et al. 2024 ), or resource acquisition methods ( Petroff et al. 2010 ; Petroff et al. 2011; Persat et al. 2015 ; Simpson 2021 ; Crockett et al. 2024 ). In this context, it is important to consider how drastic environmental changes have affected organisms with different forms of biological organization. For example, it has been suggested that an increase in viscosity during Snowball Earth may have selected for eukaryotic multicellularity ( Simpson 2021 ; Halling et al. 2024 ). As glaciations reduced temperatures and resource availability, selective pressures would favor smaller sizes in (multicellular) bacteria, which were constrained by diffusion physics, but larger sizes in motile heterotrophs that can overcome this limitation via manipulation of large-scale fluid flows, such as eukaryotes ( 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 ), with some exhibiting developmental patterns similar to eukaryotes ( Futo et al. 2021 ), others exhibit transient or facultative multicellularity, triggered by biotic or abiotic environmental signals ( Tang et al. 2023 ; Mizuno et al. 2022 ). In addition, multi-species biofilms are now considered multicellular entities that share collective metabolism and emergent group traits ( Geesink et al. 2024 ; Huelsmann et al. 2024 ). The challenge of heredity of these traits can be partially overcome by a special meta-population structure, known as ecological scaffolding ( 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 ). This suggests that most multicellular genes predate metazoans and were repurposed from those of their unicellular ancestors. While gene innovation at the origin of metazoans was limited—only 25 “core” animal-specific genes have been identified—novel regulatory mechanisms likely enabled combinatorial, spatiotemporal gene expression patterns that contributed to diverse cell types, as seen in metazoan chromatin regulation. This pattern extends to other “complex” multicellular organisms, where cell differentiation is driven not by new genes but by the selective expression/repression of genes in different tissues. Thus, the complexification at the multicellular (higher) level is accompanied by a de-complexification at the cellular (lower) level ( 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 ), and this was a major colloquium topic (see Secs. V D and V E). These models complement empirical approaches by exploring unlikely scenarios and enabling abstraction, which allows comparing different systems. 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 ). Comparisons of parallel occurrences of eukaryotic multicellularity, such as animals, brown algae, red algae, and plants, provide valuable insights into the origin of multicellular complexity ( Lotharukpong et al. 2024 ; Bourdareau et al. 2021 ; Coelho and Cock 2020 ). The colloquium participants highlighted recent progress made using genomes and transcriptomes of brown algae to identify the genetic multicellularity toolkit of the algae for the control of growth, development, and differentiation (see Sec. V G). 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 ). It must be taken into account, however, that it is also shaped by genetic drift and other neutral processes ( Bingham and Ratcliff 2024 ; Lynch et al. 2023 ). As organisms increase in size and complexity, effective population sizes necessarily decrease, reducing the efficiency of natural selection and allowing the proliferation of nonadaptive genomic elements such as introns and mobile genetic elements (Lynch et al. 2024). The colloquium participants considered that this shift is accompanied by a 10- to 100-fold increase in metabolic costs, with multicellular organisms exhibiting reduced efficiency in converting resources into biomass compared to unicellular species ( Lynch 2024 ) (see Sec. VI). The drift-barrier hypothesis explains how these constraints lead to the passive accumulation of genetic and structural complexity via constructive neutral evolution, rather than being solely driven by adaptive processes ( 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 ). Moreover, the properties of these groups depend not only on morphology ( Jacobeen et al. 2018 ; Day et al. 2022 ), but also on the mechanics of their cellular components. For example, the mechanical co-option of cell-level traits previously uninvolved in multicellular traits affects three-dimensional morphogenesis ( Milinkovitch et al. 2013 ; LeGoff and Lecuit 2016). 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 ; Bonner 2001; Tarnita et al. 2013 ), by either the coming together of unrelated cells to form an aggregative multicellular organism or the staying together of sister cells to form a clonal multicellular organism. The second framework distinguishes between simple and complex multicellularity ( Knoll 2011 ), where simple multicellular organisms tend to be small, lack spatial organization, lack division of labor, and internal resource transport is done solely through diffusion. On the other hand, complex multicellular organisms possess large size, spatial organization, and division of labor among cell types, which function in reproductive specialization ( Buss 2016 ; Michod 1999; Michod and Herron 2006 ) or often play an important role in supplementing resource diffusion through mechanisms of internal transport created by vascular cell types ( 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 ) forms colonies that are a combination of aggravative and clonal ( Ros-Rocher et al. 2024 ). Further, complex multicellularity, which has long been generally thought to be restricted to eukaryotes, seems to be present in many bacterial groups as well. For example, Vibrio splendidus 12B01 forms clonal multicellular groups with a complex life cycle and cellular differentiation between cells internal and surficial cells of the group ( Schwartzman et al. 2022 ). The motility of the internal cells may also act to supplement diffusion of resources. 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 ). Despite our general ignorance of archaea, multicellularity in archaea was thought to be rare. A recent discovery that a multicellular phenotype can be induced by physical compression in a broad phylogenetic sampling of archaea ( Rados et al. 2025 ) raises the possibility that archaean multicellularity may be common, but environmentally conditional. Moreover, given the “tissue-like” organization of cells observed by Rados et al. ( Rados et al. 2025 ), they may potentially be said to exhibit a form of complex multicellularity as well. With this new conceptual eye, hits of multicellularity are more commonly described in archaea than it seemed at first. The halophile Haloquadratum is typically noted for its unusual square cell shape, but biofilms of this are common and cells can remain connected for a period of time and form multicellular sheets. Physiological experiments on the methanogen M. maripaludis also show it will form colonies in liquid culture even when shaken (Harp Batther, personal communication, 2025). All these observations point to a largely unknown multicellular diversity among all groups of archaea. Within bacteria, multicellular group formation is essentially ubiquitous ( Shapiro 1998 ; Aguilar et al. 2007 ; Rosenberg 2009 ; Lyons and Kolter 2015 ) and present for all known evolutionary time, with stromatolites even forming reefs as early as 3.45 billion years ago ( Allwood et al. 2006 ; Allwood et al. 2009 ). Although bacterial multicellularity is largely dismissed as simple, particular examples have all the ingredients to be considered complex. Filamentous cyanobacteria can have up to three terminally differentiated cell types: the typical photosynthetic hormogonia, the nitrogen-fixing heterocyst, and the dormant resistant akinetes. Colonies of Nostoc can be macroscopic. As noted above, colonies of Vibrio splendidus 12B01 have a complex life cycle and phenotypic and metabolic differentiation between internal and surficial cells of the group ( Schwartzman et al. 2022 ). Since the Archean Eon, biofilms, mats, and stromatolites have been made by consortia of bacteria (and archaea) that mix aggregative and clonal growth and maintain a diversity of metabolisms often structured along diffusion gradients ( Nisbet and Fowler 1999 ). Archean-aged (~4-2.5 billion years ago) stromatolites can be large with column diameters up to 100 cm ( Bosak et al. 2013 ). Over geological time, the average stromatolite column diameter is stable and centimeter scale. 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 ). One possible mechanism for this could be a high extinction rate of multicellular bacteria over evolutionary time that is not quite balanced by the origination of new multicellular lineages. This would be difficult to test in the fossil record due to taxonomic uncertainty, but could be tested using new phylogenetic methods. The ubiquity of bacterial multicellularity coupled with the sense that it is constrained leads to a possible conclusion that multicellularity in bacteria is simple to gain but easy to lose. This easy come easy go evolutionary history is also supported by the ease at which bacteria can be cultured in the laboratory as populations of isolated unicells while in nature they form structured biofilms and colonies, and the common occurrence of environmental cues that can trigger multicellular development in typically unicellular strains (for example, 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 ). In stark contrast to bacteria, whose fossil record implies the continuous presence and constant origination of new multicellular groups, eukaryotic multicellularity seems to be pulsed. A low diversity of small, multicellular, or coeno-cytic eukaryotic algae of unknown affinity originate about 1.6 billion years ago ( Tang et al. 2024 ). During the late-Neoproterozoic, starting about 750 million years ago, multicellular red and green algae originate and diversify ( Yang et al. 2016 ; Del Cortona 2020) and macro algae fossils diversify and increase dramatically in size ( Bykova et al. 2020 ; Tang et al. 2024 ). Concurrently, the multicellular metazoans originate and radiate ( Erwin et al. 2011 ; Dohrmann and Wörheide 2017 ; Tang et al. 2024 ). Other multicellular eukaryotes, such as the brown algae ( Choi et al. 2024 ) and volvocine algae, evolve much later ( 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 ). Two clades of red algae appear to have evolved complex multicellularity independently from a simpler starting point at nearly the same time ( Yang et al. 2016 ). Somewhere between one and six (or more) origins of complex multicellularity are observed in green algae ( Umen 2014 ; Del Cortona 2020; Umen and Herron 2021 ) and 8-11 origins of complex multicellularity are observed in fungi ( Nagy et al. 2018 ). There is, however, apparently only one origin of complex multicellularity in the phaeophyte brown algae ( Choi et al. 2024 ) and only one origin of complex multicellularity in metazoans ( 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 ; Del Cortona 2020). Brown algae and fungi appear to have later origins within the Paleozoic ( Nagy et al. 2018 ; Choi et al. 2024 ). A comparison of eukaryotic multicellularity with the apparently even origination and diversity of multicellular bacteria over Earth history would be enlightening. At first glance, originations of simple multicellularity in photosynthetic eukaryotes is evenly distributed over their early history ( Tang et al. 2024 ), but it is unclear when simple multicellularity evolves in other eukaryotic lineages that lack a fossil record. 3. Resource transport as a defining trait of complex multicellularity One neglected attribute Knoll ( Knoll 2011 ) used to define complex multicellularity is that all complex multicellular eukaryotes evolve mechanisms for internal resource transport that allows them to reach sizes otherwise limited by diffusion. At first glance all multicellular bacteria lack internal transport beyond a reliance on diffusion. Resource concentrations at the surface of biofilms and mats can be influenced by convection of the fluid surrounding the mat ( Persat et al. 2015 ). It is possible that motile internal cells within multicellular colonies of Vibrio described by Schwartzman et al. ( Schwartzman et al. 2022 ) act to supplement diffusion inside the cell cluster by generating their own “convection,” just as spinning marine animal embryos are thought to increase internal mixing of oxygen within egg masses ( 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 ). Diffusion can be supplemented by convection or turbulence of the fluid the bacteria live in ( Persat et al. 2015 ; Fernandez et al. 2019 ; Słomka et al. 2023 ); the effect is likely to be minor due to the low Péclet number of bacteria and archaea (Vogel 1996). [The Péclet number is the ratio of convection to diffusion, which for bacteria ranges between 10 −3 and about 10 −2 ( Simpson 2021 ) and for archaea is likely lower do their likely smaller typical size and lower motility.] 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 ), indicating a reduced role for diffusion compared to bulk flow. Most unicellular eukaryotes, including ciliates and flagellates, have Péclet numbers at or above 1, the point at which flow dominates, and a majority have Péclet numbers between 10 and 1,000. This alone is enough to discern a fundamental difference in the mechanisms of resource acquisition between eukaryotes, bacteria, and archaea. Eukaryotic size and motility are sufficient that, even though small, they can manipulate their own environments to capture resources in ways bacteria fundamentally cannot. This difference in mechanisms of resource capture between eukaryotes and prokaryotes suggests the hypothesis that cold water (as would have occurred during global glaciation events known as Snowball Earths, the first about 2 billion years ago and the more recent ones between 720 and 635 million years ago) can lead to the loss of multicellularity in bacteria and the gain of it in eukaryotes due to increased viscosity decreasing the rate of diffusion and increasing the importance of bulk flows ( 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 ). However, in the transition to multicellularity, cells must revert to diffusion to communicate and share materials extracellularly between members of a group. This larger diffusion scale constrains most simple multicellular groups to small size even in resource replete environments. The origin of differentiated cell types, tissues, and other mechanisms of active resource transport acts to free the eukaryotic multicellular organisms we consider to be complex from the constraints of diffusion once again ( Knoll 2011 ). Active transport is surprisingly important to embryonic development as well. During early embryonic development, where a single large fertilized egg subdivides into ever-smaller cells, sufficient active transport within the embryo is not possible and as the embryo begins to grow its scale quickly exceeds any ability for diffusion to transport resources to internal cells. Maternally provided transcripts and yolk ( Buss 2016 ) often help the embryo energetically to survive the lag between the start of the life cycle and the embryonic development of facilitated internal transport. 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 ). At low effective population sizes (which multicellular organization exacerbates even more severely than eukaryogenesis does due to increasing cell number lowering the number of multicellular organisms in a population), drift can easily dominate over natural selection. Bacteria have genomes that are prone to gene loss due to drift ( Kuo and Ochman 2009 ; Bobay and Ochman 2017 ). In contrast, eukaryotes are observed to expand their genomes under the same conditions ( Lynch and Conery 2003 ; Lynch 2007 ; Lynch 2010 ). Bingham and Ratcliff ( Bingham and Ratcliff 2024 ) propose that this can bound the ability for prokaryotes to evolve ever-more complex multicellularity because they have to contend with an opposing tendency toward gene loss. Eukaryotes may then have an easier time evolving complexity because they accumulate genomic parts that can aid in producing ever-more complex phenotypes. 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 ) to survival on the surface of a leaf ( Remus-Emsermann and Schlechter 2018 ) or a tooth ( Baker et al. 2024 ) to food fermentation ( Michielsen et al. 2024 ) and oxidation of methane in deep sea sediments ( Metcalfe et al. 2021 ). The biogeography of the human microbiome ( Baker et al. 2024 ; McCallum and Tropini 2024 ) and the biogeochemistry of Earth are shaped by local microbial interactions ( Pfreundt et al. 2023 ; Prasad et al. 2023 ). Many ecological functions of microbes only emerge through these interactions. Where interactions involve the differentiation of cell states and divisions of metabolic or reproductive labor, we can consider the physical form to have attributes of a multicellular group. Given the importance of cellular interactions in the microbial world, why is multicellularity seemingly rare in the prokaryotes? 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 ) rather than environmental cues shifted the paradigm for prokaryotic life from solitary cells to social entities ( West et al. 2007 ). The resultant study of prokaryotic cell systems using frameworks built to understand the development (Van Gestel 2015) and system-level functioning ( Shapiro 1988 ) of complex multicellular life has led to the discovery that multicellularity shares many common attributes across domains (Van Gestel 2015). The growing interdisciplinarity of our field, as well as the strengthening of connections across domains of life, creates a window of opportunity to gain an understanding of the evolutionary origins and ecological roles of prokaryotic multicellularity. By integrating our understanding of regulation, physiology, and biophysical interaction with ecology and evolution ( West et al. 2007 ; Hallatschek et al. 2023 ; Hengge 2020 ), properties or cellular systems that underlie prokaryotic multicellularity are starting to emerge. These properties were discussed in depth during the colloquium and highlighted below are three questions and three barriers at the forefront of this topic. 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 ) and microbial communities ( Gralka et al. 2020 ). This expands the possible networks through which systems of cells can interact ( Fig. 22 ). Comparing recent work across systems suggests that the modularity with which cells can metabolically interact and the physical mechanisms coordinating metabolite exchange are attributes that differentiate cell systems along a continuum from community to multicellular group. 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 ). The metabolic constraints of decomposition break the process into a few modules that are specific and necessary and many modules that are substitutable. The modularity of metabolic exchange extends to obligate associations such as syntropy: many examples of structured microbial consortia are based on syntrophic exchange, including sulfur cycling bacterial “pink berries” ( Wilbanks et al. 2014 ), anaerobic methane oxidation in granules of archaea and bacteria ( Metcalfe et al. 2021 ), and the obligate phototrophic microbial consortium Chlorocrhomatium ( Liu et al. 2013 ). A survey of the metabolic architecture of diverse heterotrophic microbes highlights the latent potential for syntrophic interactions even without a shared evolutionary history ( Libby et al. 2019 ), suggesting that the capacity to metabolically interact may be a common basis for prokaryotic multicellularity. Differentiation into metabolically interacting subpopulations of cells can also structure interactions within multicellular groups composed of only one genotype ( 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 ), providing clues to the mechanisms underlying the remarkable reproducibility of bacterial collective behavior. An additional challenge in a plastic cell system is heritability: a hallmark of complex multicellularity in eukaryotes. As I detail below, lifecycles and cellular defense mechanisms stabilize prokaryotic multicellularity in the absence of terminal differentiation and reproductive division of labor. 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 ). The ability of taxa to undergo lifecycles with a single-cell propagule provides a mechanism to purge social cheats, as clonal multicells do ( Claessen et al. 2014 ). Life cycles readily evolve in biofilm-forming bacteria capable of dispersal, when the ability to repeatedly disperse and re-form groups is selected for ( Poltak and Cooper 2011 ; Hammerschmidt et al. 2014 ). By dissociating and re-forming groups, the fitness advantage of cheating is not held constant: once dissociated, a cheat may be less able to re-form a group, or if the group undergoes a bottleneck in formation, cheats arising at low frequency in groups may be less able to propagate to invade a new group ( 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 ) that introduce new cellular functions. Bacteria have evolved an arsenal of defenses against selfish mobile elements and viruses ( Smith et al. 2023 ). These defenses may be particularly important for obligate multicellular prokaryotes that do not disperse through a unicellular state to found new low-diversity groups because cells cannot dissociate, such as multicellular magnetostatic bacteria ( Schaible et al. 2024 ) and the consortia that form pink berries and Chlorochromatium. For example, in pink berries, evidence for the evolution of an adaptive immune-like response has been discovered in the characterization of diversity generating retroelements ( Doré et al. 2024 ). The ability of cells to respond to infection by a mobile element, a plastic response, may stabilize obligately multicellular prokaryotes ( 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 growing in three dimensions ( Martínez-Calvo et al. 2022 ), or “fountains” of Vibrio cholerae ( Hallatschek et al. 2023 ). While a range of patterns can emerge in cell collectives ( Fig. 22c ), without additional regulatory input, the structural complexity attainable by self-organization is limited. For example, many bacteria can grow as filaments, but the ability to branch in multiple dimensions or divide asymmetrically requires regulation ( Flärdh et al. 2012 ). Filaments can bundle and cluster but cannot create structural rigidity, which limits the ability of filamenting prokaryotes to resist mechanical stress in a collective or multicellular state. Among the archaea, mechanical stress triggered by compression in the halophile Haloferax volcanii cues the formation of multicellular structures that differentiate into cell types suited to move through tight spaces and remain in crevices ( Rados et al. 2025 ). Without cell type differentiation, the size of prokaryotic multicellularity would be ultimately limited by diffusion ( Jo et al. 2022 ). The ability to form structures such as simple water channels ( Jo et al. 2022 ), pockets of motile cells ( Schwartzman et al. 2022 ), and symplasmata ( Tecon and Leveau 2016 ) allows prokaryotic groups to overcome some of these barriers. Work on bacterial biofilms reveals a key role for the physical interactions in co-ordinating the metabolic exchange in a multicellular group ( Fig. 22c ). The ability of cells to produce of extracellular matrix or express specialized adhesins provides a path to constrain possible metabolic interactions ( Evans et al. 2020 ), promote repeated association ( D’Souza et al. 2018 ), or create environmental context needed to carry out biochemical transformations (Ackermann and van Vliet 2023). Biofilms can synchronize their metabolic activity, for example, creating patterns of sporulation in Bacillus subtilis through a mechanism akin to the segmentation clock of animal animals and plants ( Chou et al. 2022 ). Studies of population structure within fruiting bodies of the social bacterium Myxococcus xanthus reveal “hotspots” of genome evolution functionally tied to the physical mechanisms through which cells interact and form swarms ( Wielgoss et al. 2019 ). Together, these studies suggest that the more tightly coupled the physical and/or metabolic interaction and the more tailored the response to the environment, the greater chance of structural complexity and an interaction with hallmarks of a multicellular group. 4. Three current barriers for the field of prokaryotic multicellularity a. Identification 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 ), this approach may ultimately limit our ability to identify novelty moving forward. The forms of multicellular life we are most familiar with represent remarkably little metabolic diversity. This may mean that for eukaryotic life, most of the novelty could be achieved through physical form, whereas the lack of metabolic constraint led to different solutions in the two more metabolically diverse domains. 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 ) and ecosystem fabrication devices ( Gao et al. 2018 ) are promising leads to overcome this limitation. 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 ): this has led, for example, to the discovery organisms that oxidize ammonia to nitrate. The search for missing metabolisms focuses on the ability to integrate multipartite pathways into one cell and the constraints of combining pathways with different rate limiting steps and toxic intermediates. As discussed above, the coordination of metabolic function, within a cell or among a system of cells, is a fundamental ingredient needed to evolve multicellularity. Until we start studying the diversity of prokaryotic metabolism in the context of multicellularity, we will not be able to resolve this issue. Experiments engineering novel metabolic function already underway in yeast ( Peterson et al. 2024 ) and studies “reversing the tape” that understand the evolution of metabolism over geologic time ( Kaçar 2024 ) point to an opportunity for fruitful comparative study across domains. 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 ), from the origin of life to multicellularity. Research on the origin of life frequently focuses on the role of autocatalytic cycles, which provide a mechanism for simple chemical systems to become self-sustaining and capable of evolution ( Kauffman 2011 ; Hordijk and Hein 2010). In an analogous manner, studies on the evolutionary origins of multicellularity emphasize the role of life cycles, specifically, the ways in which groups of cells generate offspring that will also develop into groups, a process known as group reproduction (Libby and Rainey 2013; De Monte and Rainey 2014 ; Staps et al. 2021). 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 ). Additionally, understanding the selective pressures shaping different modes of group formation and reproduction offers a clearer picture of why certain multicellular life cycles dominate across diverse biological systems. 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 ; Lind et al. 2019). A useful classification of these modes distinguishes between aggregation of potentially unrelated cells and clonal reproduction, where cells remain attached after division ( Tarnita et al. 2013 ). In aggregation-based group formation, individual cells adhere to one another in response to an external signal, often triggered by environmental stress. A well-known example is Dictyostelium discoideum, a slime mold in which free-living amoebae aggregate in response to starvation, ultimately forming fruiting bodies that facilitate spore dispersal to richer hunting grounds ( Schaap 2011 ). By contrast, clonal group formation occurs when daughter cells remain attached after division, forming a cohesive unit. This mechanism is more common in systems where multicellularity provides a continuous selective advantage or in environments without reliable signals for aggregation. For example, in the snowflake yeast (Saccharomyces cerevisiae) model system ( Ratcliff et al. 2012 ), selection for rapid settling leads to the evolution of cohesive clusters of cells that remain attached, ensuring survival despite the absence of an immediately detectable external cue. Indeed, once an artificial cue is added to allow prediction of multicellular selection, aggregative forms of multicellularity can be maintained in this selective regime ( 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 ). Since aggregative group formation requires that cells associate to form groups, the resulting groups must also at some point later dissociate back into a single-cell state. Thus, life cycles with aggregative group formation usually alternate between unicellular and multicellular stages. In contrast, clonal group formation does not often feature a unicellular phase unless it is an immediate antecedent to multicellular development, i.e., two-cell groups. While the majority of life cycles with clonal groups exist predominantly in a multicellular phase, they differ in how groups reproduce. In theory, groups can fragment in exponentially many ways, depending on the number of daughter groups and the specific number of cells in each group ( Pichugin et al. 2017 ; Isaksson et al. 2023 ). However, in practice, three predominant modes are observed: (i) binary fission, where groups split evenly in half; (ii) single-cell propagules, where groups release individual cells that go on to form new groups; and (iii) complete dissociation, where groups break down entirely into their constituent cells, this last mode also being characteristic of aggregative life cycles. 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 ). Since aggregative life cycles contain unicellular stages, there are evolutionary opportunities to abandon multicellularity. Indeed, in Dictyostelium discoideum some cells fail to join the multicellular phase even in the presence of appropriate cues ( Dubravcic et al. 2014 ). Evidence suggests that these “loner” cells may be implementing a kind of bet-hedging strategy, but in so doing they also represent a lineage that has temporarily abandoned multicellularity and could be selected to continue to do so ( Tarnita et al. 2015 ). Aggregative life cycles can also fail to fix traits that are beneficial to groups but costly to individual cells ( Ratcliff et al. 2017 ). Such so-called altruistic traits are considered important for driving further multicellular complexity, as they underpin innovations such as the germ-soma distinction and cell differentiation. A key difficulty in fixing such traits arises from the relative population sizes of unicellular and multicellular stages of the life cycle ( Pentz et al. 2023 ). Since unicellular populations are typically much larger, the costs associated with altruistic traits cause them to be selected against. Conversely, in the multicellular stage where they are beneficial, the population size (number of groups) is smaller, thereby allowing drift to play a larger evolutionary role. 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 ). Not only do such traits help to drive the complexity of nascent multicellular organisms but they can also improve evolutionary stability ( Libby et al. 2016 ). The snowflake yeast model system is particularly informative in this regard. Here S. cerevisiae is incubated in a 24-hour cycle consisting of growth to saturation, followed by selection for rapid settling, for weeks to years on end. In this context, clonal groups in which daughter cells fail to completely separate from their mothers rapidly evolve on a timescale of a week and their size continually increases. After a long enough time spent in laboratory evolution, multicellular populations evolved higher rates of cell death. Outside of the multicellular context, such a trait would be costly and selected against. Yet within the multicellular context, mathematical modeling demonstrated that this trait can acquire a new functional role, enabling groups to fragment before running out of room to reproduce, thereby circumventing growth limitations imposed by physical packing constraints ( Libby et al. 2014 ). An additional benefit of such a trait is that should any cells revert to unicellularity due to mutation, their increased rate of cell death would make them less fit, reinforcing the stability of the multicellular form ( Libby and Ratcliff 2014 ). For these reasons, clonal group formation is often considered as a more likely starting point for the evolution of complex multicellular organisms ( 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 ). This limitation can act to constrain the evolution of increased group size ( Bozdag et al. 2021 ). Thus, while increasing group size may be a necessary step for the evolution of differentiation in clonal multicellular life cycles, it also introduces new challenges that may inhibit the evolution of further complexity. 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 ). Since aggregative multicellularity relies on cells coming together, different genotypes may join the same group, potentially leading to conflict. If cells are too selective in choosing aggregation partners, their ability to form groups may be hampered ( Pentz et al. 2020 ). If instead, cells are too permissive, cheating strategies can evolve, wherein some cells reap the benefits of multicellularity without paying a fair share of the cost of group formation, e.g., production of extracellular glue or other public goods. Organisms that rely on aggregation have evolved diverse strategies to mitigate genetic conflict, including kin recognition ( Nicotra 2019 ; Hirose et al. 2011 ), policing mechanisms ( Ostrowski 2019 ; Foster and Ratnieks 2000 ; Ratnieks et al. 2006 ), and restricted access to group reproduction (e.g., germ line sequestration) ( Dao et al. 2000 ). Although these various mechanisms of resolving or preventing genetic conflict may seem like barriers to multicellular complexity, many analogous solutions have evolved in complex multicellular organisms, such as immune systems that distinguish self from non-self. While there are strong arguments against aggregative life cycles leading to complex multicellularity ( Márquez-Zacarías et al. 2021 ), certain successful forms of multicellularity have evolved in spite of these challenges. One notable example is the diverse array of lichen symbioses, that is, partnerships between at least two, and often three or more, species ( Spribille et al. 2016 ; Grube and Berg 2009 ). Lichens regularly form through multispecies aggregation, yet the resulting partnership produces long-lived, complex multicellular structures capable of thriving in extreme environments ( de La Torre et al. 2010 ). The success of lichens suggests that multicellularity involving multiple species [so-called egalitarian transitions (Queller 1997)] may provide alternative pathways to complexity (Libby et al. 2021). In such cases, the multicellular phenotype emerges as a collective property distributed across species and not present in any one lineage, rather than being confined within a single lineage. Beyond lichens, there are other examples of non-canonical life cycles that do not fall neatly into the traditional clonal/aggregative framework. A recent study of the choanoflagellate Choanoeca flexa reveals that it mixes clonal and aggregative multicellularity to survive dynamic coastal environments ( Ros-Rocher et al. 2024 ). Such studies highlight the broader principle that evolutionary construction does not always proceed along a single, predictable trajectory but can instead follow multiple routes shaped by ecological interactions and selective pressures. Advancing theoretical, experimental, and comparative research will further elucidate how single cells, whether within a lineage or in symbiotic partnerships, transitioned to the diverse and complex forms of multicellular life observed today. 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 ). There are (at least) two inevitable physical facts that lead to emergent phenomena with crucial multicellular consequences. First, group formation is intrinsically a physical process, i.e., intercellular forces are required to keep cells together (Tarnita et al. 2012). The origin and nature of these forces can substantially impact the subsequent evolution of a multicellular group. Second, cells occupy space, and no two cells can occupy the same space ( Hallatschek et al. 2023 ). Thus, changes at the single-cell level that impact intercellular forces and cellular packing have emergent group-level consequences. 2. Biophysical principles govern the key processes of multicellularity Cells are subject to a plethora of physical forces from their environment ( Fig. 23a ). In the animal, plant, algal, and fungal kingdoms, multicellular organisms are distinguished by distinct cell types and a precisely organized three-dimensional structure. The key processes that enable multicellularity, i.e., cell division, adhesion, communication, differentiation, and morphogenesis, are all fundamentally shaped by physical principles. 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 ). Following division, mother and daughter cells either may remain physically connected through cytoplasmic bridges or shared cell walls, as observed in choanoflagellates ( Dayel et al. 2011 ) and snowflake yeast ( Ratcliff and Travisano 2014 ), respectively, or may adhere via extracellular matrices, as seen in Gonium ( Hanschen et al. 2016 ), a genus of volvocine algae. Alternatively, cells can maintain connections to each other through cell adhesion molecules, which, rather than being a novel feature of multicellular organisms, were co-opted from unicellular ancestors ( Suga et al. 2013 ; Sebé-Pedrós et al. 2013 ; Parra-Acero et al. 2020 ). In these unicellular ancestors, cell adhesion molecules likely played critical roles in surface attachment, in interactions with other cells during processes like mating or feeding, and in the formation of biofilms or colonies. 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 ). When this stress exceeds the strength of the mother-daughter bonds, the cluster fractures into two independently viable propagules ( Day et al. 2022 ; Jacobeen et al. 2018 ; Jacobeen et al. 2018 ). Thus, group-level reproduction, a crucial feature of multicellularity ( Okasha 2005 ), can arise as an inevitable consequence of unavoidable multicellular physics—cells cannot overlap, and intercellular forces are necessary to keep groups together—rather than requiring complex evolutionary innovations. 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 )]. are now repurposed to establish form and movement in multicellular organisms. 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 ). In response, snowflake yeast evolved to achieve larger cluster sizes by modifying their mechanical properties through a simple adaptation: they evolve from being roughly spherical to highly elongated in shape ( Jacobeen et al. 2018 ; Jacobeen et al. 2018 ). As snowflake yeast cells tend to bud at the pole opposite from their mother, longer cells create greater physical separation between cells by ensuring they grow out into free space rather than towards each other, reducing the volume fraction of cells within the cluster. With more space between cells, collisions occur less frequently, allowing clusters to grow larger before reaching the critical stress that triggers fracture. This observation demonstrates how complex multicellular traits can emerge from modifications to the physical properties of individual cells. 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 ). Similarly, intracellular mechanics, such as the linkage of the actin cortex to plasma membrane, regulate differentiation. A decrease in these linkages facilitate changes in cell shape that activate biochemical signaling pathways necessary for differentiation (De Belly 2021), while maintaining strong linkages preserves stemness and prevents differentiation ( 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 ). Thus, any trait depending on these cell packing statistics will be inherited reliably, even without complex genetic control, another example of the physics of cell packing providing complex traits for free. 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 ). Instead, physical properties and constraints provided a foundation upon which natural selection could build increasingly complex multicellular features. Furthermore, incorporating randomness, or stochasticity, into our understanding of developmental and evolutionary processes is crucial for accounting for adaptability and robustness and may play a role in fostering the emergence of new multicellular traits. The interplay between selective pressures and physical constraints may have been particularly important in early multicellular evolution, with traits often evolving through modifications of cellular properties that had robust emergent effects at the group level. Crucially, these phenomena arise from fundamental physical principles likely shared by all multicellular groups, such as the forces required to hold cells together and the spatial constraints they face. 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 ), such as cellular aggregation (e.g., self-adherence) that buffers individual cells against environmental stressors (Smukalia et al. 2008) and greater organismal size that reduces vulnerability to predation ( Tong et al. 2022 ). The evolution of multicellularity in eukaryotes occurred independently many times, which begs consideration of generalized environmental influences that may have shaped the origin and diversification of these different lineages of multicellular organisms ( 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 ), it seems reasonable to assume that such parasites were also abundant and a major selective pressure long ago when multicellular eukaryotes originated. Thus, we can presume that the environments in which early-evolved multicellular eukaryotes arose was rife with viruses that targeted pre-existing unicellular organisms, especially prokaryotes and single-cell eukaryotes. By projecting back in time using the extant distribution of viruses that infect bacteria and archaea, and tracing the evolutionary histories of certain virus genes, researchers have attempted to reconstruct the ancient virome experienced by the last universal cellular ancestor (LUCA) ( Krupovic et al. 2020 ) (see Secs. IV B and III C). Such estimates are undoubtedly difficult and likely imprecise; nevertheless, results suggest that many of the main groups of extant viruses of prokaryotes, especially double-stranded DNA phages in the realms Duplodnaviria and Varidnaviria, could have been present when eukaryogenesis occurred ( Krupovic et al. 2020 ). Modern viruses tend to be highly efficient at hijacking the metabolisms and molecular-replication machineries of their host cells, to produce many (hundreds or even thousands) of particles per infected cell. Moreover, in current-day unicellular eukaryotes and prokaryotes, virus infection typically leads to cell death. This lethality, coupled with virus growth efficiency via rapid generation times that often outpace rates of host-cell division via binary fission, creates the possibility for viruses to exert strong selection pressures on their hosts. Therefore, from these various lines of evidence we can expect that virus infection was a key environmental selection pressure throughout the historical evolution of cellular life, including within environments experienced by unicellular eukaryotes that evolutionarily transitioned to early multicellular eukaryotes and the participants of the colloquium evaluated this across all of the topics they discussed. 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 ), but it seems plausible that evolution of multicellular eukaryotes coincided with greater resistance against virus infection, when considering mechanisms such as cell differentiation, evolution of immune system defenses, and the functional and structural complexity inherent to multicellularity. 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 ). This limitation in virus infection success can arise due to differing molecules presented on the exterior surfaces of cells that are specialized for different functions, thus altering whether cell-binding targets remain available for viruses to initiate the infection process. Similarly, as cells functionally differentiate their physiologies and innate defenses may diverge as well, posing a challenge for viruses to infect various cell types in multicellular eukaryotes; these differences in virus-cell interactions are obvious in hosts such as humans with specialized immune-system cells like neutrophils, macrophages, and dendritic cells that play distinct roles in recognizing and destroying pathogens ( 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 ). Of course, somatic cells present many defenses that have evolved to reduce vulnerability to virus infection as well. Such defenses include the ultimate sacrifice of intrinsic or extrinsic apoptosis (cell death) initiated when a cell undergoes virus infection or engulfs a virus invader; here somatic cells are suicidal or purposefully destroyed, resulting in limited virus replication within cells of multicellular organisms, further protecting the germline via reduced or halted epidemiological spread of viruses between cells. 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 ). Most bacteria have a peptidoglycan-containing cell wall, which differs between gram-negative and -positive cells, and archaeal cells vary in wall types including those lacking a polysaccharide component and those that closely resemble peptidoglycan. Furthermore, many prokaryotic cells have an external capsule or a slime layer that may play a role in attachment to surfaces or in resistance to desiccation. In plants a new component of the cell wall is cellulose, and in other eukaryotes the structure and chemistry of the cell wall (if present) are more diverse. Thus, there is a wide variety of barriers to virus entry across the cellular life on Earth, which poses an obvious reason why viruses are themselves forced to become evolutionarily diverse. 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 ). Whereas prokaryotes and unicellular eukaryotes provide ample evidence for innate-immune function, including intricate strategies such as CRISPR-Cas systems and RNA interference mechanisms, these are less-complex defenses than the highly specialized cells that coordinate to participate in both innate and adaptive immunity in multicellular eukaryotes such as humans. However, recent evidence suggests that core components of relatively elaborate immune systems of multicellular eukaryotes show genetic similarities to those of bacteria ( Morehouse et al. 2020 ; Wein and Sorek 2022 ). In particular, stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP-AMP signaling during viral infection ( Ishikawa and Barber 2008 ; Burdette et al. 2011 ). It is evident that functional STING homologs are encoded within prokaryotes, located in “defense islands” that represent stretches of the genome containing various mechanistic defenses against infection by phages, viruses that specifically attack prokaryote hosts ( Morehouse et al. 2020 ). This proposed similarity suggests that some cellular defenses are ancient in origin, emphasizing that protection of the cell against invaders is likely a response to an age-old environmental selection pressure, common in the evolutionary history of cellular life. 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 ). In addition, the aforementioned apoptosis, or programmed cell death, provides a key defense mechanism that is essential to complex immune systems such that infected cells self-destruct before they risk spreading their infection to neighboring cells ( Medzhitov and Janeway 2000 ). It would be useful to conduct more studies that consider multicellular organisms like sponges and cnidarians, which represent some of the earliest animal lineages, to deduce commonalities among multicellular eukaryotes in their evolved immune systems and generalized capabilities of responding to viral pathogens ( Waldron et al. 2018 ). One goal would be to better understand how differences between prokaryotic and eukaryotic cells alone would provide automatic barriers to cell entry by infecting biological entities versus the extent that evolution of different immune responses would have likely occurred in early multicellular eukaryotes to protect against viruses that diverged from those which attacked unicellular ancestral eukaryotes. 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 particular, for which genetic and genomic tools are available ( 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 ). They exhibit a wide range of morphologies and developmental sophistication, ranging from simple filamentous forms to large, structurally complex thalli (e.g., kelps) ( Batista et al. 2024 ). Brown algae are also unique models to understand life cycle transitions because most have an alternation between complex multicellular and independent gametophyte and sporophyte generations ( Arun et al. 2019 ). This life cycle complexity, combined with their ecological diversity, makes them fascinating models for studying how multicellularity arises and is maintained and how multicellular developmental programs are connected to alternation of generations ( 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 ). For instance, in kelp species, the transition from a single cell to a complex differentiated thallus includes the formation of highly specialized structures such as holdfasts, stipes, and blades ( Liesner et al. 2025 ). These look superficially like land plant structures, but they have emerged entirely independently. Understanding these developmental processes can shed light on how cellular differentiation and specialization evolve in multicellular organisms. Questions like whether the same genes are involved in the morphological similarity or if different genes in brown algae and plants have been co-opted to perform a similar role can now be addressed because molecular tools are available to study gene function in brown algae (e.g., Badis et al. 2021 ; Coelho 2024 ; Coelho et al. 2011 ; Luthringer et al. 2024 ). Importantly, comparison of signaling mechanisms between brown algae and other multicellular lineages could offer insights into the broad molecular pathways that underlie the evolution of cellular communication. One particular striking example of convergent evolution has been the recent finding that brown algae, like animals and plants, also have a molecular “developmental hourglass” ( Fig. 24 ). Multicellular species of animals and plants differ greatly in morphology and gene expression patterns both at their earliest stages and in their adult forms, but are quite similar at intermediate times as their body plan is first established, described as an hourglass-like pattern of development. Brown algae show this pattern too. By showing that brown algae with an independent origin of multicellularity also have developed this transcriptomic hourglass pattern, these studies have established that the transcriptomic hourglass is indeed a central element of embryo development and body plan establishment in all independently originating known complex forms of multicellular life. 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 ). Examining these understudied patterns would help identify common themes in the evolution of multicellular organization, and methods such as single-cell RNA-seq combined with phylogenomics will offer the possibility to understand the molecular origins of these cell types. 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 ) together with functional tools that allow validation of candidate genes ( Badis et al. 2021 ; Farnham et al. 2013 ) gives access to understanding how brown algae use specific genes to regulate growth, development, and differentiation. Recent studies have already identified a number of key regulatory genes in brown algae that play important roles in multicellular development (reviewed in Batista et al. 2024 ). Moreover, the sequencing of several dozen genomes has revealed the potential role of gene novelty and viral-derived genes in the emergence of specific developmental patterns during brown algae evolutionary history ( Denoeud et al. 2024 ). By analyzing the genetic toolkit of brown algae, we can gain insights into how these genes were acquired and co-opted during the evolution of multicellularity and how this process might function in other multicellular lineages. 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 ). This complex process is an example of how multicellularity can be differentially important at different phases of an organism's life history. The ability of brown algae to drastically alter the size of phases of their life cycle may offer a model for understanding major changes in multicellular scale. In brown algae, both the haploid and diploid phases undergo somatic development and exist as independent entities. This feature brings about important evolutionary consequences, as organisms with life cycles that alternate between a haploid and a diploid phase, genes with phase-specific expression should differ in their evolutionary rate. Despite recent attention, the mechanisms by which such biphasic life cycles evolve and persist in this and other multicellular lineages, like green algae, remain obscure. Future studies in a range of brown algae with diverse life cycles, sexual reproduction modes, and multicellular complexity hold the promise to reveal fundamental principles underlying the emergence and evolution of complex multicellularity through comparative analysis. 2. Model systems for study of multicellularity a. Choanoeca flexa: sheet-like animal relative that unites the power of cell biology and ecology In recent years, Choanoeca flexa has emerged as a significant model system for investigating the transition to multicellularity. This choanoflagellate, phylogenetically positioned as one of the closest living relatives of animals, forms multicellular colonies with distinctive properties that provide unique insights into the evolution of complex multicellular life ( Brunet et al. 2019 ). C. flexa exhibits a mixed developmental strategy that challenges previous assumptions about choanoflagellate multicellularity. While other characterized colonial choanoflagellate species develop exclusively through clonal division, C. flexa demonstrates what has been termed clonal-aggregative multicellularity, the ability to form multicellular sheets through clonal processes, aggregative processes, or a combination of both ( Ros-Rocher et al. 2024 , bioRxiv). This phenomenon provides an opportunity to investigate the molecular mechanisms and biological consequences underlying both developmental modes within a single organism. The structural dynamics of C. flexa colonies illustrates a key early step in the transition to multicellularity: how novel multicellular traits can emerge directly from changes in cellular behaviors, without communication or coordination. These cup-shaped colonies can rapidly invert their curvature via a rhodopsin-cGMP pathway in response to changing light levels ( Brunet et al. 2019 ). This inversion requires actomyosin-mediated apical contractility and allows alternation between feeding and swimming states. Importantly, this multicellular behavior arises directly from the contraction or expansion of the apical actomyosin ring in individual cells, demonstrating how cell-level traits can be easily co-opted to create emergent multicellular functions ( Fung et al. 2023 ). C. flexa represents a potential analog for early multicellular animals. The sheet-like morphology with apical-basal polarity and direct cell-cell adhesions bears morphological similarities to simple epithelial tissues ( Brunet et al. 2019 ). The ability of C. flexa cells to sense environmental cues and directly convert them into collective contractions provides a model for how the first contractile tissues may have functioned, potentially informing hypotheses about early animal evolution that envisioned homogeneous sensory-contractile cells rather than specialized cell types ( Reyes-Rivera et al. 2022 ). A key advantage of C. flexa as a model system is the ability to study its life cycle in its natural environment. Found in splash pools on the Caribbean island of Curaçao, C. flexa's developmental patterns are regulated by salinity during natural cycles of evaporation and refilling. The organism transitions between multicellular sheets at low salinity and unicellular cysts at high salinity, a physiological response to environmental fluctuation that can be replicated in laboratory settings ( Ros-Rocher et al. 2024 , bioRxiv). This aspect allows investigation of environmentally entrained developmental plasticity and life cycle transitions. Unlike many other unicellular relatives of animals that are difficult to isolate from nature, C. flexa can be repeatedly collected from its natural environment and studied in both field and laboratory contexts. This tractability and the availability of diverse isolates enables research connecting molecular mechanisms to ecological function ( Brunet and King 2017 ). The recent assembly of the C. flexa genome and the capacity to leverage many tools used extensively in animal cell biology, e.g., CRISPR ( Brunet et al. 2019 ; Ros-Rocher et al. 2024 ), further enhance its value as a model system, allowing genomic and cell biological comparisons with other choanoflagellate and animal species. C. flexa inform broader theories about the multiple evolutionary paths to multicellularity observed across eukaryotic lineages and provide insights into how early animals may have navigated the transition to complex multicellular life ( Brunet and King 2017 ; Ros-Rocher et al. 2024 , bioRxiv). 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 ), favoring larger multicellular clusters. The experiment includes three treatment conditions to investigate the effects of different metabolic regimes: anaerobic growth with glucose, mixotrophy with glucose, and obligately aerobic growth with glycerol. Running for over 9,000 generations thus far, with populations cryogenically preserved every ~50 generations, the MuLTEE has created a “frozen fossil record” allowing resurrection of ancestors from any point in time. 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. The first step in multicellular evolution, the formation of cohesive groups, has occurred rapidly and repeatedly in experimental evolution. Under selection for rapid settling, unicellular yeast reliably evolve the “snowflake” phenotype via mutations affecting cell separation ( Ratcliff et al. 2015 ). While this was not surprising given previous shorter-term experiments, the MuLTEE demonstrates that this initial step of group formation represents a relatively minor evolutionary hurdle, arising consistently through simple genetic changes affecting cellular adhesion mechanisms. 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. Perhaps the most profound insights from the MuLTEE concern the second stage of multicellular evolution: how simple groups become evolutionary individuals in their own right. This stage has long been considered a major barrier to multicellularity because it requires groups to develop mechanisms for growth, reproduction, and the inheritance of novel multicellular traits. In extant organisms, these features are coordinated by fairly sophisticated developmental mechanisms, leaving it hard to conceptualize how they would arise de novo at the group level. 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 ). This represents a spontaneous multicellular life cycle that emerges for free and is rapidly co-opted and refined by multicellular evolution. 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 ). Furthermore, multicellular traits often show higher heritability than the unicellular traits they arise from, as group-level properties average out stochastic variation among individual cells ( Zamani-Dahaj et al. 2023 ). These findings overturn decades of thinking that the capacity for multicellular groups to gain novel, heritable multicellular traits, allowing open-ended multicellular evolution, first requires the evolution of developmental programs that act to translate mutations into multicellular traits. Stage 3: group transformation into functionally integrated organisms. The MuLTEE has begun to reveal how open-ended multicellular evolution transforms simple multicellular collectives into functionally integrated organisms. Within 3,000 generations, snowflake yeast evolved from microscopic clusters to macroscopic structures visible to the naked eye, increasing in size by 20,000-fold ( Fig. 25d ). This dramatic increase occurred through the evolution of a novel “entangled” morphology ( Fig. 25e ), where branches wrap around one another like vines, distributing forces across the organism and allowing it to remain intact even when individual cell-cell bonds break. This innovation transformed their material properties from being weaker than gelatin to as strong as wood ( Bozdag et al. 2023 ). The molecular basis of this transformation involves both genetic and epigenetic mechanisms. In addition to mutations affecting the strength of cellcell bonds and the shape of cells [longer cells underpin entanglement ( Bozdag et al. 2023 )]. The evolution of macroscopic size was associated with decreased expression of the chaperone protein Hsp90, which acts as an epigenetic regulator of the cell cycle ( Montrose et al. 2024 ). This molecular change produces elongated cells and alters budding patterns, facilitating the entangled growth form that underpins macroscopic size. 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 , bioRxiv). As multicellular clusters grow larger, diffusion limitation typically constrains nutrient delivery to interior cells, creating a theoretical upper size limit. However, evolved snowflake yeast generate metabolically driven convective fluid flows that circulate nutrients throughout the cluster ( Fig. 25g ), enabling exponential growth far beyond predicted diffusion limits. This unexpected solution allows these simple multicellular organisms to achieve macroscopic size without specialized circulatory tissues. The fluid flows arise from density gradients created by metabolic activity, driving advection currents that maintain sufficient nutrient delivery even to deeply embedded cells. 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 ). Similarly, multicellularity can drive ecological diversification through tradeoffs between growth and survival mediated by oxygen availability, leading to the stable coexistence of distinct morphotypes ( 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 ). This finding has implications for understanding macroevolutionary patterns, as whole genome duplications frequently precede major evolutionary radiations across the tree of life. Future outlook. As the MuLTEE approaches its first decade, it opens windows into evolutionary processes previously hidden by time. It provides unprecedented opportunities to observe multicellular entrenchment as cells become interdependent parts of a new whole, to witness the genetic assimilation of traits that initially arose through physical mechanisms, to document in real time the reductive evolution of mitochondria in anaerobic environments, and to uncover how novel three-dimensional architectures and developmental plans that guide morphogenesis emerge from simple growth patterns. Each of these processes represents a critical step in the transition to complex multicellularity, and the MuLTEE provides the first (and perhaps only) opportunity to observe how a lineage navigates the transition to multicellularity from the ground up. 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”) to industrial and translational applications such as biomanufacturing and regenerative medicine. In the past few years, synthetic approaches for engineering multicellular development have been flourishing. Multiple studies have leveraged the bottom-up self-organization abilities of stem cells to give rise to organoids and diverse type of embryo-like structures (gastruloids, blastoids, etc.) ( Zarkesh et al. 2022 ). In spite of the translational and fundamental insights contributed by these studies, these did not directly address past transitions to multicellularity in Earth's history and will not be the main focus of this section. Instead, we will focus on top-down approaches to the synthetic biology of multicellularity and on what they can teach us about the very first experiment in engineering multicellularity: the one nature performed. 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 ), it has been apparent that uncovering the logic of gene regulation would be fundamental to understanding multicellular development ( Britten and Davidson 1969 ). Indeed, as these mechanisms were uncovered, it seemed that changes in gene regulation might have contributed more to evolution than changes in protein sequences ( King and Wilson 1975 ; Carroll 2008 ) (but see Hoekstra and Coyne 2007 for nuances to this view). Notably, multistable gene circuits provide the mechanistic basis for persistent differential gene regulation and thus for cell differentiation in multicellular organisms. 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 ). More elaborate networks allow complex temporal features such as oscillations: an example is the classical repressilator network of three transcription factors, each of which inhibits the expression of the following ( Elowitz and Leibler 2000 ), and a more complex variant allowing mutually exclusive, multistable expression of one of two, three, or more transcription factors. This multistable network was sufficient to sustain the emergence and maintenance across division of multiple, stable gene expression states in isogenic mammalian cell cultures ( Zhu et al. 2022 ). Interestingly, that circuit relied on replicating nontrivial aspects of real-life gene regulatory networks for cell differentiation, such as activation of transcription factors by homodimerization, and inhibition by heterodimerization like those found in the pluripotency network of mammalian cells or the striated myocyte specification network. 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 ), it has been controversial whether diffusion is sufficient for observed complex patterning (Stapornwongkul et al. 2021). However, it has recently been possible to build a fully synthetic morphogen gradient by replacing an endogenous fruit fly diffusible morphogen (dpp, a BMP homolog) by an exogenous protein (a GFP), and engineering the endogenous dpp receptor to bind GFP instead ( Stapornwongkul et al. 2020 ). Remarkably, this minimal diffusion-based synthetic system was sufficient to replace the endogenous dpp circuit and to support normal patterning of the fruit fly wing. Similar approaches also allow patterning of multiple strips of cells in cell culture ( Toda et al. 2020 ). Further elaborations of these self-organized patterns might be enabled by fine engineering of cellular response to signals, such as the creation of synthetic bandpass filters ( Greber and Fussenegger 2010 ). Finally, fully synthetic reaction-diffusion systems allow the emergence of common features like stripes, spots, and spirals in cell culture ( Sekine et al. 2018 ). Similarly, engineered juxtacrine systems based on modified Delta/Notch platforms allow spontaneous emergence of “salt-and-pepper” patterns ( Matsuda et al. 2015 ). Combination of several such orthogonal juxtacrine systems (via the “SynNotch” platform) allows combinatorial, and even Boolean, outputs ( 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 )a peripheral actin network tethered to the plasma membrane by membrane-to-cortex attachment (MCA. Most dramatically, optogenetic systems now allow manipulation of the cytoskeleton by local selective illumination ( Valon et al. 2017 ), notably allowing control of cell migration ( Xu et al. 2014 ) and even of multicellular tissue folding ( 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 ) and confirmed by research on cadherins ( Halbleib and Nelson 2006 ). Sorting can be replicated in vitro by overexpressing distinct cadherins in otherwise isogenic cell cultures ( Cachat et al. 2016 ). Putting distinct cadherins downstream of SynNotch led to spontaneous cell sorting into multilayered structures via surface tension and phase separation ( 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 ): it provides feedback from form to gene expression, thus enabling robust homeostasis and self-organization. Little engineering has been done, but a tension-sensitive SynNotch has been produced and allows diverse synthetic circuits in which fibroblasts differentiate into myoblasts over distinct, specific ranges of applied tension ( 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? The first genomes of unicellular relatives of animals (choanoflagellates, filastereans, and ichthyosporeans) came with a surprise: these encoded many genes once thought to be unique to animals ( King et al. 2008 ; Suga et al. 2013 ; Grau-Bové et al. 2017 ). These included cadherins ( Abedin and King 2008 ), integrins ( Sebé-Pedrós et al. 2010 ), collagen ( Linden and King 2021 ), C-type lectins ( Levin et al. 2014 ), and multiple transcription factors typical of animal cell differentiation, such as 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 can induce mesodermal identity in frog embryos (the normal function of brachyury in vertebrates) ( Sebé-Pedrós et al. 2013 ). Recently, orthologs of the pluripotency transcription factors Sox2 and POU were discovered in a choanoflagellate, and their overexpression in mouse cell lines was found to produce induced pluripotent stem cells, like their mammalian counterparts ( 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 ); integrins (which mediate cell-matrix adhesion in animal tissues) seem to mediate adhesion to the external environment in Capsaspora ( Parra-Acero et al. 2020 ); and homologs of transcription factors involved in animal cell specification control generic “cellular modules” in choanoflagellates. This is the case for Rfx, which specifies ciliated cells in animal and orchestrates flagellum biogenesis in choanoflagellates ( Brunet and King 2017 ), and for Myc, which specifies stem and progenitor cells in animals and might set proliferation rate (by limiting the speed of protein synthesis) in choanoflagellates ( Young et al. 2011 ). These observations suggest that a “modular structure” of pre-existing transcriptional networks in unicellular ancestors might have facilitated division of labor during cell type diversification after the evolution of multicellularity ( 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 ). This suggests that many, if not most, eukaryotic lineages might be equally well equipped to evolve complex multicellularity in terms of pre-existing molecular toolkit when given the opportunity. The fact that “complex” multicellular organisms only evolved in a handful of lineages might be due to ecological first-mover effects, rather than to any genetic specificity of their ancestors ( 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. In what environments did multicellularity originate? Did it evolve because of specific selective pressures, or was it initially a neutral event ( Lynch et al. 2014 )? Attempts at correlating facultative multicellularity with environmental factors have revealed few interpretable correlations ( Cornwallis et al. 2023 ), leaving a key space open for experimental evolution to investigate the environmental factors of the evolutionary transition to multicellularity. 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 ) and the evolution of larger colony sizes in ichthyosporeans (close relatives of animals) ( Dudin et al. 2022 ). Similarly, selection on visible macroscopic clustering of E. coli (originally induced by hypersaline stress) allowed the genetic assimilation of this previously environmentally induced multicellular phenotype into a constitutive phenotype ( 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 ) and 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 ). Counterintuitively, however, oxygen consumption actually hindered the evolution of larger size in snowflake yeast at intermediate oxygen levels (compared to high levels or when grown anaerobically) as limited diffusion of oxygen into the interiors of large colonies slowed down their growth, thus selectively favoring small colonies in intermediate oxygen levels ( Bozdag et al. 2023 ). This effect could be partially circumvented by heterologous expression of oxygen-storage proteins ( Wong et al. 2025 ). More recently, the higher viscosity of cold water in the aftermath of global Snowball Earth glaciation events has been proposed to have selected for multicellular flagellate colonies, capable of generating a more powerful collective fluid flows for feeding and transport even in highly viscous water ( Simpson 2021 ). Indeed, artificially increasing viscosity by addition of agar selected for multicellular forms of Chlamydomonas ( 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 )]. These are summarized in 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 contexts), or bacteria (with biology often distant from that specifically involved in past origins of multicellularity). As functional genetic tools continue to become available in a growing range of phylogenetically relevant single-cell organisms ( Booth et al. 2018 ; Booth and King 2020 ; Parra-Acero et al. 2018 ; Suga and Ruiz-Trillo 2013 ), the time seems ripe for the application of the principles of synthetic biology to these taxa, to approach the dream of “re-running the tape” of the origin of animals, plants, fungi, and algae at multiple spatial and temporal scales. 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 ). This means that just as we can rely on principles of chemistry, physics, and geology to guide our understanding of early-life scenarios, we can use evolutionary theory to refine the focus on situations that are likely to be possible from the standpoint of evolutionary processes, as opposed to being overly reliant on exuberant verbal musings. 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 ). Modern-day organisms prevail because mutation rates per nucleotide site are almost always smaller than 10 −8 per generation ( Lynch et al. 2023 ). In the earliest stages of life, error rates were likely much higher, on the order of 10 −2 -10 −1 ( Johnston et al. 2001 ; Attwater et al. 2013 ; Zhang et al. 2013 ; Wachowius and Holliger 2019 ), implying an upper limit to genome size of ~100 nucleotides. Physicist Eigen ( Eigen 1971 ; Eigen and Schuster 1977 ) recognized this problem in postulating that an error catastrophe occurs beyond a critical mutation rate that makes it impossible to maintain the “master” genome sequence and its embodied information. However, a population can survive the loss of the very best genome, as the key to persistence is simply that the average genome be capable of replacing itself. Still, there will always be a minimum mutation rate above which the cumulative deleterious-mutation load is so large that the error threshold is exceeded. Indeed, this basic concept underlies strategies of therapeutic lethal mutagenesis in the management of viral diseases ( 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 ). If the molecules responsible for genome replication are unlinked to the nucleotide sites responsible for general operations, the strength of selection on fidelity is weakened because the inflictor of damage quickly becomes dissociated from the damage it induced. This suggests that prior to genome-size expansion, genome-encoded mechanisms responsible for replication would have been linked to the client genes responsible for the remaining genome functions. In principle, this might have started through physical linkage of multiple functions on single chromosomes or replicons. However, complete association would also be ensured without physical linkage if protocells endowed with membranes reproduced in a purely asexual fashion, i.e., without exchange among protocells. Alternatively, if different tasks were carried out by unlinked molecules before the advent of membranes, these would need to remain spatially associated on physical structures without rampant diffusion such that “anti-mutator genes” would still remain associated with and profit from their less mutationally incapacitated products ( 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 ), but two key modifications are needed for early-life issues. First, natural selection facilitates the entry of anti-mutator mechanisms provided the improvement in fitness is greater than the power of random-genetic drift (the stochastic forces resulting from finite population size). However, existing theory assumes a constant genome size, so extension to early-life issues will need to allow for the progressive appearance of novel functional genes and a dynamic genome size in parallel with mutation-rate modifiers. In principle, the two processes can lead to reinforcement, with genome size and replication fidelity coevolving. Second, whereas genome replication rates are typically decoupled from cell-division times in contemporary cells laden with other cellular infrastructure, in the earliest cells composed of little more than a membrane and a few ribozymes, genome-size expansion would likely increase baseline replication times. Thus, there is a need to consider the balance between improved functionality and replication fidelity, which enhances the production of appropriate progeny genotypes, and reduced rates of entry of such progeny into the population, which reduces the rate of adaptation (discussed further below). 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 ). If in the earliest stages of life's origin, single RNA molecules served as both genomes and phenotypes and nothing else, the individuality requirement might have been met, but such a link would have been broken as soon as individual genomes began generating side products for enhancing rates of resource acquisition and genomic replication. Thus, whereas physical structures such as the individual pores of towers emanating from hydrothermal vents are sometimes viewed as logical steps towards life from the standpoint of metabolism, there remains the problem of the diffusion of products into the shared public-goods environment. This then leads to the supposition that the establishment of membrane-bound genomes must have been an essential early step in the origin of life, perhaps a step between the emergence of sequence-based RNA biology and the emergence of full reliance on DNA and proteins ( 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 ). Upon entry into the protein world, the introduction of mechanisms that enabled cells to sequester and eventually synthesize lipids would have been a game changer by enabling cells to colonize environments lacking free lipids. 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 ; Chillón and Marcia 2021). The tendency for single-stranded RNAs, especially functional ones, to fold into structures containing stems and loops might have been a challenge to evolving multigenic chromosomes in the RNA world. However, the existence of double-stranded and negative-sense RNA viruses indicates that genomes based on this kind of molecule are possible, perhaps with one strand constituting the template for genomic replication and the other comprising the functional ribozymes ( 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 ). In addition, unless rates of compensatory mutation are quite high, recurrent and permanent stochastic loss of optimal fitness classes can lead to mutational meltdown and eventual extinction of asexual populations due to Muller's ratchet (Lynch et al 1993; Gabriel et al. 1993 ). Such processes are directly related to the issue of error catastrophes noted above ( 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 ). Either way, as with all mutations, novel insertions initiate as single copies within single individuals and hence are vulnerable to early loss by random genetic drift and/or subsequent loss by stochastic inactivation or deletion, the rates of which would have been extraordinarily high in the RNA world with poor replication fidelity. Permanent maintenance of a novel gene requires one or more forms of positive selection: (i) endowment with a new beneficial function promoted by positive selection, (ii) continued self-proliferation of the invading element by subsequent self-proliferation (as in mobile-genetic elements), or (iii) self-preservation by a toxin-antitoxin system (as in many bacterial plasmids). 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 ). Origin-of-life research could therefore profit from the extension of theory that has been developed for the expansion of selfish mobile-genetic elements in the context of different population-genetic environments ( 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 origin of an entirely new function is a rare event, early biology represents a different environment. The successful establishment of new genes requires at a minimum that the selective advantage of any novel function exceeds its rate of mutational inactivation. While simple, unoptimized genomes would have had wide-open possibilities for rapid improvement, this could nonetheless have posed a challenge in a high-mutation-rate world. For the case of gene duplications, considerable theory has already been developed to ascertain the likelihood of preservation of gene copies by neofunctionalization ( Lynch et al. 2001 ). There are however special considerations that will need to be incorporated into early-life scenarios for genome expansion. For example, the genomes of today's bacterial cells contain 1-10 million nucleotides, and duplicate genes (or other exogenous DNAs) can be strongly selected against just based on their fractional energetic burden on the host cell ( Lynch and Marinov 2015 ). In the earliest stages of evolution, genomes would have contained just hundreds to thousands of nucleotides, and in the absence of one of the above preservation mechanisms, the addition of new genes would be even more strongly selected against. For example, a 100-bp addition to a genome of length 1,000 bp would likely result in a 10% increase in genomic doubling time, whereas this same addition to a 10 7 -bp genome would incur just a 10 −6 selective disadvantage. On the other hand, with so much room for improvement, available beneficial mutations might also have had selective advantages far beyond those typically experienced by today's organisms. 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 ). However, the assembly of simple peptides with functional significance cannot be ruled out, as laboratory studies indicate that, without enzymatic intervention, RNA molecules can facilitate the assembly of noncoded peptide chains ( 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 ) to introduce the concept of statistical proteins. We can be certain that his view is correct, as even in today's cells the translation-error rate remains on the order of 0.001 per translated codon and thus even modern proteins are, to an extent, “statistical” ( 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 ), to achieve an understanding of how the code might have initiated and expanded to the 64-codon language in the LUCA and beyond, there is a need for formal evolutionary models allowing for incremental growth of the code. The simplest starting point would be a refinement of gene-duplication models ( Force et al. 1999 ; Lynch et al. 2001 ), with duplicated ancestral tRNAs having generalized functions incrementally becoming more tailored to specific amino-acid cargos by subfunctionalization or being modified to allow the entry of new amino acids by neofunctionalization, discussed more fully below. Whereas the full framework for such analysis is well established, special attention will need to be given to the tripartite nature of this particular problem: coevolution of codons on template molecules and anticodons on tRNA molecules and of the anticodons and the amino-acid specificity tags on the tRNAs. This then raises the issue of the evolution of “molecular languages,” also discussed further below. 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 -10 7 times greater than replication-error rates, with absolute values on the order of 10 −5 per transcribed nucleotide and 10 −3 per translated codon, respectively ( Lynch et al. 2023 ). Such rates almost certainly would have been higher prior to the establishment of refined mechanisms for transcription and translation. 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 ). Many DNA viruses encode their own thymidylate synthases, ribonucleotide reductases (essential to making deoxyribonucleotides), and polymerases, and some such viruses have U-DNA genomes. In principle, a relatively passive virus might have taken control of its host cell, reverse transcribing the host RNA genome and gradually integrating the host genes into its own genome, eventually leading to complete DNA takeover, and in doing so perhaps achieving a reduction in the mutation rate as a side benefit that allowed for further genome expansion. 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 ), and there are two unrelated classes of thymidylate synthases among bacteria and archaea ( Myllykallio et al. 2002 ). There are major differences between the proteins involved in DNA replication in the bacterial and archaeal lineages ( Leipe et al. 1999 ), and both the RNA and DNA polymerases in mitochondria are clearly derived from viruses ( Moreira 2000 ; Filée et al. 2002 ). Other key proteins with major disparities among deep lineages include DNA ligases, topoisomerases, reverse transcriptase, and helicases ( 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 ) demonstrates a rapidity of early evolutionary change that some find challenging. This predicted origin time could be altered if early rates of molecular evolution were substantially greater than those subsequent to the LUCA, but two significant items remain: (i) the accumulation of ~2,000 genes by the first appearance of the LUCA and (ii) the refinement of the features of those genes by natural selection. This is not, however, necessarily at all shocking. If we suppose a span of 200 Ma from the origin of the RNA world to the establishment of the LUCA, we require an average accumulation rate of ~10 genes/Ma. Such rates are not out of the ordinary, given that rates of gene duplications in contemporary species are on the order of 0.1%-1% per gene per 10 6 generations ( Lynch 2007 ). At those rates, just 100 genes would be expected to spawn 0.1-1 duplicates per 10 6 generations, so just 10-100 generations per year would achieve a rate of ~10 duplicated genes/Ma. Because the initial rate of genome growth was likely superlinear, possibly accompanied by whole-genome duplications, and because the preservation of duplicate genes by neofunctionalization was likely much higher at the dawn of life than it is in today's organisms with more saturated gene functions, these are conservative estimates. 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 /N), where Ne is known as the genetic effective population size. The ratio N e /N is almost always smaller than 1.0 often by orders of magnitude, as advantageous haplotypes leave many more progeny than others, leading to a genetic discounting of many members of the population through repeated bottlenecks. Multiplying the three factors together yields 2suN e as expected, the pace of evolutionary change at the molecular level. This simple and intuitive expression is directly proportional to the selective advantage, the mutational rate of origin, and the effective number of individuals. 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 = 2 v , as both sites can incur matching mutations, and this is the same as the rate of loss of a pre-existing stem pair. The co-occurrences of specific sets of n structural pairs of mutations have probabilities of u = (2 v ) n . In modern-day organisms with u < 10 −8 , the probability of such multinucleotide mutations is thus generally very small, resulting in stepwise molecular evolution. However, with v ≈ 0.01 in the early RNA world, then with n = 4, the rate of arrival of specific tetranucleotide mutations would be ~6 ×10 −6 , so even with a tiny molecular population size of N = 10 6 , multiple newborns of each such complex mutation would arise each generation. Thus, in the earliest stages of life, despite the potential loss of adaptation by mutational degeneration, the rate of emergence of complex adaptions would have been far beyond what occurs in today's organisms. 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 ). In the earliest stages of life, however, the room for improvement would have been much larger. Moreover, if a particular mutant type alters the environment in such a way as to increase the population size, the probability of fixation of a beneficial type can substantially exceed 2s. 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 might have been extraordinarily high in the RNA world, but it is also plausible that N e was exceptionally low, as the high rate of appearance of background deleterious mutations would have greatly obscured the signal of individual beneficial mutations. This issue can be resolved by extending theory to cases in which mutation rates are many orders of magnitude greater than in today's organisms. 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 and s suggests a potential capacity of early life to evolve at rates many orders of magnitude beyond what is possible in today's sluggish organisms. This speculative supposition can readily be evaluated using the machinery provided by population-genetic theory. 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 ), the population vaults a barrier of potentially low fitness, never being imperiled in the process. For example, if u for a particular kind of complex mutation in the RNA world were to be 10 6 -fold higher than in today's organisms (a huge understatement for the tetranucleotide mutation case described above), this would imply a 10 12 -fold increase in the rate of stochastic tunneling. Presented with possibility, it appears that dramatic accelerations in the mutual establishment of multiple adaptations, involving the simultaneous fixation of multiple interacting genomic sites, may have occurred in the earliest stage of life. 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 years. Even if one imagines the replication time of the earliest molecules in the RNA world to have been as long as one year, given the above parameters and given that there may have been many parallel sites of developing RNA worlds, it seems clear that provided the availability of key resources, an early RNA world could have become established and substantially refined on a very short timescale. These crude examples simply illustrate the ways in which conventional evolutionary theory, which applies even to competing populations of naked molecules, can be used to examine the plausibility of various scenarios for the earliest stages in the emergence of life. 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 per nucleotide site per replication [based on known rates in today's prokaryotes ( Lynch et al. 2023 )]. Most molecular features of cells had become highly refined, so average selective advantages of downstream modifications would have been much smaller than those prior to the emergence of the LUCA. Numerous new morphological features of cells emerged in descendant eukaryotic lineages, although metabolic changes were less common. As the average population-genetic environment had clearly changed since that experienced in the first 0.5-1.0 billion years of evolution on the planet, this raises questions about the mechanisms of evolution in the apparently explosive origin of the eukaryotic domain. 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 ). Features that appear to be designed to reduce the sensitivity to malfunctions, i.e., to enhance robustness, may in fact be no more efficient than their simpler predecessors in ancestral species ( Frank 2007 ; Frank 2023 ; Lynch 2012 ), as the addition of novel lines of defense leads to the relaxation on prior functions. 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 ). Gene-structural complexity is also greatly magnified in eukaryotic lineages, especially in multicellular lineages, but there is no evidence that embellishments such as intron colonization and expansion of untranslated regions on transcripts were advanced via advantages bestowed upon host genes ( Lynch 2007 ). Rather, each such addition to eukaryotic genes imposes problems associated with energetic costs and mutational vulnerability. In this brief synopsis, we consider some of the key questions and approaches relevant to resolving the issue of how eukaryotic cellular complexity can arise by nonadaptive processes. 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 ), and this may have been particularly common in the emergence of early life. Duplication is a more likely route to gene birth in more recent epochs. Either way, the potential fates of new genes are threefold: mutational inactivation and/or deletion (nonfunctionalization), preservation by the origin of a novel function (neofunctionalization) with the ancestral copy maintaining the original function, or partitioning of prior independently mutable tasks of the ancestral gene (subfunctionalization) ( Force et al. 1999 ). Owing to various errors during genome replication, gene duplications arise at high rates in all organisms, but in prokaryotes, the most common fate is the rapid purging of one copy from the genome. A likely reason for this is the strong fractional contribution of the energetic cost of an additional gene to the total energy budget of a prokaryotic cell in the absence of the emergence of a novel beneficial function ( Lynch and Marinov 2015 ). This view provides a simple explanation for the genomic streamlining of microbial genomes. 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 ), and natural selection is frequently unable to purge copies. In addition, owing to the more complex gene structure and expression patterns, eukaryotic genes have an elevated probability of preservation by subfunctionalization, a process that is driven entirely by divergent degenerative mutations appearing in each copy. Of course, the capacity for such partitioning of tasks relies on the prior existence of gene subfunctions, but these too arise by localized (smaller scale) duplications in the regulatory regions of eukaryotic genes, which can lead to modular patterns of gene expression ( Force et al. 2005 ). The net consequence is that eukaryotic genomes are subject to the passive accumulation of more complex and numerous genes than typically found in prokaryotes, some of which do indeed eventually acquire novel functions, but with the entire process initially made possible by nonadaptive means. Considerable theory has been developed to help understand how the alternative fates of duplicate genes are influenced by the population-genetic environment (Colbourne 2011). 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 ). Whereas one might argue that this resulted from some sort of expansive opening of novel ecological opportunities made possible by the new eukaryotic cell plan, this alone does not explain the speciation process, which requires the establishment of reproductive isolating barriers, ultimately in the form of genomic incompatibilities. 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 ). Imagine a pair of gene duplicates present on different chromosomes in an ancestral species. If reciprocal copies are silenced in a pair of incipient sister taxa, a change in the genetic map will have occurred, resulting in hybrids being presence/absence heterozygotes at both loci. In this case, by random segregation during meiosis, 1/4 of the gametes produced by such individuals will be completely lacking in gene function. Because gene duplication is an ongoing process across the genomes of eukaryotes, such incompatibilities will continue to accumulate over time, but the effect may have been unusually intense at the base of the eukaryotic lineage. Substantial evidence suggests that a massive amount of gene duplication occurred prior to the emergence of the LECA, and the possibility of one or more whole-genome duplications cannot be ruled out ( Lynch 2024 ). Given that hundreds to thousands of genes would have been subject to random reciprocal silencing during this early period of genomic expansion, the early and rapid proliferation of the main eukaryotic groups was likely facilitated by passive population-genetic processes that would have existed regardless of ecological circumstances. 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 ). Moreover, arguments for the adaptive value of complexity beg the question as to why prokaryotes, with billions of years and millions of lineages, have not gone down similar pathways. 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 ). If, for example, some protein A begins to bind to another protein B in a way that does not significantly abrogate the functions of either, an A:B interface will have been initiated, providing a potentially protective environment for the accumulation of previously forbidden mutations. For example, mutations to hydrophobic residues that would ordinarily be harmful on protein surfaces might be rendered neutral in this context. However, once established, the presence of such mutations may lock the complex into a permanent state, as exposure would result in malfunctional proteins. The so-called onionskin model for the accreted growth of the ribosomal proteins is often cited as the archetype of such exuberant growth. 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 ). However, the arguments made by these authors do not hold up to scrutiny from the perspective of evolutionary genetics, bioenergetics, and molecular and cellular biology ( Lynch and Marinov 2015 ; Lynch and Marinov 2017 ; Chiyomaru and Takemoto, 2020 ; Pittis and Gabaldon 2016; Gabaldón 2018 ; Hampl 2019). Whereas it is certainly correct that the mitochondrion is the primary engine of ATP production in eukaryotes, which relinquished the use of ATP synthase on external cell membranes, when cell size is taken into consideration, eukaryotes are not endowed with any particularly special bioenergetic capacity, and the colonization of novel genes is made easier, not more difficult, by drift processes with the expansion of cell size based on population-genetic consideration alone. 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 ). Analogous situations, commonly referred to as developmental systems drift, have been uncovered in studies of the developmental pathways of animals ( 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 ), understanding how molecular languages become established in the first place remains an entirely open question. Although it is likely that periods of redundancy are required, the paths by which regulatory rewiring become established via population-genetic processes are virtually unexplored. All of these issues are central to understanding the emergence of diverse lineages, as the loss of communication capacity between the molecules in related lineages constitutes a barrier to gene flow. 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 ) or redox gradients driving abiotic carbon fixation ( Vasiliadou et al. 2019 ; Lane and Martin 2012 ). However, the role of these plausible environments in driving abiotic processes remains very poorly constrained. 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 ) of the last universal common ancestor. Modern organisms also represent only the current survivors of a vast evolutionary experiment, leaving much of the diversity and innovation of early-life processes lost to time and perhaps only a subset of all innovations that occurred surviving to this day ( De Duve 2005 ). These constraints force researchers to rely on indirect evidence and theoretical models, which struggle to reconstruct the complexity of prebiotic processes in a way that does not simply recapitulate the assumptions that went into them. 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 ) could be used to explore the interplay between compartmentalization and catalytic efficiency. 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 ), researchers can identify conserved elements that offer clues to the structure and function of ancestral states. For example, studying the ancient folds of proteins involved in modern metabolic cycles could reveal their deepest evolutionary origins and inform hypotheses about the first enzymatic functions. 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 ). These experiments stand to reveal how compartmentalization and chemical heterogeneity contributed to the emergence of complexity and metabolism in prebiotic systems. 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 ). As always, models also provide a means of testing hypotheses in silico, saving time and resources in the laboratory. 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 ), but key alternative settings need to be explored as well. High-throughput platforms for testing vesicle permeability, growth/replication, and compartmentalization could provide crucial data on the transition from chemical networks to individuated cellular systems. Systems chemistry approaches that integrate prebiotic chemistry with physical processes such as wet-dry cycling, mineral templating, and alternative chemical backbones could expand our understanding of life's potential diversity. For instance, studying systems that use alternative energy sources or non-phosphorus-based backbones could reveal new pathways for prebiotic evolution. 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 ). Inferring the earliest events in the evolution of eukaryotes over such a vast amount of time since their origin, coupled with the fact that many early events may not be captured or are rare in the fossil record of this time, is a major challenge. Though there has been impressive work documenting ancient microfossil diversity and interpreting planetary conditions during the period in which eukaryotes likely arose, there is much still to be learned here by continuing to uncover more diversity and pin down the time of major evolutionary events. 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 ). Beyond community sequencing, advances in single-cell technologies now open the way for their study; isolation via fluorescence-activated cell sorting allows single cells to be studied regardless of cultivability via microscopy and molecular probing, or even single-cell genome/transcriptomesequencing. Continued exploration of uncultivable lineages stands to greatly expand our knowledge of the diversity of eukaryotes as well as their poorly cultivable Asgard archaeal relatives. Advancing this field requires refining research strategies and prioritizing hypothesis-driven inquiry. 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 ). Staining or other analysis of lipid components and vesicles in Asgard archaea and endosymbiotic bacteria can reveal whether membrane compartmentalization was already emerging before eukaryotes evolved. Live cell imaging with fluorescent markers allows tracking of cell division, vesicle formation, and cytoskeletal organization in Asgard archaea, considered the closest prokaryotic relatives of eukaryotes. Live-cell imaging may also enhance the study of symbiotic interactions between archaea and bacteria and in the study of phagocytosis-like mechanisms. 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 ) and within the next five years the pace of progress will likely increase dramatically, accelerating an already rapid expansion of structure inference without time-consuming crystallography. The field is poised for investment and dramatic breakthroughs using this technology. 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 gene evolution. Eukaryotic genes also evolve faster, potentially obscuring their prokaryotic origins. Structural homology methods may help distinguish true eukaryotic innovations from fast-evolving prokaryote-sourced genes. Gene duplication and subfunctionalization contributed to eukaryotic functions (e.g., GTPases, kinases, and transcription factors), but the extent of duplication and loss before the LECA remains unclear. A refined LECA reconstruction could clarify these processes and their role in eukaryogenesis. 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 ). 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. Such a transition, or a more complex symbiosis without extensive membrane replacement, could also be tied to the origin of the endomembrane system. Ascertaining the provenance of membrane structures and chemicals in eukaryogenesis via genetic manipulation of prokaryotes and study of eukaryotic membrane proteins stands to narrow down the field of possible eukaryotic origin models. 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 ). These approaches could be explored and standardized using large amounts of pre-existing data, particularly from natural history collections containing vast numbers of preserved specimens spanning the breadth of multicellular diversity. However, it should be kept in mind that there is substantial disagreement among biologists as to the very definition of complexity and whether this is a direct target of selection. 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 ) to the electrical networks formed by cable bacteria, prokaryotes display a remarkable array of multicellular innovations. However, these forms of multicellularity are often cryptic and environmentally dependent, requiring specialized tools and approaches to detect and characterize. This raises the possibility that we have systematically underestimated the prevalence and complexity of prokaryotic multicellularity in nature. 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/ ) for eukaryotes, at least as a curated database for genomes and/or transcriptomes. Colloquium participants believe that one of the biggest problems such a center could solve is providing access to eukaryotic data sets in a format that is universally accessible to others, in a comprehensively standardized and maintained form. In particular, converting the data of transcriptomes to protein annotation was cited as particularly helpful by researchers. 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 ), and providing them with long-term stability would ensure their continued productivity. 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, and Leishmania) belong to poorly understood eukaryotic lineages that deserve deeper study. Studying their evolution and comparative genomics could reveal metabolic vulnerabilities for targeted therapies. 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, and Hemimastigophora (cytoskeletal and flagellar structures). Metamonads and anaerobic protists (useful in the study of mitochondria evolution). Carpediemonas-like organisms (close to the LECA). Marine and unicellular eukaryotes with unique genomic features (Ancyromonads, Telonemids (plastic evolution), Centrohelids, and Heliomonads (starlike axopodia). Plastid-bearing lineages with cryptic evolutionary histories, i.e., Glaucophytes and Apicomplexan relatives, e.g., Chromera, Vitrella, and Perkinsids (plastids, chloroplast evolution, plastid loss, and secondary endosymbiosis). Early-diverging fungi and opisthokonts, e.g., Rozellids and Microsporidia (genome reduction and parasitism relationship to genome evolution) Fonticulids and Nuclearids (insights into ancestors of fungi). 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 show viability up to ~30% archaeal phospholipids in membranes. However, higher percentages of archaeal lipids are deleterious. Experimental evolution should help ascertain if the full membrane phospholipids can be replaced, especially in the context of natural competition with native bacteria. Experiments replacing archaeal by bacterial phospholipids and assessing the effects on the archaeal fitness would be more relevant. Alternatively, the bacterial-like membranes of eukaryotes might have been directly inherited from a bacterial host. In this case, it would be expected that other components related to membranes, signaling, and exchange with the external environment have predominantly bacterial origins, even if a degree of chimerism exists with archaeal proteins (membrane-bending proteins seem mostly of archaeal origin in eukaryotes). Studying the phylogenetic origin of these different pathways and processes ancestrally present in eukaryotes will be required. 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 is technically challenging and very labor intensive. 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. 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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 Given the extent of gene transfer, inferring the root of the ribosomal phylogeny from the root determined using ancient gene duplications observed in other gene families remains controversial (see Doolittle and Brunet 2016 for discussion). The fact that the duplicated genes used to root their respective phylogenies include elongation factors, aaRSs, and signal recognition particles can be seen as an emerging consensus signal for the translation machinery. © 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 DOI: 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. doi: 10.1128/AAMCol.Jun.2025 PDF version of this title (12M) 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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