Archaeal cells are typically enveloped by glycosylated S-layer proteins. Archaeal protein glycosylation provides valuable insights not only into their adaptation to their niches but also into their evolutionary trajectory. Notably, thermophilic Thermoproteota modify proteins with N-glycans that include two GlcNAc units at the reducing end, resembling the \"core structure\" preserved across eukaryotes. Recently, Asgard archaea, now classified as members of the phylum Promethearchaeota, have offered unprecedented opportunities for understanding the role of archaea in eukaryogenesis. Despite the presence of genes indicative of protein N-glycosylation in this archaeal group, these have not been experimentally investigated. Here we performed a glycoproteome analysis of the firstly isolated Asgard archaeon Promethearchaeum syntrophicum. Over 700 different proteins were identified through high-resolution LC-MS/MS analysis, however, there was no evidence of either the presence or glycosylation of putative S-layer proteins. Instead, N-glycosylation in this archaeon was primarily observed in an extracellular solute-binding protein, possibly related to chemoreception or transmembrane transport of oligopeptides. The glycan modification occurred on an asparagine residue located within the conserved N-X-S/T sequon, consistent with the pattern found in other archaea, bacteria, and eukaryotes. Unexpectedly, three structurally different N-glycans lacking the conventional core structure were identified in this archaeon, presenting unique compositions that included atypical sugars. Notably, one of these sugars was likely HexNAc modified with a threonine residue, similar to modifications previously observed in mesophilic methanogens within the Methanobacteriati. Our findings advance our understanding of Asgard archaea physiology and evolutionary dynamics.

SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.

Steroids are indispensable components of the eukaryotic cellular membrane and the acquisition of steroid biosynthesis was a key factor that enabled the evolution of eukaryotes. The polycyclic carbon structures of steroids can be preserved in sedimentary rocks as chemical fossils for billions of years and thus provide invaluable clues to trace eukaryotic evolution from the distant past. Steroid biosynthesis consists of (1) the production of protosteroids and (2) the subsequent modifications toward \"modern-type\" steroids such as cholesterol and stigmasterol. While protosteroid biosynthesis requires only two genes for the cyclization of squalene, complete modification of protosteroids involves ~10 additional genes. Eukaryotes universally possess at least some of those additional genes and thus produce modern-type steroids as major final products. The geological biomarker records suggest a prolonged period of solely protosteroid production in the mid-Proterozoic before the advent of modern-type steroids in the Neoproterozoic. It has been proposed that mid-Proterozoic protosteroids were produced by hypothetical stem-group eukaryotes that presumably possessed genes only for protosteroid production, even though in modern environments protosteroid production as a final product is found exclusively in bacteria. The host identity of mid-Proterozoic steroid producers is crucial for understanding the early evolution of eukaryotes. In this perspective, we discuss how geological biomarker data and genetic data complement each other and potentially provide a more coherent scenario for the evolution of steroids and associated early eukaryotes. We further discuss the potential impacts that steroids had on the evolution of aerobic metabolism in eukaryotes, which may have been an important factor for the eventual ecological dominance of eukaryotes in many modern environments.

Microbial hydrogen (H2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H2-metabolizing enzymes.

The division of organisms on the Tree of Life into either a three-domain (3D) tree or a two-domain (2D) tree has been disputed for a long time. Ever since the discovery of Archaea by Carl Woese in 1977 using 16S ribosomal RNA sequence as the evolutionary marker, there has been a great advance in our knowledge of not only the growing diversity of Archaea but also the evolutionary relationships between different lineages of living organisms. Here, we present this perspective to summarize the progress of archaeal diversity and changing notion of the Tree of Life. Meanwhile, we provide the latest progress in genomics/physiology-based discovery of Asgard archaeal lineages as the closest relative of Eukaryotes. Furthermore, we propose three major directions for future research on exploring the \"next one\" closest Eukaryote relative, deciphering the function of archaeal eukaryotic signature proteins and eukaryogenesis from both genomic and physiological aspects, and understanding the roles of horizontal gene transfer, viruses, and mobile elements in eukaryogenesis.

Malate dehydrogenase (MDH) is a ubiquitous enzyme involved in cellular respiration across all domains of life. MDH\'s ubiquity allows it to act as an excellent model for considering the history of life and how the rise of aerobic respiration and eukaryogenesis influenced this evolutionary process. Here, we present the diversity of the MDH family of enzymes across bacteria, archaea, and eukarya, the relationship between MDH and lactate dehydrogenase (LDH) in the formation of a protein superfamily, and the connections between MDH and endosymbiosis in the formation of mitochondria and chloroplasts. The development of novel and powerful DNA sequencing techniques has challenged some of the conventional wisdom underlying MDH evolution and suggests a history dominated by gene duplication, horizontal gene transfer, and cryptic endosymbiosis events and adaptation to a diverse range of environments across all domains of life over evolutionary time. The data also suggest a superfamily of proteins that do not share high levels of sequential similarity but yet retain strong conservation of core function via key amino acid residues and secondary structural components. As DNA sequencing and \'big data\' analysis techniques continue to improve in the life sciences, it is likely that the story of MDH will continue to refine as more examples of superfamily diversity are recovered from nature and analyzed.

Isoprenoids and their derivatives, essential for all cellular life on Earth, are particularly crucial in archaeal membrane lipids, suggesting that their biosynthesis pathways have ancient origins and play pivotal roles in the evolution of early life. Despite all eukaryotes, archaea, and a few bacterial lineages being known to exclusively use the mevalonate (MVA) pathway to synthesize isoprenoids, the origin and evolutionary trajectory of the MVA pathway remain controversial. Here, we conducted a thorough comparison and phylogenetic analysis of key enzymes across the four types of MVA pathway, with the particular inclusion of metagenome assembled genomes (MAGs) from uncultivated archaea. Our findings support an archaeal origin of the MVA pathway, likely postdating the divergence of Bacteria and Archaea from the Last Universal Common Ancestor (LUCA), thus implying the LUCA\'s enzymatic inability for isoprenoid biosynthesis. Notably, the Asgard archaea are implicated in playing central roles in the evolution of the MVA pathway, serving not only as putative ancestors of the eukaryote- and Thermoplasma-type routes, but also as crucial mediators in the gene transfer to eukaryotes, possibly during eukaryogenesis. Overall, this study advances our understanding of the origin and evolutionary history of the MVA pathway, providing unique insights into the lipid divide and the evolution of early life.

All organisms living on Earth descended from a single, common ancestral population of cells, known as LUCA-the last universal common ancestor. Since its emergence, the diversity and complexity of life have increased dramatically. This chapter focuses on four key biological innovations throughout Earth\'s history that had a significant impact on the expansion of phylogenetic diversity, organismal complexity, and ecospace habitation. First is the emergence of the last universal common ancestor, LUCA, which laid the foundation for all life-forms on Earth. Second is the evolution of oxygenic photosynthesis, which resulted in global geochemical and biological transformations. Third is the appearance of a new type of cell-the eukaryotic cell-which led to the origin of a new domain of life and the basis for complex multicellularity. Fourth is the multiple independent origins of multicellularity, resulting in the emergence of a new level of complex individuality. A discussion of these four key events will improve our understanding of the intertwined history of our planet and its inhabitants and better inform the extent to which we can expect life at different degrees of diversity and complexity elsewhere.

Inner membranes of mitochondria are extensively folded, forming cristae. The observed overall correlation between efficient eukaryotic ATP generation and the area of internal mitochondrial inner membranes both in unicellular organisms and metazoan tissues seems to explain why they evolved. However, the crucial use of molecular oxygen (O2) as final acceptor of the electron transport chain is still not sufficiently appreciated. O2 was an essential prerequisite for cristae development during early eukaryogenesis and could be the factor allowing cristae retention upon loss of mitochondrial ATP generation. Here I analyze illuminating bacterial and unicellular eukaryotic examples. I also discuss formative influences of intracellular O2 consumption on the evolution of the last eukaryotic common ancestor (LECA). These considerations bring about an explanation for the many genes coming from other organisms than the archaeon and bacterium merging at the start of eukaryogenesis.

Both the concept of a Darwinian tree of life (TOL) and the possibility of its accurate reconstruction have been much criticized. Criticisms mostly revolve around the extensive occurrence of lateral gene transfer (LGT), instances of uptake of complete organisms to become organelles (with the associated subsequent gene transfer to the nucleus), as well as the implications of more subtle aspects of the biological species concept. Here we argue that none of these criticisms are sufficient to abandon the valuable TOL concept and the biological realities it captures. Especially important is the need to conceptually distinguish between organismal trees and gene trees, which necessitates incorporating insights of widely occurring LGT into modern evolutionary theory. We demonstrate that all criticisms, while based on important new findings, do not invalidate the TOL. After considering the implications of these new insights, we find that the contours of evolution are best represented by a TOL.