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Plastids evolved from a cyanobacterium that was engulfed by a heterotrophic eukaryotic host and became a stable organelle. Some of the resulting eukaryotic algae entered into a number of secondary endosymbioses with diverse eukaryotic hosts. These events had major consequences on the evolution and diversification of life on Earth. Although almost all plastid diversity derives from a single endosymbiotic event, the analysis of nuclear genomes of plastid-bearing lineages has revealed a mosaic origin of plastid-related genes. In addition to cyanobacterial genes, plastids recruited for their functioning eukaryotic proteins encoded by the host nucleus and also bacterial proteins of noncyanobacterial origin. Therefore, plastid proteins and plastid-localised metabolic pathways evolved by tinkering and using gene toolkits from different sources. This mixed heritage seems especially complex in secondary algae containing green plastids, the acquisition of which appears to have been facilitated by many previous acquisitions of red algal genes (the ‘red carpet hypothesis’).

Asgard archaea are considered to be the closest known relatives of eukaryotes. Their genomes contain hundreds of eukaryotic signature proteins (ESPs), which inspired hypotheses on the evolution of the eukaryotic cell 1 – 3 . A role of ESPs in the formation of an elaborate cytoskeleton and complex cellular structures has been postulated 4 – 6 , but never visualized. Here we describe a highly enriched culture of ‘ Candidatus Lokiarchaeum ossiferum’, a member of the Asgard phylum, which thrives anaerobically at 20 °C on organic carbon sources. It divides every 7–14 days, reaches cell densities of up to 5 × 10 7 cells per ml and has a significantly larger genome compared with the single previously cultivated Asgard strain 7 . ESPs represent 5% of its protein-coding genes, including four actin homologues. We imaged the enrichment culture using cryo-electron tomography, identifying ‘ Ca . L. ossiferum’ cells on the basis of characteristic expansion segments of their ribosomes. Cells exhibited coccoid cell bodies and a network of branched protrusions with frequent constrictions. The cell envelope consists of a single membrane and complex surface structures. A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions. The twisted double-stranded architecture of the filaments is consistent with F-actin. Immunostaining indicates that the filaments comprise Lokiactin—one of the most highly conserved ESPs in Asgard archaea. We propose that a complex actin-based cytoskeleton predated the emergence of the first eukaryotes and was a crucial feature in the evolution of the Asgard phylum by scaffolding elaborate cellular structures. Culture and analysis of ‘ Candidatus  Lokiarchaeum ossiferum’—a member of the Asgard phylum—reveals an elaborate cell architecture with extensive membranous protrusions.

Ammonia oxidation, as the first step of nitrification, constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase, is lacking, in particular for the environmentally abundant ammonia-oxidizing archaea (AOA). Here the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Besides the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modeling and in-gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase but also reveals clear differences in extracellular domains of the enzyme. These data open avenues for further structure-function studies of this ecologically important nitrification complex.

The recently established phylogeny of amoA provides a finer resolution than previous studies, allowing clustering of AOA beyond the order level and thus revealing novel clades. While the 16S rRNA gene is mostly appreciated in microbiome studies, this novel phylogeny is in limited use. A highly resolved taxonomy for ammonia-oxidizing archaea (AOA) based on the alpha subunit of ammonia monooxygenase ( amoA ) was recently established, which uncovered novel environmental patterns of AOA, challenging previous generalizations. However, many microbiome studies target the 16S rRNA gene as a marker; thus, the usage of this novel taxonomy is currently limited. Here, we exploited the phylogenetic congruence of archaeal amoA and 16S rRNA genes to link 16S rRNA gene classification to the novel amoA taxonomy. We screened publicly available archaeal genomes and contigs for the co-occurring amoA and 16S rRNA genes and constructed a 16S rRNA gene database with the corresponding amoA clade taxonomy. Phylogenetic trees of both marker genes confirmed congruence, enabling the identification of clades. We validated this approach with 16S rRNA gene amplicon data from peatland soils. We succeeded in linking 16S rRNA gene amplicon sequence variants belonging to the class Nitrososphaeria to seven different AOA ( amoA ) clades, including two of the most frequently detected clades ( Nitrososphaerales γ and δ clades) for which no pure culture is currently available. Water status significantly impacted the distribution of the AOA clades as well as the whole AOA community structure, which was correlated with pH, nitrate, and ammonium, consistent with previous clade predictions. Our study emphasizes the need to distinguish among AOA clades with distinct ecophysiologies and environmental preferences, for a better understanding of the ecology of the globally abundant AOA. IMPORTANCE The recently established phylogeny of amoA provides a finer resolution than previous studies, allowing clustering of AOA beyond the order level and thus revealing novel clades. While the 16S rRNA gene is mostly appreciated in microbiome studies, this novel phylogeny is in limited use. Here, we provide an alternative path to identifying AOA with this novel and highly resolved amoA taxonomy by using 16S rRNA gene sequencing data. We constructed a 16S rRNA gene database with the associated amoA clade taxonomy based on their phylogenetic congruence. With this database, we were able to assign 16S rRNA gene amplicons from peatland soils to different AOA clades, with a level of resolution provided previously only by amoA phylogeny. As 16S rRNA gene amplicon sequencing is still widely employed in microbiome studies, our database may have a broad application for interpreting the ecology of globally abundant AOA.

Abstract Photosynthetic eukaryotes and their relatives are the result of an intricate evolutionary history involving a series of plastid acquisitions through endosymbiosis, multiple reversions to heterotrophy, and sometimes total plastid losses. Among these events, one of the most debated is the emergence and diversification of the CASH lineages (Cryptophyta, Alveolata, Stramenopiles, and Haptophyta). Although they all include species bearing a complex plastid that derived from the endosymbiosis of a red alga, their phylogenetic relationships remain controversial, and the timing and number of plastid acquisitions are still undetermined. The inner metabolism of all plastids is mostly supported by nuclear-encoded proteins, and consequently, mechanisms allowing the relocation of those proteins have evolved or were recycled at each endosymbiotic event. Thus, the study of the composition and origins of those translocation machineries provides important clues for understanding how photosynthetic lineages have emerged and might be related. In CASH species, the SELMA complex, composed of about 20 proteins, is dedicated to the transport of preproteins across the periplastidial membrane, the second outermost membrane of complex red plastids. In this work, we present a comprehensive genomic survey and phylogenetic analysis of the proteins composing the SELMA complex. We confirm the presence, homology, and monophyletic origin of SELMA in the four CASH lineages and use these observations to infer a scenario for the serial transmission of secondary red plastids that differs from previous hypotheses and sheds new light on the evolution of photosynthetic eukaryotes.

Marine sediments represent a vast habitat for complex microbiomes. Among these, ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are one of the most common, yet little explored, inhabitants, which seem extraordinarily well adapted to the harsh conditions of the subsurface biosphere. We present 11 metagenome-assembled genomes of the most abundant AOA clades from sediment cores obtained from the Atlantic Mid-Ocean ridge flanks and Pacific abyssal plains. Their phylogenomic placement reveals three independently evolved clades within the order Nitrosopumilales, of which no cultured representative is known yet. In addition to the gene sets for ammonia oxidation and carbon fixation known from other AOA, all genomes encode an extended capacity for the conversion of fermentation products that can be channeled into the central carbon metabolism, as well as uptake of amino acids probably for protein maintenance or as an ammonia source. Two lineages encode an additional (V-type) ATPase and a large repertoire of DNA repair systems that may allow to overcome the challenges of high hydrostatic pressure. We suggest that the adaptive radiation of AOA into marine sediments occurred more than once in evolution and resulted in three distinct lineages with particular adaptations to this extremely energy-limiting and high-pressure environment.

Endosymbiosis has been common all along eukaryotic evolution, providing opportunities for genomic and organellar innovation. Plastids are a prominent example. After the primary endosymbiosis of the cyanobacterial plastid ancestor, photosynthesis spread in many eukaryotic lineages via secondary endosymbioses involving red or green algal endosymbionts and diverse heterotrophic hosts. However, the number of secondary endosymbioses and how they occurred remain poorly understood. In particular, contrasting patterns of endosymbiotic gene transfer have been detected and subjected to various interpretations. In this context, accurate detection of endosymbiotic gene transfers is essential to avoid wrong evolutionary conclusions. We have assembled a strictly selected set of markers that provides robust phylogenomic evidence suggesting that nuclear genes involved in the function and maintenance of green secondary plastids in chlorarachniophytes and euglenids have unexpected mixed red and green algal origins. This mixed ancestry contrasts with the clear red algal origin of most nuclear genes carrying similar functions in secondary algae with red plastids.

A highly resolved taxonomy for ammonia-oxidizing archaea (AOA) based on the alpha subunit of ammonia monooxygenase (amoA) was recently established, which uncovered novel environmental patterns of AOA, challenging previous generalizations. However, many microbiome studies target the 16S rRNA gene as a marker; thus, the usage of this novel taxonomy is currently limited. Here, we exploited the phylogenetic congruence of archaeal amoA and 16S rRNA genes to link 16S rRNA gene classification to the novel amoA taxonomy. We screened publicly available archaeal genomes and contigs for the co-occurring amoA and 16S rRNA genes and constructed a 16S rRNA gene database with the corresponding amoA clade taxonomy. Phylogenetic trees of both marker genes confirmed congruence, enabling the identification of clades. We validated this approach with 16S rRNA gene amplicon data from peatland soils. We succeeded in linking 16S rRNA gene amplicon sequence variants belonging to the class Nitrososphaeria to seven different AOA (amoA) clades, including two of the most frequently detected clades (Nitrososphaerales γ and δ clades) for which no pure culture is currently available. Water status significantly impacted the distribution of the AOA clades as well as the whole AOA community structure, which was correlated with pH, nitrate, and ammonium, consistent with previous clade predictions. Our study emphasizes the need to distinguish among AOA clades with distinct ecophysiologies and environmental preferences, for a better understanding of the ecology of the globally abundant AOA. IMPORTANCE The recently established phylogeny of amoA provides a finer resolution than previous studies, allowing clustering of AOA beyond the order level and thus revealing novel clades. While the 16S rRNA gene is mostly appreciated in microbiome studies, this novel phylogeny is in limited use. Here, we provide an alternative path to identifying AOA with this novel and highly resolved amoA taxonomy by using 16S rRNA gene sequencing data. We constructed a 16S rRNA gene database with the associated amoA clade taxonomy based on their phylogenetic congruence. With this database, we were able to assign 16S rRNA gene amplicons from peatland soils to different AOA clades, with a level of resolution provided previously only by amoA phylogeny. As 16S rRNA gene amplicon sequencing is still widely employed in microbiome studies, our database may have a broad application for interpreting the ecology of globally abundant AOA.

Ammonia oxidation as the first step of nitrification constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase is lacking, in particular for the environmentally abundant ammonia oxidizing archaea (AOA). Here, the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Beside the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modelling and in gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase indicating that AmoY and AmoZ serve an important structural and functional role. These data open avenues for further structure-function studies of this ecologically important key nitrification complex. Competing Interest Statement The authors have declared no competing interest.