BE3 is the major branching enzyme isoform required for amylopectin synthesis in Chlamydomonas reinhardtii.

Starch-branching enzymes (BEs) are essential for starch synthesis in both plants and algae where they influence the architecture and physical properties of starch granules. Within Embryophytes, BEs are classified as type 1 and type 2 depending on their substrate preference. In this article, we report the characterization of the three BE isoforms encoded in the genome of the starch producing green algae Chlamydomonas reinhardtii: two type 2 BEs (BE2 and BE3) and a single type 1 BE (BE1). Using single mutant strains, we analyzed the consequences of the lack of each isoform on both transitory and storage starches. The transferred glucan substrate and the chain length specificities of each isoform were also determined. We show that only BE2 and BE3 isoforms are involved in starch synthesis and that, although both isoforms possess similar enzymatic properties, BE3 is critical for both transitory and storage starch metabolism. Finally, we propose putative explanations for the strong phenotype differences evidenced between the C. reinhardtii be2 and be3 mutants, including functional redundancy, enzymatic regulation or alterations in the composition of multimeric enzyme complexes.

Metagenome-derived virus-microbe ratios across ecosystems.

It is generally assumed that viruses outnumber cells on Earth by at least tenfold. Virus-to-microbe ratios (VMR) are largely based on counts of fluorescently labelled virus-like particles. However, these exclude intracellular viruses and potentially include false positives (DNA-containing vesicles, gene-transfer agents, unspecifically stained inert particles). Here, we develop a metagenome-based VMR estimate (mVRM) that accounts for DNA viruses across all stages of their replication cycles (virion, intracellular lytic and lysogenic) by using normalised RPKM (reads per kilobase of gene sequence per million of mapped metagenome reads) counts of the major capsid protein (MCP) genes and cellular universal single-copy genes (USCGs) as proxies for virus and cell counts, respectively. After benchmarking this strategy using mock metagenomes with increasing VMR, we inferred mVMR across different biomes. To properly estimate mVMR in aquatic ecosystems, we generated metagenomes from co-occurring cellular and viral fractions (>50 kDa-200 µm size-range) in freshwater, seawater and solar saltern ponds (10 metagenomes, 2 control metaviromes). Viruses outnumbered cells in freshwater by ~13 fold and in plankton from marine and saline waters by ~2-4 fold. However, across an additional set of 121 diverse non-aquatic metagenomes including microbial mats, microbialites, soils, freshwater and marine sediments and metazoan-associated microbiomes, viruses, on average, outnumbered cells by barely two-fold. Although viruses likely are the most diverse biological entities on Earth, their global numbers might be closer to those of cells than previously estimated.

Archaeal overdominance close to life-limiting conditions in geothermally influenced hypersaline lakes at the Danakil Depression, Ethiopia.

The Dallol protovolcanic area on the Danakil Depression (Afar region, Ethiopia) exhibits unique hydrothermal manifestations in hypersaline context, yielding varied polyextreme physicochemical conditions. Previous studies identified a wide archaeal diversity in less extreme brines but failed to identify microorganisms thriving in either high-chaotropicity, low-water-activity brines or hyperacidic-hypersaline Na-Fe-rich brines. Recently, we accessed several small lakes under intense degassing activity adjacent to the Round Mountain, west to the Dallol dome [Western Canyon Lakes (WCL); WCL1-5]. They exhibited intermediate parameter combinations (pH ~ 5, 34%-41% (weight/volume) NaCl-dominated salts with relatively high levels of chaotropic Mg-Ca salts) that should allow to better constrain life limits. These lakes were overwhelmingly dominated by Archaea, encompassing up to 99% of prokaryotic 16S rRNA gene amplicon sequences in metabarcoding studies. The majority belonged to Halobacteriota and Nanohaloarchaeota, the latter representing up to half of prokaryotic sequences. Optical and epifluorescence microscopy showed active cells in natural samples and diverse morphotypes in enrichment cultures. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy revealed tiny cells (200-300 nm diameter) epibiotically associated with somewhat larger cells (0.6-1 µm) but also the presence of silica-dominated precipitates of similar size and shape, highlighting the difficulty of distinguishing microbes from mineral biomorphs in this kind of low-biomass systems.

Small freshwater ecosystems with dissimilar microbial communities exhibit similar temporal patterns.

Despite small freshwater ecosystems being biodiversity reservoirs and contributing significantly to greenhouse fluxes, their microbial communities remain largely understudied. Yet, microorganisms intervene in biogeochemical cycling and impact water quality. Because of their small size, these ecosystems are in principle more sensitive to disturbances, seasonal variation and pluri-annual climate change. However, how microbial community composition varies over space and time, and whether archaeal, bacterial and microbial eukaryote communities behave similarly remain unanswered. Here, we aim to unravel the composition and intra/interannual temporal dynamic patterns for archaea, bacteria and microbial eukaryotes in a set of small freshwater ecosystems. We monitored archaeal and bacterial community composition during 24 consecutive months in four ponds and one brook from northwestern France by 16S rRNA gene amplicon sequencing (microbial eukaryotes were previously investigated for the same systems). Unexpectedly for oxic environments, bacterial Candidate Phyla Radiation (CPR) were highly diverse and locally abundant. Our results suggest that microbial community structure is mainly driven by environmental conditions acting over space (ecosystems) and time (seasons). A low proportion of operational taxonomic units (OTUs) (<1%) was shared by the five ecosystems despite their geographical proximity (2-9 km away), making microbial communities almost unique in each ecosystem and highlighting the strong selective influence of local environmental conditions. Marked and similar seasonality patterns were observed for archaea, bacteria and microbial eukaryotes in all ecosystems despite strong turnovers of rare OTUs. Over the 2-year survey, microbial community composition varied despite relatively stable environmental parameters. This suggests that biotic associations play an important role in interannual community assembly.

Core microbial communities of lacustrine microbialites sampled along an alkalinity gradient.

Microbialites are usually carbonate-rich sedimentary rocks formed by the interplay of phylogenetically and metabolically complex microbial communities with their physicochemical environment. Yet, the biotic and abiotic determinants of microbialite formation remain poorly constrained. Here, we analysed the structure of prokaryotic and eukaryotic communities associated with microbialites occurring in several crater lakes of the Trans-Mexican volcanic belt along an alkalinity gradient. Microbialite size and community structure correlated with lake physicochemical parameters, notably alkalinity. Although microbial community composition varied across lake microbialites, major taxa-associated functions appeared quite stable with both, oxygenic and anoxygenic photosynthesis and, to less extent, sulphate reduction, as major putative carbonatogenic processes. Despite interlake microbialite community differences, we identified a microbial core of 247 operational taxonomic units conserved across lake microbialites, suggesting a prominent ecological role in microbialite formation. This core mostly encompassed Cyanobacteria and their typical associated taxa (Bacteroidetes, Planctomycetes) and diverse anoxygenic photosynthetic bacteria, notably Chloroflexi, Alphaproteobacteria (Rhodobacteriales, Rhodospirilalles), Gammaproteobacteria (Chromatiaceae) and minor proportions of Chlorobi. The conserved core represented up to 40% (relative abundance) of the total community in lakes Alchichica and Atexcac, displaying the highest alkalinities and the most conspicuous microbialites. Core microbialite communities associated with carbonatogenesis might be relevant for inorganic carbon sequestration purposes.

Functional shifts in microbial mats recapitulate early Earth metabolic transitions.

Phototrophic microbial mats dominated terrestrial ecosystems for billions of years, largely causing, through cyanobacterial oxygenic photosynthesis, but also undergoing, the Great Oxidation Event approximately 2.5 billion years ago. Taking a space-for-time approach based on the universality of core metabolic pathways expressed at ecosystem level, we studied gene content and co-occurrence networks in high-diversity metagenomes from spatially close microbial mats along a steep redox gradient. The observed functional shifts suggest that anoxygenic photosynthesis was present but not predominant under early Precambrian conditions, being accompanied by other autotrophic processes. Our data also suggest that, in contrast to general assumptions, anoxygenic photosynthesis largely expanded in parallel with the subsequent evolution of oxygenic photosynthesis and aerobic respiration. Finally, our observations might represent space-for-time evidence that the Wood-Ljungdahl carbon fixation pathway dominated phototrophic mats in early ecosystems, whereas the Calvin cycle probably evolved from pre-existing variants before becoming the dominant contemporary form of carbon fixation.

Secondary Plastids of Euglenids and Chlorarachniophytes Function with a Mix of Genes of Red and Green Algal Ancestry.

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.

The Chlamydomonas mex1 mutant shows impaired starch mobilization without maltose accumulation.

The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.

Unveiling microbial interactions in stratified mat communities from a warm saline shallow pond.

Modern phototrophic microbial mats are complex communities often used as analogs of major Precambrian ecosystems. Characterizing biotic, notably metabolic, interactions among different microbial mat members is essential to gain insights into the ecology and biogeochemistry of these systems. We applied 16S/18S rRNA metabarcoding approaches to characterize the structure of archaea, bacteria and protist communities from microbial mats collected along strong physicochemical (oxygen, salinity, temperature, depth) gradients in a shallow pond at the salar de Llamara (Chile). All mats were highly diverse, including members of virtually all known high-rank eukaryotic and prokaryotic taxa but also many novel lineages. Bacterial candidate divisions accounted for almost 50% of sequences in deeper mats, while Archaea represented up to 40% of sequences in some mat layers. Molecular phylogenetic analyses revealed six novel deeply divergent archaeal groups, along abundant and diverse Pacearchaeota and Woesearchaeota. Multivariate statistical analyses showed that local environmental conditions strongly influenced community composition. Co-occurrence network structure was markedly different between surface mats located in the oxygenated zone and mats located in transition and anoxic water layers. We identified potential biotic interactions between various high- and low-rank taxa. Notably, a strong positive correlation was observed between Lokiarchaeota and the poorly known candidate bacterial division TA06.

An Early-Branching Freshwater Cyanobacterium at the Origin of Plastids.

Photosynthesis evolved in eukaryotes by the endosymbiosis of a cyanobacterium, the future plastid, within a heterotrophic host. This primary endosymbiosis occurred in the ancestor of Archaeplastida, a eukaryotic supergroup that includes glaucophytes, red algae, green algae, and land plants [1-4]. However, although the endosymbiotic origin of plastids from a single cyanobacterial ancestor is firmly established, the nature of that ancestor remains controversial: plastids have been proposed to derive from either early- or late-branching cyanobacterial lineages [5-11]. To solve this issue, we carried out phylogenomic and supernetwork analyses of the most comprehensive dataset analyzed so far including plastid-encoded proteins and nucleus-encoded proteins of plastid origin resulting from endosymbiotic gene transfer (EGT) of primary photosynthetic eukaryotes, as well as wide-ranging genome data from cyanobacteria, including novel lineages. Our analyses strongly support that plastids evolved from deep-branching cyanobacteria and that the present-day closest cultured relative of primary plastids is Gloeomargarita lithophora. This species belongs to a recently discovered cyanobacterial lineage widespread in freshwater microbialites and microbial mats [12, 13]. The ecological distribution of this lineage sheds new light on the environmental conditions where the emergence of photosynthetic eukaryotes occurred, most likely in a terrestrial-freshwater setting. The fact that glaucophytes, the first archaeplastid lineage to diverge, are exclusively found in freshwater ecosystems reinforces this hypothesis. Therefore, not only did plastids emerge early within cyanobacteria, but the first photosynthetic eukaryotes most likely evolved in terrestrial-freshwater settings, not in oceans as commonly thought.

Resilience of Freshwater Communities of Small Microbial Eukaryotes Undergoing Severe Drought Events.

Small and shallow aquatic ecosystems such as ponds and streams constitute a significant proportion of continental surface waters, especially in temperate zones. In comparison with bigger lakes and rivers, they harbor higher biodiversity but they also exhibit reduced buffering capacity face to environmental shifts, such that climate global change can affect them in a more drastic way. For instance, many temperate areas are predicted to undergo droughts with increasing frequency in the near future, which may lead to the temporal desiccation of streams and ponds. In this work, we monitored temporal dynamics of planktonic communities of microbial eukaryotes (cell size range: 0.2-5 µm) in one brook and one pond that experienced recurrent droughts from 1 to 5 consecutive months during a temporal survey carried out monthly for 2 years based on high-throughput 18S rDNA metabarcoding. During drought-induced desiccation events, protist communities present in the remaining dry sediment, though highly diverse, differed radically from their planktonic counterparts. However, after water refill, the aquatic protist assemblages recovered their original structure within a month. This rapid recovery indicates that these eukaryotic communities are resilient to droughts, most likely via the entrance in dormancy. This property is essential for the long-term survival and functional stability of small freshwater ecosystems.

Metagenome-based diversity analyses suggest a significant contribution of non-cyanobacterial lineages to carbonate precipitation in modern microbialites.

Cyanobacteria are thought to play a key role in carbonate formation due to their metabolic activity, but other organisms carrying out oxygenic photosynthesis (photosynthetic eukaryotes) or other metabolisms (e.g., anoxygenic photosynthesis, sulfate reduction), may also contribute to carbonate formation. To obtain more quantitative information than that provided by more classical PCR-dependent methods, we studied the microbial diversity of microbialites from the Alchichica crater lake (Mexico) by mining for 16S/18S rRNA genes in metagenomes obtained by direct sequencing of environmental DNA. We studied samples collected at the Western (AL-W) and Northern (AL-N) shores of the lake and, at the latter site, along a depth gradient (1, 5, 10, and 15 m depth). The associated microbial communities were mainly composed of bacteria, most of which seemed heterotrophic, whereas archaea were negligible. Eukaryotes composed a relatively minor fraction dominated by photosynthetic lineages, diatoms in AL-W, influenced by Si-rich seepage waters, and green algae in AL-N samples. Members of the Gammaproteobacteria and Alphaproteobacteria classes of Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacterial taxa, followed by Planctomycetes, Deltaproteobacteria (Proteobacteria), Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi. Community composition varied among sites and with depth. Although cyanobacteria were the most important bacterial group contributing to the carbonate precipitation potential, photosynthetic eukaryotes, anoxygenic photosynthesizers and sulfate reducers were also very abundant. Cyanobacteria affiliated to Pleurocapsales largely increased with depth. Scanning electron microscopy (SEM) observations showed considerable areas of aragonite-encrusted Pleurocapsa-like cyanobacteria at microscale. Multivariate statistical analyses showed a strong positive correlation of Pleurocapsales and Chroococcales with aragonite formation at macroscale, and suggest a potential causal link. Despite the previous identification of intracellularly calcifying cyanobacteria in Alchichica microbialites, most carbonate precipitation seems extracellular in this system.

Bacterial gene import and mesophilic adaptation in archaea.

It is widely believed that the archaeal ancestor was hyperthermophilic, but during archaeal evolution, several lineages - including haloarchaea and their sister methanogens, the Thaumarchaeota, and the uncultured Marine Group II and Marine Group III Euryarchaeota (MGII/III) - independently adapted to lower temperatures. Recent phylogenomic studies suggest that the ancestors of these lineages were recipients of massive horizontal gene transfer from bacteria. Many of the acquired genes, which are often involved in metabolism and cell envelope biogenesis, were convergently acquired by distant mesophilic archaea. In this Opinion article, we explore the intriguing hypothesis that the import of these bacterial genes was crucial for the adaptation of archaea to mesophilic lifestyles.

Marked seasonality and high spatial variability of protist communities in shallow freshwater systems.

Small eukaryotes have key roles in aquatic ecosystems, influencing their local environment, global biogeochemical cycles and climate. Their impact depends on community structure, which varies along time. However, very few studies take into account temporal variation. This is especially true for small, shallow freshwater systems, which remain largely understudied despite their wide variety, global surface and intense microbial activity. We have monthly followed changes in the community structure of small microbial eukaryotes (0.2-5 µm cell diameter) for 2 years in four ponds and one brook located in North-Western France based on massive 18S rDNA amplicon 454 pyrosequencing. We detected a total of 3742 stringently defined operational taxonomic units (OTUs) encompassing all recognized eukaryotic supergroups and lineages of uncertain affiliation. Although geographically close, protist communities in the five ecosystems were contrasting, with very few shared OTUs, suggesting that environmental selection mainly drives community structure. The temporal dynamics of different high-rank taxa appeared complex and rapid at monthly scales. Despite this, a clear and reproducible seasonality was observed. As expected, low-abundance OTUs dominated the community. Although some of them appeared sporadically or remained at low frequencies during the survey, others occasionally reached relatively high abundances, sometimes recurrently. This shows that at least a fraction of low-abundance eukaryotes constitutes a seed bank. The annual proportion of primary producers, free-living heterotrophs and parasites appeared remarkably constant among the different ecosystems, suggesting underlying trends of ecosystem carrying capacity for these functional groups.

Colonization of plant substrates at hydrothermal vents and cold seeps in the northeast Atlantic and Mediterranean and occurrence of symbiont-related bacteria.

Reducing conditions with elevated sulfide and methane concentrations in ecosystems such as hydrothermal vents, cold seeps or organic falls, are suitable for chemosynthetic primary production. Understanding processes driving bacterial diversity, colonization and dispersal is of prime importance for deep-sea microbial ecology. This study provides a detailed characterization of bacterial assemblages colonizing plant-derived substrates using a standardized approach over a geographic area spanning the North-East Atlantic and Mediterranean. Wood and alfalfa substrates in colonization devices were deployed for different periods at 8 deep-sea chemosynthesis-based sites in four distinct geographic areas. Pyrosequencing of a fragment of the 16S rRNA-encoding gene was used to describe bacterial communities. Colonization occurred within the first 14 days. The diversity was higher in samples deployed for more than 289 days. After 289 days, no relation was observed between community richness and deployment duration, suggesting that diversity may have reached saturation sometime in between. Communities in long-term deployments were different, and their composition was mainly influenced by the geographical location where devices were deployed. Numerous sequences related to horizontally-transmitted chemosynthetic symbionts of metazoans were identified. Their potential status as free-living forms of these symbionts was evaluated based on sequence similarity with demonstrated symbionts. Results suggest that some free-living forms of metazoan symbionts or their close relatives, such as Epsilonproteobacteria associated with the shrimp Rimicaris exoculata, are efficient colonizers of plant substrates at vents and seeps.

Extending the conserved phylogenetic core of archaea disentangles the evolution of the third domain of life.

Initial studies of the archaeal phylogeny relied mainly on the analysis of the RNA component of the small subunit of the ribosome (SSU rRNA). The resulting phylogenies have provided interesting but partial information on the evolutionary history of the third domain of life because SSU rRNA sequences do not contain enough phylogenetic signal to resolve all nodes of the archaeal tree. Thus, many relationships, and especially the most ancient ones, remained elusive. Moreover, SSU rRNA phylogenies can be heavily biased by tree reconstruction artifacts. The sequencing of complete genomes allows using a variety of protein markers as an alternative to SSU rRNA. Taking advantage of the recent burst of archaeal complete genome sequences, we have carried out an in-depth phylogenomic analysis of this domain. We have identified 200 new protein families that, in addition to the ribosomal proteins and the subunits of the RNA polymerase, form a conserved phylogenetic core of archaeal genes. The accurate analysis of these markers combined with desaturation approaches shed new light on the evolutionary history of Archaea and reveals that several relationships recovered in recent analyses are likely the consequence of tree reconstruction artifacts. Among others, we resolve a number of important relationships, such as those among methanogens Class I, and we propose the definition of two new superclasses within the Euryarchaeota: Methanomada and Diaforarchaea.

Complex communities of small protists and unexpected occurrence of typical marine lineages in shallow freshwater systems.

Although inland water bodies are more heterogeneous and sensitive to environmental variation than oceans, the diversity of small protists in these ecosystems is much less well known. Some molecular surveys of lakes exist, but little information is available from smaller, shallower and often ephemeral freshwater systems, despite their global distribution and ecological importance. We carried out a comparative study based on massive pyrosequencing of amplified 18S rRNA gene fragments of protists in the 0.2-5 µm size range in one brook and four shallow ponds located in the Natural Regional Park of the Chevreuse Valley, France. Our study revealed a wide diversity of small protists, with 812 stringently defined operational taxonomic units (OTUs) belonging to the recognized eukaryotic supergroups (SAR--Stramenopiles, Alveolata, Rhizaria--Archaeplastida, Excavata, Amoebozoa, Opisthokonta) and to groups of unresolved phylogenetic position (Cryptophyta, Haptophyta, Centrohelida, Katablepharida, Telonemida, Apusozoa). Some OTUs represented deep-branching lineages (Cryptomycota, Aphelida, Colpodellida, Tremulida, clade-10 Cercozoa, HAP-1 Haptophyta). We identified several lineages previously thought to be marine including, in addition to MAST-2 and MAST-12, already detected in freshwater, MAST-3 and possibly MAST-6. Protist community structures were different in the five ecosystems. These differences did not correlate with geographical distances, but seemed to be influenced by environmental parameters.

Rooting the domain archaea by phylogenomic analysis supports the foundation of the new kingdom Proteoarchaeota.

The first 16S rRNA-based phylogenies of the Archaea showed a deep division between two groups, the kingdoms Euryarchaeota and Crenarchaeota. This bipartite classification has been challenged by the recent discovery of new deeply branching lineages (e.g., Thaumarchaeota, Aigarchaeota, Nanoarchaeota, Korarchaeota, Parvarchaeota, Aenigmarchaeota, Diapherotrites, and Nanohaloarchaeota) which have also been given the same taxonomic status of kingdoms. However, the phylogenetic position of some of these lineages is controversial. In addition, phylogenetic analyses of the Archaea have often been carried out without outgroup sequences, making it difficult to determine if these taxa actually define lineages at the same level as the Euryarchaeota and Crenarchaeota. We have addressed the question of the position of the root of the Archaea by reconstructing rooted archaeal phylogenetic trees using bacterial sequences as outgroup. These trees were based on commonly used conserved protein markers (32 ribosomal proteins) as well as on 38 new markers identified through phylogenomic analysis. We thus gathered a total of 70 conserved markers that we analyzed as a concatenated data set. In contrast with previous analyses, our trees consistently placed the root of the archaeal tree between the Euryarchaeota (including the Nanoarchaeota and other fast-evolving lineages) and the rest of archaeal species, which we propose to class within the new kingdom Proteoarchaeota. This implies the relegation of several groups previously classified as kingdoms (e.g., Crenarchaeota, Thaumarchaeota, Aigarchaeota, and Korarchaeota) to a lower taxonomic rank. In addition to taxonomic implications, this profound reorganization of the archaeal phylogeny has also consequences on our appraisal of the nature of the last archaeal ancestor, which most likely was a complex organism with a gene-rich genome.

What was the real contribution of endosymbionts to the eukaryotic nucleus? Insights from photosynthetic eukaryotes.

Eukaryotic genomes are composed of genes of different evolutionary origins. This is especially true in the case of photosynthetic eukaryotes, which, in addition to typical eukaryotic genes and genes of mitochondrial origin, also contain genes coming from the primary plastids and, in the case of secondary photosynthetic eukaryotes, many genes provided by the nuclei of red or green algal endosymbionts. Phylogenomic analyses have been applied to detect those genes and, in some cases, have led to proposing the existence of cryptic, no longer visible endosymbionts. However, detecting them is a very difficult task because, most often, those genes were acquired a long time ago and their phylogenetic signal has been heavily erased. We revisit here two examples, the putative cryptic endosymbiosis of green algae in diatoms and chromerids and of Chlamydiae in the first photosynthetic eukaryotes. We show that the evidence sustaining them has been largely overestimated, and we insist on the necessity of careful, accurate phylogenetic analyses to obtain reliable results.

Pangenome evidence for extensive interdomain horizontal transfer affecting lineage core and shell genes in uncultured planktonic thaumarchaeota and euryarchaeota.

Horizontal gene transfer (HGT) is an important force in evolution, which may lead, among other things, to the adaptation to new environments by the import of new metabolic functions. Recent studies based on phylogenetic analyses of a few genome fragments containing archaeal 16S rRNA genes and fosmid-end sequences from deep-sea metagenomic libraries have suggested that marine planktonic archaea could be affected by high HGT frequency. Likewise, a composite genome of an uncultured marine euryarchaeote showed high levels of gene sequence similarity to bacterial genes. In this work, we ask whether HGT is frequent and widespread in genomes of these marine archaea, and whether HGT is an ancient and/or recurrent phenomenon. To answer these questions, we sequenced 997 fosmid archaeal clones from metagenomic libraries of deep-Mediterranean waters (1,000 and 3,000 m depth) and built comprehensive pangenomes for planktonic Thaumarchaeota (Group I archaea) and Euryarchaeota belonging to the uncultured Groups II and III Euryarchaeota (GII/III-Euryarchaeota). Comparison with available reference genomes of Thaumarchaeota and a composite marine surface euryarchaeote genome allowed us to define sets of core, lineage-specific core, and shell gene ortholog clusters for the two archaeal lineages. Molecular phylogenetic analyses of all gene clusters showed that 23.9% of marine Thaumarchaeota genes and 29.7% of GII/III-Euryarchaeota genes had been horizontally acquired from bacteria. HGT is not only extensive and directional but also ongoing, with high HGT levels in lineage-specific core (ancient transfers) and shell (recent transfers) genes. Many of the acquired genes are related to metabolism and membrane biogenesis, suggesting an adaptive value for life in cold, oligotrophic oceans. We hypothesize that the acquisition of an important amount of foreign genes by the ancestors of these archaeal groups significantly contributed to their divergence and ecological success.