Different distance-based threshold selection approaches were used to assess and compare use of the internal transcribed spacer (ITS) region to distinguish among 901 Cortinarius species represented by >3000 collections. Sources of error associated with genetic markers and selection approaches were explored and evaluated using MOTUs from genus and lineage based-alignments. Our study indicates that 1%-2% more species can be distinguished by using the full-length ITS barcode as compared to either the ITS1 or ITS2 regions alone. Optimal threshold values for different picking approaches and genetic marker lengths inferred from a subset of species containing major lineages ranged from 97.0% to 99.5% sequence similarity using clustering optimization and UNITE SH, and from 1% to 2% sequence dissimilarity with CROP. Errors for the optimal cutoff ranged from 0% to 70%, and these can be reduced to a maximum of 22% when excluding species lacking a barcode gap. A threshold value of 99% is suitable for distinguishing species in the majority of lineages in the genus using the entire ITS region but only 90% of the species could be identified using just the ITS1 or ITS2 region. Prior identification of species, lacking barcode gaps and their subsequent separate analyses, maximized the accuracy of threshold approaches.

The evolution of obligate host-association of bacterial symbionts and pathogens remains poorly understood. The Rickettsiales are an alphaproteobacterial order of obligate endosymbionts and parasites that infect a wide variety of eukaryotic hosts, including humans, livestock, insects and protists. Induced by their host-associated lifestyle, Rickettsiales genomes have undergone reductive evolution, leading to small, AT-rich genomes with limited metabolic capacities. Here we uncover eleven deep-branching alphaproteobacterial metagenome assembled genomes from aquatic environments, including data from the Tara Oceans initiative and other publicly available datasets, distributed over three previously undescribed Rickettsiales-related clades. Phylogenomic analyses reveal that two of these clades, Mitibacteraceae and Athabascaceae, branch sister to all previously sampled Rickettsiales. The third clade, Gamibacteraceae, branch sister to the recently identified ectosymbiotic ‘Candidatus Deianiraea vastatrix’. Comparative analyses indicate that the gene complement of Mitibacteraceae and Athabascaceae is reminiscent of that of free-living and biofilm-associated bacteria. Ancestral genome content reconstruction across the Rickettsiales species tree further suggests that the evolution of host association in Rickettsiales was a gradual process that may have involved the repurposing of a type IV secretion system.

In the ongoing debates about eukaryogenesis—the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors—members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2–4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

SummaryAmplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost‐effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read‐curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full‐length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high‐throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full‐length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost‐effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.

The endosymbiotic origin of plastids from cyanobacteria gave eukaryotes photosynthetic capabilities and launched the diversification of countless forms of algae. These primary plastids are found in members of the eukaryotic supergroup Archaeplastida. All known archaeplastids still retain some form of primary plastids, which are widely assumed to have a single origin. Here, we use single-cell genomics from natural samples combined with phylogenomics to infer the evolutionary origin of the phylum Picozoa, a globally distributed but seemingly rare group of marine microbial heterotrophic eukaryotes. Strikingly, the analysis of 43 single-cell genomes shows that Picozoa belong to Archaeplastida, specifically related to red algae and the phagotrophic rhodelphids. These picozoan genomes support the hypothesis that Picozoa lack a plastid, and further reveal no evidence of an early cryptic endosymbiosis with cyanobacteria. These findings change our understanding of plastid evolution as they either represent the first complete plastid loss in a free-living taxon, or indicate that red algae and rhodelphids obtained their plastids independently of other archaeplastids.

Halobacteria (henceforth: Haloarchaea) are predominantly aerobic halophiles that are thought to have evolved from anaerobic methanogens. This remarkable transformation most likely involved an extensive influx of bacterial genes. Whether it entailed a single massive transfer event or a gradual stream of transfers remains a matter of debate. To address this, genomes that descend from methanogen-to-halophile intermediates are necessary. Here, we present five such near-complete genomes of Marine Group IV archaea (Hikarchaeia), the closest known relatives of Haloarchaea. Their inclusion in gene tree-aware ancestral reconstructions reveals an intermediate stage that had already lost a large number of genes, including nearly all of those involved in methanogenesis and the Wood-Ljungdahl pathway. In contrast, the last Haloarchaea common ancestor gained a large number of genes and expanded its aerobic respiration and salt/UV resistance gene repertoire. Our results suggest that complex and gradual patterns of gain and loss shaped the methanogen-to-halophile transition.

Chlamydiae is a bacterial phylum composed of obligate animal and protist endosymbionts. However, other members of the Planctomycetes–Verrucomicrobia–Chlamydiae superphylum are primarily free living. How Chlamydiae transitioned to an endosymbiotic lifestyle is still largely unresolved. Here we reconstructed Planctomycetes–Verrucomicrobia–Chlamydiae species relationships and modelled superphylum genome evolution. Gene content reconstruction from 11,996 gene families suggests a motile and facultatively anaerobic last common Chlamydiae ancestor that had already gained characteristic endosymbiont genes. Counter to expectations for genome streamlining in strict endosymbionts, we detected substantial gene gain within Chlamydiae. We found that divergence in energy metabolism and aerobiosis observed in extant lineages emerged later during chlamydial evolution. In particular, metabolic and aerobic genes characteristic of the more metabolically versatile protist-infecting chlamydiae were gained, such as respiratory chain complexes. Our results show that metabolic complexity can increase during endosymbiont evolution, adding an additional perspective for understanding symbiont evolutionary trajectories across the tree of life.

Altitudinal gradients provide valuable information about the effects of environmental variables on changes in species richness and composition as well as the distribution of below ground fungal communities. Since most knowledge in this respect has been gathered on aboveground communities, we focused our study towards the characterization of belowground fungal communities associated with two different ages of Norway spruce (Picea abies) trees along an altitudinal gradient. By sequencing the internal transcribed spacer (ITS) region on the Illumina platform, we investigated the fungal communities in a floristically and geologically relatively well explored forest on the slope of Mt. Iseler of the Bavarian Alps. From fine roots and rhizosphere of a total of 90 of Norway spruce trees from 18 plots we detected 1285 taxa, with a range of 167 to 506 (average 377) taxa per plot. Fungal taxa are distributed over 96 different orders belonging to the phyla Ascomycota, Basidiomycota, Chrytridiomycota, Glomeromycota, and Mucoromycota. Overall the Agaricales (438 taxa) and Tremellales (81 taxa) belonging to the Basidiomycota and the Hypocreales (65 spp.) and Helotiales (61 taxa) belonging to the Ascomycota represented the taxon richest orders. The evaluation of our multivariate generalized mixed models indicate that the altitude has a significant influence on the composition of the fungal communities (p < 0.003) and that tree age determines community diversity (p < 0.05). A total of 47 ecological guilds were detected, of which the ectomycorrhizal and saprophytic guilds were the most taxon-rich. Our ITS amplicon Illumina sequencing approach allowed us to characterize a high fungal community diversity that would not be possible to capture with fruiting body surveys alone. We conclude that it is an invaluable tool for diverse monitoring tasks and inventorying biodiversity, especially in the detection of microorganisms developing very ephemeral and/or inconspicuous fruiting bodies or lacking them all together. Results suggest that the altitude mainly influences the community composition, whereas fungal diversity becomes higher in mature/older trees. Finally, we demonstrate that novel techniques from bacterial microbiome analyses are also useful for studying fungal diversity and community structure in a DNA metabarcoding approach, but that incomplete reference sequence databases so far limit effective identification.