The microsporidium Vairimorpha (Nosema) ceranae is an emerging threat to honey bees (Apis mellifera), known to disrupt gut microbiota and suppress immune responses, potentially contributing to colony losses. Fungal extracts have recently gained interest as sources of bioactive compounds with antimicrobial and immunomodulatory potential. In this study, we explored the effects of different dietary supplements-sugar syrup, HiveAlive™, and a novel Ganoderma australe extract (GanoBee)-on gut bacterial composition and immune-related gene expression in honey bees subjected to experimental exposure to V. ceranae 1 x 104 spores per bee. The GanoBee diet altered the gut microbiota, notably reducing the relative abundance of Rhizobiaceae (Bartonella apis) and increasing Frischella compared to other treatments. While alpha diversity was not significantly affected by diet or exposure to V. ceranae, beta diversity differed significantly in bees fed with GanoBee. Additionally, the expression of the antimicrobial peptide genes abaecin and hymenoptaecin was elevated in both exposed and unexposed bees fed with GanoBee, depending on the sampling day. However, the establishment of V. ceranae infection appeared limited, likely due to low spore viability, and mortality in control bees was higher than expected. The low Vairimorpha ceranae infection levels observed in this study are likely attributable to reduced spore viability caused by storage conditions and/or suboptimal environmental conditions within the laboratory cages. Post hoc analyses indicated that the high viscosity of GanoBee-supplemented diets likely contributed to the elevated bee mortality observed, underscoring a critical limitation of the experimental design related to diet formulation and delivery method. These physical factors complicate the interpretation of treatment efficacy and highlight the importance of optimizing feeding protocols to avoid confounding effects. Despite these constraints, GanoBee demonstrated promising potential as a modulator of gut microbiota composition and immune-related gene expression, supporting the need for further research under improved and carefully controlled experimental conditions.

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.

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 eukaryotes 1 . However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved 2–4 . 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.

Our knowledge of archaeal diversity and evolution has expanded rapidly in the past decade. However, hardly any genomes of the phylum Korarchaeota have been obtained due to the difficulty in accessing their natural habitats and – possibly – their limited abundance. As a result, many aspects of Korarchaeota biology, physiology and evolution remain enigmatic. Here, we expand this phylum with five high-quality metagenome-assembled genomes. This improved taxon sampling combined with sophisticated phylogenomic analyses robustly places Korarchaeota at the base of TACK and Asgard clades, revisiting the phylum’s long-assumed position. Furthermore, we observe a clear split between terrestrial and marine thermal clades. Gene tree-aware ancestral reconstructions suggest that the last Korarchaeota common ancestor was a thermophilic autotroph. In contrast, Korarchaeaceae, the lineage where environmental transitions occurred, shifted towards a heterotrophic lifestyle. Terrestrial Korarchaeota gained many cas and CARF genes indicating they may need to manage viral infections. Together, our study provides new insights into these early diverging Archaea and suggests that gradual gene gain and loss shaped their adaptation to different thermal environments. Importance Korarchaeota are an ancient group of archaea, but their biology, physiology and evolution have remained obscure. Analysis of five novel Korarchaeota MAGs, and publicly available reference data provides robust phylogenomic evidence that Korarchaeota are placed at the base of Asgard archaea and TACK, revisiting the phylum’s long-assumed position. Gene content reconstruction suggests a versatile thermophilic and autotrophic last Korarchaeota common ancestor. Environmental distribution surveying of public databases places all Korarchaeota in thermophilic environments and indicates that their habitat is limited to hydrothermal vents and hot springs. Our modeling indicates at least two transitions linked to habitat switching between these environments in the evolutionary history of Korarchaeota. Both are linked to a significant alteration of the inferred ancestral gene content, including a shift towards a heterotrophic and potential scavenging lifestyle. Furthermore, hot spring Korarchaeota acquired various genes participating in resistance to viruses, suggesting they may need to manage frequent viral threats.

Bacterioplankton of the SAR11 clade are the most abundant marine microorganisms and consist of numerous subclades spanning order-level divergence (Pelagibacterales). The assignment of the earliest diverging subclade V (a.k.a. HIMB59) to the Pelagibacterales is highly controversial, with multiple recent phylogenetic studies placing them completely separate from SAR11. Other than through phylogenomics, subclade V has not received detailed examination due to limited genomes from this group. Here, we assessed the ecogenomic characteristics of subclade V to better understand the role of this group in comparison to the Pelagibacterales. We used a new isolate genome, recently released single-amplified genomes and metagenome-assembled genomes, and previously established SAR11 genomes to perform a comprehensive comparative genomics analysis. We paired this analysis with the recruitment of metagenomes spanning the open ocean, coastal, and brackish systems. Phylogenomics, average amino acid identity, and 16S rRNA gene phylogeny indicate that SAR11 subclade V is synonymous with the ubiquitous AEGEAN-169 clade and support the contention that this group represents a taxonomic family. AEGEAN-169 shared many bulk genome qualities with SAR11, such as streamlining and low GC content, but genomes were generally larger. AEGEAN-169 had overlapping distributions with SAR11 but was metabolically distinct from SAR11 in its potential to transport and utilize a broader range of sugars as well as in the transport of trace metals and thiamin. Thus, regardless of the ultimate phylogenetic placement of AEGEAN-169, these organisms have distinct metabolic capacities that likely allow them to differentiate their niche from canonical SAR11 taxa. IMPORTANCE One goal of marine microbiologists is to uncover the roles various microorganisms are playing in biogeochemical cycles. Success in this endeavor relies on differentiating groups of microbes and circumscribing their relationships. An early-diverging group (subclade V) of the most abundant bacterioplankton, SAR11, has recently been proposed as a separate lineage that does not share a most recent common ancestor. But beyond phylogenetics, little has been done to evaluate how these organisms compare with SAR11. Our work leverages dozens of new genomes to demonstrate the similarities and differences between subclade V and SAR11. In our analysis, we also establish that subclade V is synonymous with a group of bacteria established from 16S rRNA gene sequences, AEGEAN-169. Subclade V/AEGEAN-169 has clear metabolic distinctions from SAR11 and their shared traits point to remarkable convergent evolution if they do not share a most recent common ancestor.

Native Andean–Patagonian Nothofagus forests harbour a unique diversity of microorganisms with diverse ecological roles. Although ectomycorrhizal associations constitute an important fragment of the biota associated with these forests, the factors affecting such communities are largely unknown. We investigated the biodiversity, relative abundance, and composition of ectomycorrhizal fungal communities in relation to two host tree species and ages and the soil properties in six monospecific and mixed evergreen–deciduous Nothofagus forests. We used the internal transcribed spacer (ITS2) region by sequencing 9,600 ectomycorrhizae (ECM) root tips for the identification of fungi. In total, 1,125 fungal taxa at the genus level distributed over 131 orders were identified. The phyla Ascomycota (34.5%) and Basidiomycota (62.1%) were the most abundant, whereas Mucoromycota (3.1%), Chytridiomycota, Cryptomycota, Olpidiomycota, and Zoopagomycota occurred less frequently. The highest taxon diversity was found in old trees, whereas young trees often exhibited a lower diversity of the associated fungi. The fungal taxa were grouped into seven broad ecological categories, of which saprotrophic associations were most common, followed by pathotrophic, pathotrophic–saprotrophic–symbiotrophic, pathotrophic–saprotrophic, and symbiotrophic associations. We did not detect significant differences in the number of taxa in each category between young and old N . dombeyi and N . obliqua . Overall, the scale of the Illumina sequencing approach allowed us to detect a fungal taxa diversity that would not be possible to find through surveys of fruiting bodies alone and that have never been observed in Nothofagus forests before. Our findings suggest the impact of the proximity between sites, the similarity of the soil conditions, and anthropogenic use of the forests on the belowground fungal community’s diversity and composition. Furthermore, there were differences between above- and belowground occurrences of the edible mushrooms B . loyo and Ramaria spp. However, future research, including on EMC tips found beneath fairy rings could provide significantly better correlations with the occurrence of aboveground fruiting body.

Bistorta vivipara is a widespread herbaceous perennial plant with a discontinuous pattern of distribution in arctic, alpine, subalpine and boreal habitats across the northern Hemisphere. Studies of the fungi associated with the roots of B. vivipara have mainly been conducted in arctic and alpine ecosystems. This study examined the fungal diversity and specificity from root tips of B. vivipara in two local mountain ecosystems as well as on a global scale. Sequences were generated by Sanger sequencing of the internal transcribed spacer (ITS) region followed by an analysis of accurately annotated nuclear segments including ITS1-5.8S-ITS2 sequences available from public databases. In total, 181 different UNITE species hypotheses (SHs) were detected to be fungi associated with B. vivipara, 73 of which occurred in the Bavarian Alps and nine in the Swabian Alps–with one SH shared among both mountains. In both sites as well as in additional public data, individuals of B. vivipara were found to contain phylogenetically diverse fungi, with the Basidiomycota, represented by the Thelephorales and Sebacinales, being the most dominant. A comparative analysis of the diversity of the Sebacinales associated with B. vivipara and other co-occurring plant genera showed that the highest number of sebacinoid SHs were associated with Quercus and Pinus, followed by Bistorta. A comparison of B. vivipara with plant families such as Ericaceae, Fagaceae, Orchidaceae, and Pinaceae showed a clear trend: Only a few species were specific to B. vivipara and a large number of SHs were shared with other co-occurring non-B. vivipara plant species. In Sebacinales, the majority of SHs associated with B. vivipara belonged to the ectomycorrhiza (ECM)-forming Sebacinaceae, with fewer SHs belonging to the Serendipitaceae encompassing diverse ericoid–orchid–ECM–endophytic associations. The large proportion of non-host-specific fungi able to form a symbiosis with other non-B. vivipara plants could suggest that the high fungal diversity in B. vivipara comes from an active recruitment of their associates from the co-occurring vegetation. The non-host-specificity suggests that this strategy may offer ecological advantages; specifically, linkages with generalist rather than specialist fungi. Proximity to co-occurring non-B. vivipara plants can maximise the fitness of B. vivipara, allowing more rapid and easy colonisation of the available habitats.

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.

Bistorta vivipara is a widespread herbaceous perennial plant with a discontinuous pattern of distribution in arctic, alpine, subalpine and boreal habitats across the northern Hemisphere.Studies of the fungi associated with the roots of B. vivipara have mainly been conducted in arctic and alpine ecosystems.This study examined the fungal diversity and specificity from root tips of B. vivipara in two local mountain ecosystems as well as on a global scale.Sequences were generated by Sanger sequencing of the internal transcribed spacer (ITS) region followed by an analysis of accurately annotated nuclear segments including ITS1-5.8S-ITS2sequences available from public databases.In total, 181 different UNITE species hypotheses (SHs) were detected to be fungi associated with B. vivipara, 73 of which occurred in the Bavarian Alps and nine in the Swabian Alps -with one SH shared among both mountains.In both sites as well as in additional public data, individuals of B. vivipara were found to contain phylogenetically diverse fungi, with the Basidiomycota, represented by the Thelephorales and Sebacinales, being the most dominant.A comparative analysis of the diversity of the Sebacinales associated with B. vivipara and other co-occurring plant genera showed that the highest number of sebacinoid SHs were associated with Quercus and Pinus, followed by Bistorta.A comparison of B. vivipara with plant families such as Ericaceae, Fagaceae, Orchidaceae, and Pinaceae showed a clear trend: Only a few species were specific to B. vivipara and a large number of SHs were shared with other co-occurring non-B.vivipara plant species.In Sebacinales, the majority of SHs associated with B. vivipara belonged to the ectomycorrhiza (ECM)-forming Sebacinaceae, with fewer SHs belonging to the Serendipitaceae encompassing diverse ericoid-orchid-ECM-endophytic associations.The large proportion of non-host-specific fungi able to form a symbiosis with other non-B.vivipara plants could suggest that the high fungal diversity in B. vivipara comes from an active recruitment of their associates

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.

Initial single gene alignments and trees for 320 markers. this still includes paralogs and contamination removed before concatenation

1Department of Organismal Biology, Program in Systematic Biology, Uppsala University, Uppsala, Sweden 2Department of Cell and Molecular Biology, Program in Molecular Evolution, Uppsala University, Uppsala, Sweden 3Biodesign Center for Mechanisms of Evolution, School of Life Sciences, Arizona State University, Tempe, AZ, USA 4Ocean EcoSystems Biology, RD3, GEOMAR Helmholtz Centre for Ocean Research Kiel, Germany 5Monterey Bay Aquarium Research Institute, Moss Landing, CA, USA 6Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada 7Centre for Ecology and Evolution in Microbial Model Systems – EEMiS, Linnaeus University, Kalmar, Sweden 8Laboratory of Microbiology, Wageningen University and Research, NL-6708 WE Wageningen, The Netherlands 9Science for Life Laboratory, Uppsala University, Uppsala, Sweden

cleaned alignments and fasta files for 317 phylogenetic marker genes. Supermatrix alignments and reconstructed tree

The evolution of obligate host-association of bacterial symbionts and pathogens remains poorly understood. The Rickettsiales represent an order of obligate alphaproteobacterial 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. We describe several genomes of deep-branching, environmental alphaproteobacteria that branch basal to previously sampled Rickettsiales, and whose genome content are reminiscent of free-living and biofilm-associated lifestyles. Ancestral genome content reconstruction across the Rickettsiales tree revealed that the free-living to host-association transition of this group occurred more recently than previously anticipated, and likely involved the repurposing of a type IV secretion system. One-Sentence Summary Deep-branching Rickettsiales provide insights into the evolution of obligate host-associated lifestyle

MotivationMetagenomic and metatranscriptomic sequencing analyses have become increasingly popular tools for producing massive amounts of short-read data, often used for the reconstruction of draft genomes or the detection of (active) genes in microbial communities. Unfortunately, sequence assemblies of such datasets generally remain a computationally challenging task. Frequently, researchers are only interested in a specific group of organisms or genes; yet, the assembly of multiple datasets only to identify candidate sequences for a specific question is sometimes prohibitively slow, forcing researchers to select a subset of available datasets to address their question. Here we present PhyloMagnet, a workflow to screen meta-omics datasets for taxa and genes of interest using gene-centric assembly and phylogenetic placement of sequences.ResultsUsing PhyloMagnet, we could identify up to 87% of the genera in anin vitromock community with variable abundances, while the false positive predictions per single gene tree ranged from 0% to 23%. When applied to a group of metagenomes for which a set of MAGs have been published, we could detect the majority of the taxonomic labels that the MAGs had been annotated with. In a metatranscriptomic setting the phylogenetic placement of assembled contigs corresponds to that of transcripts obtained from transcriptome assembly.AvailabilityPhyloMagnet is built using Nextflow, available atgithub.com/maxemil/PhyloMagnetand is developed and tested on Linux. It is released under the open source GNU GPL license and documentation is available atphylomagnet.readthedocs.io. Version 0.5 of PhyloMagnet was used for all benchmarks experiments.

Understanding the impacts of agricultural practices on belowground fungal communities is crucial in order to preserve biological diversity in agricultural soils and enhance their role in agroecosystem functioning. Although fungal communities are widely distributed, relatively few studies have correlated agricultural production practices. We investigated the diversity, composition and ecological functionality of fungal communities in roots of winter wheat (Triticum aestivum) growing in conventional and organic farming systems. Direct and nested polymerase chain reaction (PCR) amplifications spanning the internal transcribed spacer (ITS) region of the rDNA from pooled fine root samples were performed with two different sets of fungal specific primers. Fungal identification was carried out through similarity searches against validated reference sequences (RefSeq). The R package 'picante' and FUNGuild were used to analyse fungal community composition and trophic mode, respectively. Either by direct or cloning sequencing, 130 complete ITS sequences were clustered into 39 operational taxonomic units (OTUs) (25 singletons), belonging to the Ascomycota (24), the Basidiomycota (14) and to the Glomeromycota (1). Fungal communities from conventional farming sites are phylogenetically more related than expected by chance. Constrained ordination analysis identified total N, total S and Pcal that had a significant effect on the OTU's abundance and distribution, and a further correlation with the diversity of the co-occurring vegetation could be hypothesised. The functional predictions based on FUNGuild suggested that conventional farming increased the presence of plant pathogenic fungi compared with organic farming. Based on diversity, OTU distribution, nutrition mode and the significant phylogenetic clustering of fungal communities, this study shows that fungal communities differ across sampling sites, depending on agricultural practices. Although it is not fully clear which factors determine the fungal communities, our findings suggest that organic farming systems have a positive effect on fungal communities in winter wheat crops.

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.

Joran Martijn , Anders E. Lind, Max E. Schön, Ian Spiertz, Lina Juzokaite, Ignas Bunikis, Olga V. Pettersson and Thijs J. G. Ettema 1,3* Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, SE-75123, Uppsala, Sweden. Science for Life Laboratory, Uppsala University, SE-75185, Uppsala, Sweden. Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Stippeneng 4, 6708WE, Wageningen, The Netherlands.

Fungi belonging to the Entorrhizales (Entorrhizomycota) comprise biotrophic pathogens associated with roots of the Cyperaceae and Juncaceae plant species. They are nearly globally distributed but rarely studied due to a hidden lifestyle without causing visible effects on host plants. Therefore, the evolutionary origin and phylogenetic relationships of the group are still poorly understood and it is not known whether species diversification was the result of co-evolution with their hosts or the result of host jumps. To infer hypotheses about the evolutionary history of the Entorrhizales, divergence times were estimated and plant-fungal tanglegrams calculated. Relaxed molecular clock analyses suggest that the Entorrhizomycota originated around the Neoproterozoic-Palaeozoic and diverged during the Late Cretaceous-Paleogene into the extant orders Entorrhizales and Talbotiomycetales. The split of the major lineages within the Entorrhizales took place in the Eocene, somewhat later than the divergence of the host families Cyperaceae and Juncaceae. Topology- and distance-based co-phylogenetic analyses of the fungi and their hosts revealed a large number of co-speciation and lineage sorting events in early fungal speciation, which resulted in a phylogenetic split corresponding to species infecting Cyperaceae or Juncaceae. Given that this split is congruent with spore differences, Entorrhiza s. str. is emended for species infecting hosts in the Cyperaceae, and a new genus Juncorrhiza is described for species restricted to hosts in the Juncaceae. Additionally, three new species are described: Entorrhiza fuirenae, Juncorrhiza maritima and J. oxycarpi.

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.