Towards a balanced view of the bacterial tree of life

Abstract

The bacterial tree of life has recently undergone significant expansion, chiefly from candidate phyla retrieved through genome-resolved metagenomics. Bypassing the need for genome availability, we present a snapshot of bacterial phylogenetic diversity based on the recovery of high-quality SSU rRNA gene sequences extracted from nearly 7000 metagenomes and all available reference genomes. We illuminate taxonomic richness within established bacterial phyla together with environmental distribution patterns, providing a revised framework for future phylogeny-driven sequencing efforts.

Keywords: Bacteria; Bacterial diversity; Candidate Phyla Radiation (CPR); Metagenomics; Microbial dark matter (MDM); Novel bacterial lineages; Small subunit (SSU) rRNA; Tree of life.

Fig. 1

Bacterial SSU rRNA gene-based maximum-likelihood phylogenetic tree. Shown are representative taxa based on all SSU rRNA genes ≥ 1200 bp extracted from 6744 metagenomes deposited in IMG/M [10] and reference genomes (isolates, single amplified genomes, and metagenome-assembled genomes), which were consecutively de-replicated at 97% (97% OTUs) and then clustered at 85% sequence similarity thresholds (85% clusters). Bacterial phyla represented by at least two 85% clusters are shown, and for each phylum, bootstrap support is indicated if greater than 50. Branches in the tree are colored in turquoise if clusters contain SSU rRNA genes extracted from genome sequences. Sizes of pie charts correlate with the total number of 97% OTUs in the respective phylum (log2 transformed) and are divided based on percentage of 97% OTUs assigned to any of the three categories: (1) OTUs consisting of metagenomic SSU rRNA sequences with at least one member found in the SILVA database (MG & SILVA, yellow), (2) OTUs consisting solely of metagenomic sequences derived from this study (MG only, red), and (3) OTUs containing SSU rRNA genes extracted from reference genomes. Eighty-five percent clusters marked with filled black circles would likely have been missed by amplicon studies using the Earth Microbiome primer set (515f/806r) [14], with the size of the filled circles correlating with the mismatch score (indicated are mismatch scores greater than 1 [4]). The tree was rooted based on a representative set of 15 archaeal SSU rRNA gene sequences

Fig. 2

Environmental distribution of the four bacterial phyla with the highest taxonomic richness. For each phylum (left column), the proportion of all 97% OTUs derived from metagenomes exclusively (middle column, black) or observed previously either in the SILVA database or by genome sequencing (middle column, white) is indicated together with their environmental origin (right column). In instances where SSU rRNA gene sequences in 97% OTUs originated from different environmental categories, all categories were considered with a maximum count of one per environment. Connections between columns are only shown if representing more than ten 97% OTUs. Stacked bars illustrate total number of 97% OTUs in the respective habitat and either containing SSU rRNA gene sequences which include SILVA representatives (green), sequences derived from metagenomes exclusively (red), or from reference genomes (turquoise). We excluded the environment type “engineered” from this figure due to the heterogeneous origins of these samples