Phylogenetically Novel Uncultured Microbial Cells Dominate Earth Microbiomes

Abstract

To describe a microbe's physiology, including its metabolism, environmental roles, and growth characteristics, it must be grown in a laboratory culture. Unfortunately, many phylogenetically novel groups have never been cultured, so their physiologies have only been inferred from genomics and environmental characteristics. Although the diversity, or number of different taxonomic groups, of uncultured clades has been studied well, their global abundances, or numbers of cells in any given environment, have not been assessed. We quantified the degree of similarity of 16S rRNA gene sequences from diverse environments in publicly available metagenome and metatranscriptome databases, which we show have far less of the culture bias present in primer-amplified 16S rRNA gene surveys, to those of their nearest cultured relatives. Whether normalized to scaffold read depths or not, the highest abundances of metagenomic 16S rRNA gene sequences belong to phylogenetically novel uncultured groups in seawater, freshwater, terrestrial subsurface, soil, hypersaline environments, marine sediment, hot springs, hydrothermal vents, nonhuman hosts, snow, and bioreactors (22% to 87% uncultured genera to classes and 0% to 64% uncultured phyla). The exceptions were human and human-associated environments, which were dominated by cultured genera (45% to 97%). We estimate that uncultured genera and phyla could comprise 7.3 × 10²⁹ (81%) and 2.2 × 10²⁹ (25%) of microbial cells, respectively. Uncultured phyla were overrepresented in metatranscriptomes relative to metagenomes (46% to 84% of sequences in a given environment), suggesting that they are viable. Therefore, uncultured microbes, often from deeply phylogenetically divergent groups, dominate nonhuman environments on Earth, and their undiscovered physiologies may matter for Earth systems. IMPORTANCE In the past few decades, it has become apparent that most of the microbial diversity on Earth has never been characterized in laboratory cultures. We show that these unknown microbes, sometimes called "microbial dark matter," are numerically dominant in all major environments on Earth, with the exception of the human body, where most of the microbes have been cultured. We also estimate that about one-quarter of the population of microbial cells on Earth belong to phyla with no cultured relatives, suggesting that these never-before-studied organisms may be important for ecosystem functions. Author Video: An author video summary of this article is available.

Keywords: environmental microbiology; phylogeny; uncultured microbes.

FIG 1

Fractions of 16S rRNA genes from bacteria (top panel) and archaea (bottom panel) in public databases from primer-amplified metagenomes (with and without read depths) and metatranscriptomes at different percent identities with their closest cultured relative. Vertical dashed lines represent estimated cutoff levels for different taxonomic levels of novelty relative to all cultures (indicated at the top of the panel) (9). Primer-amplified bacterial sequences showed 30% to up to 100% similarity to their closest cultured relative but were removed for clarity.

FIG 2

Proportion of 16S rRNA gene sequences in each category of phylogenetic novelty relative to cultures for each environment, by amplicons, metagenomes (without scaffold read depth), and metatranscriptomes. Closed circles represent primer-amplified amplicons, open circles represent metagenomes, and crosses represent transcriptomes. Total numbers of sequences and studies are listed in Table S2.

FIG 3

Proportion of 16S rRNA gene sequences by scaffold read depth averaged across all metagenomes. Each single data point represents the abundance of reads in that similarity bin from a single metagenome. Rows represent different similarity bins.

FIG 4

Rank abundance plots by taxonomic genus assignments for metagenomic data (top three rows) and metatranscriptomic data (bottom two rows). Listed in each box are the top 10 most abundant genera for that environment in the format of phylum_lowest identified taxonomic group, with asterisks (*) denoting archaea. Data are colored for uncultured phyla (teal), uncultured class to genus (pink) or cultured phyla (tan). Taxonomic-based genera that had sequences from multiple phylogeny-based percent identity bins were labeled with the color of the bin with the most sequences.