Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

Abstract

Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

Vast Differences in Strain-Level Diversity in the Gut Microbiota of Two Closely Related Honey Bee Species

“…Despite extensive horizontal gene transfer in bacteria [ 3 ], recent studies have shown that bacteria exist as discrete populations, which can be identified based on metagenomic read recruitment [ 25 , 26 , 27 , 28 ] or gANI (genomic average nucleotide identity) [ 11 , 29 , 30 ]. Bacteria belonging to the same population generally have more than 95% gANI with each other, whereas bacteria from different populations typically have less than 90% gANI [ 11 , 30 ]. These populations have therefore also been referred to as “sequence-discrete populations” (SDPs) or even “species” [ 11 , 27 , 30 ], although the latter term continues to be controversial for bacteria.…”

“…Bacteria belonging to the same population generally have more than 95% gANI with each other, whereas bacteria from different populations typically have less than 90% gANI [ 11 , 30 ]. These populations have therefore also been referred to as “sequence-discrete populations” (SDPs) or even “species” [ 11 , 27 , 30 ], although the latter term continues to be controversial for bacteria. For the western honeybee, Apis mellifera , a recent metagenomic study identified 1–4 SDPs per 16S rRNA phylotype in the gut microbiota [ 7 ].…”

Huge and variable diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems

“…Additionally, we do not attribute this observed genome similarity to index hopping during sequencing (see Supplementary Note 2). Even at the species level (>95% ANI 39 ), most pairs of analyzed sites share no more than one species in common (SI Fig 3). The overall lack of similarity between these ten groundwater sites--at the phylum level, species level, and strain level-indicates a high degree of specialization based on local hydrogeochemical conditions.…”

Insights into rumen microbial biosynthetic gene cluster diversity through genome-resolved metagenomics

“…Collection, and rumen-specific MAGs Recent analyses support a 95% ANI threshold to delineate microbial species 51,52 . The ANI values of MAGs from the current study and genomes from the GTDB (a curated and dereplicated collection of 22,441 genomes in the GTDB-Tk FastANI database 21 ), Hungate1000 project 3 , and Stewart et al 4,5 were compared in a pairwise fashion with FastANI (version 1.1) 51 .…”