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.
Highlights
Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them
Sets of microbial genomes without the selection biases introduced by isolation were generated from metagenomic studies of three environments: infant fecal samples (1,163 metagenomes collected from 160 hospitalized premature infants over 5 years) [33], the ocean (234 metagenomes collected from the global Tara Oceans Expedition over 7 years) [34], and a meadow soil ecosystem (60 metagenomes collected from three depths at five locations for five time points across a grassland meadow) [26]
Clustering was observed based on both average nucleotide identity and genome alignment fraction, estimated rates of horizontal gene transfer fell to near zero at the 95% average nucleotide identities (ANI) boundary in all tested environments, and genome-wide dN/dS ratios consistently leveled near values of 0.15 at around 98% ANI in most environments (Fig. 2)
Summary
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. 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. Attempts have been made to remove the bias from this reference genome set in searching for naturally distinct bacterial populations, for example, by sampling five genomes from each species with at least five genomes in the database [10], but selective cultivation and sequencing cause biases that are difficult to account for. Whole-genome comparisons of dN/dS data nearly always result in values below 1 [24], indicative of purifying selection and likely due to the continuous removal of slightly deleterious nonsynonymous mutations over time
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