Towards estimating the number of strains that make up a natural bacterial population
What a strain is and how many strains make up a natural bacterial population remain elusive concepts despite their apparent importance for assessing the role of intra-population diversity in disease emergence or response to environmental perturbations. To advance these concepts, we sequenced 138 randomly selected Salinibacter ruber isolates from two solar salterns and assessed these genomes against companion short-read metagenomes from the same samples. The distribution of genome-aggregate average nucleotide identity (ANI) values among these isolates revealed a bimodal distribution, with significantly lower occurrence of values between 99.2% and 99.8% relative to ANI >99.8% or <99.2%, revealing a natural "gap" in the sequence space within species. Accordingly, we used this ANI gap to define genomovars and a higher ANI value of >99.99% and shared gene-content >99.0% to define strains. Using these thresholds and extrapolating from how many metagenomic reads each genomovar uniquely recruited, we estimated that -although our 138 isolates represented about 80% of the Sal. ruber population- the total population in one pond is composed of 5,500 to 11,000 genomovars, the great majority of which appear to be rare in situ. These data also revealed that the most frequently recovered isolate in lab media was often not the most abundant genomovar in situ, suggesting that cultivation biases are significant, even in cases that cultivation procedures are thought to be robust. Preliminary analyses of available genomes revealed that the thresholds used for defining strains and distinct intra-species units (genomovars) may be broadly applicable to additional bacterial species. Significance StatementStrains are the smallest distinguishable units within a microbial species. Strains that carry unique gene content often underly the emergence of disease outbreaks and the response of the species to environmental perturbations. Therefore, a major challenge in microbiome research across environmental and clinical settings is to evaluate how many strains of the same species coexist in nature and how dominant strains emerge from this diversity. Unfortunately, the available theoretical concept of strain is not directly applicable to culture-independent surveys. Here, we provide such a practical definition for strain and use it to show that that the number of strains making up a natural bacterial population may be large, in the order of a few thousands, but not infinite.