Procaryotic species and subspecies delineation using average nucleotide identity and gene order conservation

Two genome-based parameters, gene order conservation (GOC) and average nucleotide identity (ANI) were studied to define how accurately two species, or two subspecies could be distinguished on the basis of these parameters. The first examined species was Lactococcus lactis since its two subspecies, the subsp. lactis and the subsp. cremoris have complete genome sequences available. Comparative analysis of DNA sequences of two subspecies of Streptococcus equi, S. equi subsp. equi and S. equi subsp. zooepidemicus were performed. In addition, S. thermophilus and S. salivarius, which formerly were classified into one species, were also compared. The GOC values are 66% or higher if the strains belong to the same species and there is no difference in GOC data of strains belonging to either the same subspecies or different subspecies. However, if two different species are compared, the GOC value decreases below 50% enabling an accurate separation of two species. The GOC value of 66% corresponds to a DNA-DNA hybridisation value of 70%. Unlike GOC, ANI measurements allow distinction of subspecies. If the strains belong to the same subspecies, ANI values are higher than 96%. However, when strains from different subspecies are compared the values are between 88% and 90%. Therefore, the average nucleotide identity can be used to delineate the boundary between subspecies. All genomic data and results of phylogenetic analyses obtained here by comparing S. salivarius and S. thermophilus support their previous taxonomic classification that they are actually two subspecies and not two separate species.