International Journal of Systematic and Evolutionary Microbiology, Vol 52, 573-586, Copyright © 2002 by Society for General Microbiology

A mathematical method for determining genome divergence and species delineation using AFLP

C. Mougel, J. Thioulouse, G. Perriere and X. Nesme
Ecologie Microbienne UMR-CNRS 5557 and INRA, Bat. G. Mendel, Universite Claude Bernard-Lyon 1, 43 bd du 11 novembre 1918, 69622 Villeurbanne cedex, France

The delineation of bacterial species is presently achieved using direct DNA--DNA relatedness studies of whole genomes. It would be helpful to obtain the same genomically based delineation by indirect methods, provided that descriptions of individual genome composition of bacterial genomes are obtained and included in species descriptions. The amplified fragment length polymorphism (AFLP) technique could provide the necessary data if the nucleotides involved in restriction and amplification are fundamental to the description of genomic divergences. Firstly, in order to verify that AFLP analysis permits a realistic exploration of bacterial genome composition, the strong correspondence between predicted and experimental AFLP data was demonstrated using Agrobacterium strain C58 as a model system. Secondly, a method is proposed for determining current genome mispairing and evolutionary genome divergences between pairs of bacteria, based on arbitrary sampling of genomes by using AFLP. The measure of current genome mispairing was validated by comparison with DNA--DNA relatedness data, which itself correlates with base mispairing. The evolutionary genome divergence is the estimated rate of nucleotide substitution that has occurred since the strains diverged from a common ancestor. Current genome mispairing and evolutionary genome divergence were used to compare members of Agrobacterium, used as a model of closely related genomic species. A strong and highly significant correlation was found between calculated genome mispairing and DNA--DNA relatedness values within genomic species. The canonical 70% DNA--DNA hybridization value used to delineate genomic species was found to correspond to a range of current genome mispairing of 13--13.6%. These limits correspond to 0.097 and 0.104 nucleotide substitutions per site, respectively. In addition, experimental data showed that the large Ti and cryptic plasmids of Agrobacterium had little effect on the estimation of genome divergence. Evolutionary genome divergence was used for phylogenetic inferences. Data showed that members of the same genomic species clustered consistently, as supported by bootstrap resampling. On the basis of these results, it is proposed that the genomic delineation of bacterial species could be based, in future, on phylogenetic groups supported by bootstraps and genome descriptions of individual strains, obtained by AFLP analysis, recorded in accessible databases; this approach might eventually replace DNA--DNA hybridization studies.