Species boundaries and molecular markers for classification among 16SrI phytoplasmas informed by genome analysis

Phytoplasmas are diverse plant-pathogenic bacteria that greatly impact agriculture worldwide. The current classification system for these uncultivated bacteria is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains that belong to the 16SrI group within the genus 9Candidatus Phytoplasma9 and investigated the possible species boundaries. We found that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units equivalent to species. Strains assigned to the same species share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different species share