Based on the annotations described above, we enumerated the Legionella pan-genome and how accessory genes defined species relationships. We identified 12,977 ortholog gene families in all 43 genomes based on OrthoMCL clustering with a BLASTP identity cutoff of 10−5. Supplementary Figure 2A shows the barplot listing the number of gene clusters found in each genome. There were 1140 core gene families in all 43 genomes, representing the core genes in Legionella species, which were used in downstream phylogenetic, recombination and positive selection analyses.