Protein sequence of five B. subtilis strains and 10 Bacillus species were downloaded from EnsEMBLBacteria(release 19).

Those five strains were B. subtilis subsp. subtilis str. 168, B. subtilis BSn5, B. subtilis subsp. subtilis str. RO-NN-1, B. subtilis subsp. spizizenii TU-B-10 and B. subtilis subsp. spizizenii str. W23, which was same as the strains listed in Paul et al 2013.

Those 10 species were B.alcalophilus, B.azotoformans, B.cellulosilyticus, B.cereus, B.clausii, B.coagulans, B.halodurans, B.megaterium, B.pumilus, B.selenitireducens.

Five strains of B. subtilis one-to-one orthologs were identified by reciprocal best hits implemented in BLASTP with default parameters. 334 of core genes which length longer than 200 amino acid listed in Paul et al 2013 were found.

We used B.subtilis subsp. subtilis str. 168 as the reference genome and extracted best hits from the other 10 species with protein sequence identity and length coverage values of at least 70%. 266 of 334 core genes had at least one hit in one of the ten species.

Multiple protein sequence alignments were generated by ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) with default parameters. Sites with gap in any of the 5 strains were removed.

In fig ab, variable sites of each protein were defined by the total number of mismatch sites of alignment.
In fig cd, variable sites of each protein were defined by the length of alignment.
