J. Microbiol. Biotechnol. (2017), 27(0), 1–5 https://doi.org/10.4014/jmb.1712.12006 Research Article Review jmb Fig. S1. Circular genomic map of B. s. subtilis KCTC 3135T by CLgenomics ver. 1.55. From the outer circle to the inward circle, the circle indicates rRNA and/or tRNA, reverse CDS, forward CDS, GC skew and the GC ratio. A 2017 ⎪ Vol. 27⎪ No. 0 2Name et al. Fig. S2. OrthoANI distance Neighbor-Joining tree with 60 strains (extended OrthoANI tree). A neighbor-joining tree based on the OrthoANI distance matrix. B. subtilis with three subspecies contains 59 genomes and S. aureus subsp. aureus DSM 20231T was used as an outgroup. The scale bar indicates the sequence divergence. J. Microbiol. Biotechnol. Title 3 Fig. S3. The tarABD MLST NJ tree and heatmap with 60 strains (extended NJ tree). A) Phylogenetic tree using the tarABD Multilocus sequence analysis (MLSA) Neighbor-Joining (NJ) tree and B) heatmap based on the similarities of the tarABD from B. subtilis genome to the reference tar or tag genes. B. subtilis with three subspecies contains 59 genomes and S. aureus subsp. aureus DSM 20231T was used for outgroup. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. The brand nodes also recovered maximum-likelihood (ML) and maximum-parsimony (MP) trees are marked by the filled black circles. The bar indicates nucleotide substitution rate in given length of the scale. The color indicated the tblastn nucleotide similarities to reference tar (red) or tag (green) genes. A 2017 ⎪ Vol. 27⎪ No. 0 4Name et al. Fig. S4. Neighbor-Joining tree with 59 strains based on concatenated sequence of seven housekeeping genes. Seven housekeeping genes (glpF, ilvD, pta, purH, pycA, rpoD, tpiA) are used for Multilocus sequence analysis (MLSA) Neighbor-Joining (NJ) phylogenetic tree. B. subtilis with three subspecies contains 59 genomes. B. subtilis subsp. inaquosorum J-5 was used for outgroup. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. The bar indicates nucleotide substitution rate in given length of the scale. J. Microbiol. Biotechnol. Title 5 Fig. S5. Neighbor-Joining tree based on the concatenated sequence of seven conserved genes and tarABD. Seven housekeeping genes (glpF, ilvD, pta, purH, pycA, rpoD, tpiA) and the tarABD are concatenated for Multilocus sequence analysis (MLSA) Neighbor-Joining (NJ) phylogenetic tree. B. subtilis with three subspecies contains 59 genomes. B. subtilis subsp. inaquosorum J-5 was used for outgroup. Bootstrap values (>70%) based on 1000 replicates are shown at branch nodes. The bar indicates nucleotide substitution rate in given length of the scale. A 2017 ⎪ Vol. 27⎪ No. 0 Supplementary Table 1. Bacillus subtilis genomes used in thist study assembly accession sequence accession number Species strain name (RefSeq) (chromosome) B. subtili s subsp. subtilis 168G GCF_001703495.1 CP016852.1 168 GCF_000009045.1 AL009126.3 29R7-12 GCF_001902555.1 CP017763.1 3NA GCF_000827065.1 CP010314.1 6051-HGW GCF_000344745.1 CP003329.1 AG1839 GCF_000699525.1 CP008698.1 BAB-1 GCF_000349795.1 CP004405.1 BEST195 GCF_000209795.2 AP011541.2 BEST7003 GCF_000523045.1 AP012496.1 BS16045 GCF_001720505.1 CP017112.1 BS34A GCF_000952895.1 LN680001.1 BS38 GCF_001746575.1 CP017314.1 BS49 GCF_000953615.1 LN649259.1 BSD-2 GCF_001465815.1 CP013654.1 BSn5 GCF_000186745.1 CP002468.1 BSP1 GCF_000321395.1 CP003695.1 CGMCC 2108 GCF_001565875.1 CP014471.1 CU1050 GCF_001541905.1 CP014166.1 D12-5 GCF_001596535.1 CP014858.1 delta6 GCF_001660525.1 CP015975.1 HJ0-6 GCF_001704095.1 CP016894.1 HJ5 GCF_000973605.1 CP007173.1 HRBS-10TDI13 GCF_001747445.1 CP015222.1 JH642 substr. AG174 GCF_000699465.1 CP007800.1 JS GCF_000259365.1 CP003492.1 KCTC 1028 GCF_000971925.1 CP011115.1 KCTC 3135T GCF_001697265.1 CP015375.1 KH2 GCF_001890405.1 CP018184.1 LM 4-2 GCF_000978495.1 CP011101.1 OH 131.1 GCF_000706705.1 CP007409.1 PS832 GCF_000789295.1 CP010053.1 PY79 GCF_000497485.1 CP006881.1 QB928 GCF_000293765.1 CP003783.1 RO-NN-1 GCF_000227485.1 CP002906.1 SG6 GCF_000782835.1 CP009796.1 SZMC 6179J GCF_001604995.1 CP015004.1 TO-A JPC GCF_001037985.1 CP011882.1 TOA GCF_000737405.1 CP005997.1 UD1022 GCF_001015095.1 CP011534.1 VV2 GCF_001808235.1 CP017676.1 XF-1 GCF_000338735.1 CP004019.1 YP1 GCF_000877815.1 CP010014.1 B. subtilis subsp. spizizenii ATCC 6633 GCF_000177595.1 contigs file (incomplete) BST GCF_000743215.1 contigs file (incomplete) DV1-B-1 GCF_000245035.1 contigs file (incomplete) HUK15 GCF_001566945.1 contigs file (incomplete) JCM 2499 GCA_001312785.1a contigs file (incomplete) MJ01 GCF_001889625.1 CP018173.1 NRS 231 GCF_000816805.1 CP010434.1 RFWG1A3 GCF_000931815.1 contigs file (incomplete) RFWG1A4 GCF_000931825.1 contigs file (incomplete) RFWG4C10 GCF_000931835.1 contigs file (incomplete) RFWG5B15 GCF_000931845.1 contigs file (incomplete) T30 GCF_000959025.1 CP011051.1 TU-B-10T GCF_000227465.1 CP002905.1 W23 GCF_000146565.1 CP002183.1 B. subtilis subsp. inaquosorum DE111 GCF_001534785.1 CP013984.1 J-5 GCF_001889385.1 CP018295.1 KCTC 13429T GCF_000332645.1 contigs file (incomplete) S. aureus subsp. aureus (outgroup) DSM 20231T GCF_001027105.1 CP011526.1 a GenBank assembly accession, RefSeq assembly accession non-available. Supplementary Table 2. The tar and the tag genes tblastn similarities with 60 genomes Genes Query Target Identity Alignment length E-value Clade B_subtilis_subsp_spizizenii_T30 tarA CAC86111.1 CP011051.1_cds_BIS30_07525_1422 100 257 0 tarB CAC86110.1 CP011051.1_cds_BIS30_07530_1423 99.74 383 0 tarD CAC86112.1 CP011051.1_cds_BIS30_07520_1421 100 129 8.00E-93 tarF CAC86113.1 CP011051.1_cds_BIS30_07515_1420 100 394 0 tagA AAA22844.1 CP011051.1_cds_BIS30_07525_1422 59.53 257 3.00E-110 tagB AAA22845.1 CP011051.1_cds_BIS30_07530_1423 49.21 378 3.00E-134 tagD AAA22843.1 CP011051.1_cds_BIS30_07520_1421 76.74 129 9.00E-72 tagF NP_391453.1 CP011051.1_cds_BIS30_07515_1420 64.21 380 1.00E-178 B_subtilis_subsp_spizizenii_W23 tarA CAC86111.1 CP002183.1_cds_BSUW23_17555_3487 100 257 0 tarB CAC86110.1 CP002183.1_cds_BSUW23_17560_3488 99.74 383 0 tarD CAC86112.1 CP002183.1_cds_BSUW23_17550_3486 100 129 8.00E-93 tarF CAC86113.1 CP002183.1_cds_BSUW23_17545_3485 100 394 0 tagA AAA22844.1 CP002183.1_cds_BSUW23_17555_3487 59.53 257 4.00E-110 tagB AAA22845.1 CP002183.1_cds_BSUW23_17560_3488 49.21 378 3.00E-134 tagD AAA22843.1 CP002183.1_cds_BSUW23_17550_3486 76.74 129 1.00E-71 tagF NP_391453.1 CP002183.1_cds_BSUW23_17545_3485 64.21 380 1.00E-178 B_subtilis_subsp_spizizenii_RFWG1A4 tarA CAC86111.1 GCF_000931825.1_03517 100 257 0 tarB CAC86110.1 GCF_000931825.1_03518 99.48 383 0 tarD CAC86112.1 GCF_000931825.1_03516 100 77 8.00E-52 tarF CAC86113.1 GCF_000931825.1_03515 72.18 248 1.00E-134 tagA AAA22844.1 GCF_000931825.1_03517 59.53 257 4.00E-110 tagB AAA22845.1 GCF_000931825.1_03518 49.47 378 6.00E-136 tagD AAA22843.1 GCF_000931825.1_03516 83.12 77 7.00E-43 tagF NP_391453.1 GCF_000931825.1_03515 58.3 235 2.00E-96 B_subtilis_subsp_spizizenii_NRS_231 tarA CAC86111.1 CP010434.1_cds_SD85_17845_3413 100 257 0 tarB CAC86110.1 CP010434.1_cds_SD85_17850_3414 99.74 383 0 tarD CAC86112.1 CP010434.1_cds_SD85_17840_3412 100 129 8.00E-93 tarF CAC86113.1 CP010434.1_cds_SD85_17835_3411 100 394 0 tagA AAA22844.1 CP010434.1_cds_SD85_17845_3413 59.53 257 4.00E-110 tagB AAA22845.1 CP010434.1_cds_SD85_17850_3414 49.21 378 3.00E-134 tagD AAA22843.1 CP010434.1_cds_SD85_17840_3412 76.74 129 1.00E-71 tagF NP_391453.1 CP010434.1_cds_SD85_17835_3411 64.21 380 1.00E-178 B_subtilis_subsp_spizizenii_JCM_2499 tarA CAC86111.1 GCA_001312785.1_00738 100 257 0 tarB CAC86110.1 GCA_001312785.1_00737 99.74 383 0 tarD CAC86112.1 GCA_001312785.1_00739 100 129 8.00E-93 tarF CAC86113.1 GCA_001312785.1_00740 99.65 286 0 tagA AAA22844.1 GCA_001312785.1_00738 59.53 257 4.00E-110 tagB AAA22845.1 GCA_001312785.1_00737 49.21 378 3.00E-134 tagD AAA22843.1 GCA_001312785.1_00739 76.74 129 1.00E-71 tagF NP_391453.1 GCA_001312785.1_00740 69.96 283 4.00E-141 B_subtilis_subsp_spizizenii_BST tarA CAC86111.1 GCF_000743215.1_02629 100 257 0 tarB CAC86110.1 GCF_000743215.1_02628 99.74 383 0 tarD CAC86112.1 GCF_000743215.1_02630 100 129 8.00E-93 tarF CAC86113.1 GCF_000743215.1_02631 100 394 0 tagA AAA22844.1 GCF_000743215.1_02629 59.53 257 4.00E-110 tagB AAA22845.1 GCF_000743215.1_02628 49.21 378 3.00E-134 tagD AAA22843.1 GCF_000743215.1_02630 76.74 129 1.00E-71 tagF NP_391453.1 GCF_000743215.1_02631 64.21 380 1.00E-178 B_subtilis_subsp_spizizenii_ATCC_6633 tarA CAC86111.1 GCF_000177595.1_03132 100 257 0 tarB CAC86110.1 GCF_000177595.1_03131 99.74 383 0 tarD CAC86112.1 GCF_000177595.1_03133 100 129 8.00E-93 tarF CAC86113.1 GCF_000177595.1_03134 100 394 0 tagA AAA22844.1 GCF_000177595.1_03132 59.53 257 4.00E-110 tagB AAA22845.1 GCF_000177595.1_03131 49.21 378 3.00E-134 tagD AAA22843.1 GCF_000177595.1_03133 76.74 129 1.00E-71 tagF NP_391453.1 GCF_000177595.1_03134 64.21 380 1.00E-178 B_subtilis_subsp_spizizenii_MJ01 tarA CAC86111.1 CP018173.1_cds_BAX60_02740_545 97.28 257 0 tarB CAC86110.1 CP018173.1_cds_BAX60_02745_546 95.04 383 0 tarD CAC86112.1 CP018173.1_cds_BAX60_02735_544 96.12 129 4.00E-90 tarF CAC86113.1 CP018173.1_cds_BAX60_02730_543 64.97 394 0 tagA AAA22844.1 CP018173.1_cds_BAX60_02740_545 59.53 257 1.00E-110 tagB AAA22845.1 CP018173.1_cds_BAX60_02745_546 48.94 378 7.00E-135 tagD AAA22843.1 CP018173.1_cds_BAX60_02735_544 78.29 129 5.00E-73 tagF NP_391453.1 CP018173.1_cds_BAX60_02730_543 55.99 384 5.00E-162 B_subtilis_subsp_inaquosorum_KCTC_13429T tarA CAC86111.1 GCF_000332645.1_00225 96.89 257 0 tarB CAC86110.1 GCF_000332645.1_00226 93.99 383 0 tarD CAC86112.1