Colonies of ΔtasA were inoculated as described above and incubated for 5 days. Fluorescent protrusions from the edge of the colony were isolated on LB for single colonies. Liquid cultures of single colonies were frozen and restreaked on LB, and the biofilm on solid surface assay was performed as described above for phenotype validation.

Genomes of nine representative strains, in addition to four ancestral strains, were sequenced. DNA was extracted using a DNeasy blood and tissue kit (QIAGEN). Libraries were generated using a Nextera XT DNA sample preparation kit (Illumina). Sequencing of 100–base pair single-read reads (50 cycles) was performed on an Illumina HiSeq 2500 sequencer (Illumina), using TruSeq rapid mode run reagent kits: two TruSeq Rapid SBS Kits HS (Illumina) and TruSeq Rapid SR Cluster Kit (Illumina).

Sequencing reads were aligned separately for each sample to the reference genome of B. subtilis NCIB 3610 (GenBank: NZ_CM000488) that was downloaded from National Center for Biotechnology Information. The reads were aligned using Novoalign 2.08.01 (Novocraft Technologies Sdn Bhd, www.novocraft.com) with the default parameters and [−r Random]. Detection of mutations (mismatches and insertions) was performed by comparing the alignments of each sample to the alignments of the ancestor B. subtilis sample that was also sequenced. Genomic positions that consistently differed between both alignments (>70%) were recorded as mutations. Genomic positions with no aligned reads but with aligned reads in the ancestor sample were recorded as deletions. Mutations in additional strains were identified using PCR and Sanger sequencing.

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