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Supplementary Methods
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Description of Homologous Recombination (HR) hypotheses at the polymorphic
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Mph region (Mph Cluster A gene) in the B. cereus group.
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According to the differences in phylogenetic trees constructed from six housekeeping genes (Fig.
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3), the 16 Kbp, 5.5 Kbp, and 4 Kbp surrounding regions of the Mph Cluster A gene, and the Mph
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gene polymorphic region (Suppl. Fig. S3), seven homologous recombination hypotheses involving
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the polymorphic Mph region were proposed. For all hypotheses, the recombinant is defined as the
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strain in which the HR event have occurred (or its direct ancestor), the minor parent is defined as
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the strain from which the acquired sequence belongs to, and the major parent is defined as the
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closest strain to the recombinant which did not undergone the HR event.
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1- B. cereus Rock 1-3 isolate have acquired the Mph allele from a strain related to B. cereus
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Rock 3-28 isolate.
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Phylogenetic information
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B. cereus Rock 3-28 and B. cereus Rock 1-3 are sister strains in the polymorphic Mph region
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phylogeny (Suppl. Fig. S3D), with B. cereus Rock 4-18 as an outgroup, while B. cereus Rock 3-28
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is an outgroup of B. cereus Rock 4-18 /B. cereus Rock 1-3 strains in all other phylogenies (Fig. 3
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and Suppl. Fig. S3A-C).
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In the 16 Kbp phylogeny (Suppl. Fig. S3A), B. cereus Rock 3-28 is a sister group of B.cereus
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VD115, while it is the sister group of B. cereus Rock 4-18/B. cereus Rock 1-3 in the housekeeping
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genes phylogeny (Fig. 3) and in both 5 Kbp phylogenies (Suppl. Fig. S3B-C).
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Hypothesis
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This means that B. cereus Rock 1-3 and B. cereus Rock 3-28 Mph genes are more similar to each
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other than to the B. cereus Rock 4-18 Mph gene, while the overall sequence of B. cereus Rock 1-3
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is more similar to B. cereus Rock 4-18 than to B. cereus Rock 3-28. A HR event thus probably
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occurred at the polymorphic Mph region, with B. cereus Rock 1-3 as the recombinant, a strain
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related to B. cereus Rock 3-28 as the minor parent, and a strain related to B. cereus Rock 4-18 as
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the major parent.
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The inconsistency of B. cereus Rock 3-28 position in the 16 Kbp tree compared to other trees
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may be explained by another HR event which may have occurred outside of the 5.5 kbp region but
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in the 16 Kbp region, involving B. cereus Rock 3-28 as the recombinant, a strain related to B.
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cereus VD115 as the minor parent, and a strain related to B. cereus Rock 3-28 as the major parent.
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RDP analysis
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Analysis A:
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Included isolates: B. cereus Rock 1-3, B. cereus Rock 3-28, B. cereus Rock 4-18, B. cereus VD156
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(outgroup). Analysis performed on the 16 Kbp region with the polymorphic Mph region included.
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Four methods provided support with p-value < 10-3 for the Mph replacement in B. cereus Rock
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1-3 by an allele related to the B. cereus Rock 3-28 allele (Suppl. Table S3). All methods infer a ~1
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Kbp recombination event including ~ 500 bp from the Mph polymorphic region.
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Analysis B:
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Included isolates: B. cereus Rock 1-3, B. cereus Rock 3-28, B. cereus Rock 4-18, B. cereus
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VD115, B. cereus HuB5-5 and B. cereus ST196 (outgroup). Analysis performed on the 16 Kbp
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region without the polymorphic Mph region.
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Five methods provided highly significant support for a ~2 Kbp recombination event that
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occurred in B. cereus Rock 3-28 with B. cereus VD115 as the minor parent (Suppl. Table S2). All
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methods locate the recombination event between positions ~14300 and ~16300 in the alignment,
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thus outside of the 5.5 Kbp region.
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2- B. cereus HuB5-5 isolate have lost the Mph allele after RH with a strain related to B.
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cereus VD115 isolate.
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Phylogenetic information
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B. cereus HuB5-5 is included in a monophyletic group of Mph-carrying isolates (including B.
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cereus Rock 1-3 and B. cereus Rock 4-18) in the housekeeping genes phylogeny (Fig. 3), while B.
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cereus HuB5-5 branches as a sister group of B. cereus Rock 1-3 and B. cereus Rock 4-18 in the 16
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Kbp and both 5.5 Kbp phylogenies (Suppl. Fig. S3A-C).
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Hypothesis
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As B. cereus HuB5-5 do not carry the polymorphic Mph region, this strain may have lost it
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through RH with another strain not carrying the Mph gene. B. cereus HuB5-5 is the recombinant,
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and any of the B. cereus Rock 1-3 / B. cereus Rock 3-28 / B. cereus Rock 4-18 may be the major
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parent. B. cereus HuB5-5 is located in the same group with 100% bootstrap support in all
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phylogenies, suggesting that the minor parent also belongs to this group, thus related to B. cereus
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VD115.
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RDP analysis
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Included isolates: B. cereus HuB5-5, B. cereus Rock 1-3, B. cereus Rock 3-28, B. cereus Rock
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4-18, B. cereus VD115, and B. cereus ST196 (outgroup). Analysis performed on the 16 Kbp
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region without the polymorphic Mph region.
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The RDP analysis did not returned any putative HR event in B. cereus HuB5-5 in the region of
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interest, which does not support the Mph gene removal through HR with a B. cereus VD115
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relative (Suppl. Table S2). Moreover no HR with B. cereus ST196 was inferred, excluding a
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potential event with any of the known Mph-free isolates.
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A potential explanation would be that a HR event larger than the 16 Kbp region have occurred
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between B. cereus HuB5-5 and a strain related to the B. cereus VD115 isolate, but such a putative
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RH event cannot be inferred by RDP with the present 16 kbp window.
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3- B. cereus Rock 3-42 isolate have acquired the Mph allele from a strain related to B.
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thuringiensis BGSC 4BA1 isolate.
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Phylogenetic information
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B. cereus Rock 3-42 groups with B. cereus ATCC 4342 with 100% bootstrap value in the
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housekeeping genes phylogeny (Fig. 3), while it branches as the sister group of B. anthracis
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isolates in the 16 kbp and both 5.5 Kbp phylogenies (Suppl. Fig. S3A-C). In the polymorphic Mph
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region phylogeny, it branches as the sister group of B. thuringiensis BGSC 4BA1 (Suppl. Fig.
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S3D).
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Hypothesis
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The inconsistent topology between housekeeping genes phylogeny and other phylogenies suggests
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that B. cereus Rock 3-42 may have acquired the polymorphic Mph region from a relative of B.
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anthracis strains. The major parent would be B. cereus ATCC 4342, and the minor parent would
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be any of the B. anthracis strains. The fact that B. cereus Rock 3-42 still branches with B.
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anthracis strains when the polymorphic Mph region is removed (Suppl. Fig. S3C) suggests that
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the RH event may span the whole 5.5 Kbp region. A second HR event then may have occurred in
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the polymorphic Mph region with B. cereus Rock 3-42 as the recombinant, a B. anthracis strain as
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the major parent, and B. thuringiensis BGSC 4BA1 as the minor parent.
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RDP analysis
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Analysis A:
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Included isolates: B. cereus Rock 3-42, B. anthracis BF1, B. thuringiensis BGSC 4BA1, B.
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thuringiensis BGSC 4AW1, B. cereus ATCC 4342, and B. cereus MM3 (outgroup). Analysis
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performed on the 16 Kbp region without the polymorphic Mph region.
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The RDP analysis did not returned any putative HR event in B. cereus Rock 3-42 in the region
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of interest, which does not support the Mph gene acquisition through HR from a B. anthracis
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relative.
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Again, a potential explanation would be that B. cereus Rock 3-42 has acquired the Mph gene
through a recombination larger than the 16 Kbp region.
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Analysis B:
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Included isolates: B. cereus Rock 3-42, B. anthracis BF1, B. thuringiensis BGSC 4BA1, B.
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thuringiensis BGSC 4AW1, B. cereus BGSC 6E1, and B. cereus VD156 (outgroup). Analysis
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performed on the 16 Kbp region with the polymorphic Mph region included.
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Four methods provided support for a ~2 Kbp recombination event that occurred in B. cereus
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Rock 3-42 with B. thuringiensis BGSC 4BA1 as the minor parent (Suppl. Table S2). The
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recombined section spans the whole polymorphic Mph region for most methods.
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4- B. cereus R309803 isolate have acquired the Mph gene from a strain related to B. cereus
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VD156 isolate.
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Phylogenetic information
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B. cereus R309803 groups as the root of Clade 1 and Clade 2 in the housekeeping genes
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phylogeny (Fig. 3) and the 16 Kbp phylogeny (Suppl. Fig. S3A), while it branches as the sister
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group of B. cereus VD156 and B. thuringiensis BGSC 4BD1 in all other phylogenies (Suppl. Fig.
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S3B-D).
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Hypothesis
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B. cereus R309803 is the recombinant with B. cereus BAG2x1-1 as the major parent, and has
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acquired the Mph gene from a minor parent related to B. cereus VD156.
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RDP analysis
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Included isolates: B. cereus R309803, B. cereus VD156, B. cereus BAG2x1-1, B. cereus ST196
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(outgroup). Analysis performed on the 16 Kbp region without the polymorphic Mph region.
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Five methods provided highly significant support for a ~3.5 Kbp recombination event that
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occurred in B. cereus R309803 with B. cereus VD156 as the minor parent (Suppl. Table S2). The
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recombined section spans the whole polymorphic Mph region.
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5- B. cereus m1293 and B. cereus LCT BC244 isolates have acquired the Mph gene from a
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strain related to the group B. anthracis/B. cereus Rock 3-42 /B. thuringiensis BGSC 4BA1/B.
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thuringiensis BGSC 4AW1/B. cereus BGSC 6E1 isolates (hereafter referred to the Ba++
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group).
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Phylogenetic information
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B. cereus m1293/B. cereus LCT BC244 is the sister group of B. cereus VD102/B. cereus BDRD
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ST26 in the housekeeping gene phylogeny (Fig. 3) and the 16 Kbp phylogeny (Suppl. Fig. S3A)
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but become the sister group of Ba++ in the 5.5 Kbp phylogeny including the polymorphic Mph
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region (Suppl. Fig. S3B). When the polymorphic region is removed, B. cereus m1293/B. cereus
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LCT BC244 branches back in the B. cereus VD102/B. cereus BDRD ST26 group (Suppl. Fig.
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S3C).
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Hypothesis
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The group composed of B. cereus m1293 and B. cereus LCT BC244 is the recombinant, with B.
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cereus BDRD ST26 as the major parent, and have acquired the Mph gene from a minor parent
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related to the Ba++ group. B. cereus VD102 is not chosen as the major parent because it is a
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potential recombinant, see Hypothesis 6 below.
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RDP analysis
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Included isolates: B. cereus m1293, B. cereus BDRD ST26, B. thuringiensis BGSC 4AW1
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(representative of the Ba++ group), B. cereus MM3 (outgroup). Analysis performed on the 16 Kbp
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region without the polymorphic Mph region.
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All six methods provide highly significant support for a ~2 Kbp recombination event that
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occurred in B. cereus m1293 and B. cereus BDRD ST26, with B. thuringiensis BGSC 4AW1 as
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the minor parent (Suppl. Table S2). The recombined section spans the whole polymorphic Mph
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region.
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6- B. cereus VD102 isolate have lost the Mph gene after RH with a strain related to B. cereus
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BDSD ST26.
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Phylogenetic information
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B. cereus VD102 is the sister group of B. cereus m1293/B. cereus CT BC244 in the housekeeping
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gene phylogeny (Fig. 3) and the 16 Kbp phylogeny (Suppl. Fig. S3A) but in the 5.5 Kbp
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phylogeny without the polymorphic Mph region, B. cereus VD102 branches as the sister group of
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B. cereus LCT BC244 with high bootstrap support, with B. cereus m1293 as the outgroup (Suppl.
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Fig. S3C).
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Hypothesis
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B. cereus VD102 may be the closest relative of B. cereus LCT BC244, and may have lost the Mph
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gene through HR with a Mph-free Clade 1 strain. Because of the extreme nucleotide similarity
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between B. cereus m1293, B. cereus LCT BC244 and B. cereus VD102, the presence of the
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polymorphic Mph region in B. cereus m1293 and B. cereus LCT BC244 may have artificially
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inflated their genetic similarity compared to B. cereus VD102. B. cereus VD102 would thus be the
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recombinant, with B. cereus LCT BC244 as the major parent and any of the Mph-free Clade 1
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strain as the minor parent.
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RDP analysis
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Included isolates: B. cereus LCT BC244, B. cereus VD102, B. cereus BDRD ST26, B. cereus
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ATCC 4342, B. cereus F, B. cereus BAG6X1-1 (outgroup). Analysis performed on the 16 Kbp
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region without the polymorphic Mph region.
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Four methods provided significant support for a ~1.5-2 Kbp recombination event that occurred
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in B. cereus VD102, with B. cereus ATCC 4342 as the minor parent (Suppl. Table S2). The
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recombined section spans the whole polymorphic Mph region.
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The same recombination event is also detected in B. cereus BDRD ST26 by RDP and
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GENECONV methods, but with a corrected p-value significant only for GENECONV. This event
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thus does not fulfill our prerequisites and is therefore considered as an artefactual result.
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7- B. cereus BGSC 6E1 isolate have acquired the Mph allele from a strain related to B.
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thuringiensis BGSC 4AW1.
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Phylogenetic information
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In all phylogenies except the polymorphic Mph gene phylogeny, B. cereus BGSC 6E1 branches as
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the root of the Ba++ group, while in the polymorphic Mph gene phylogeny, B. cereus BGSC 6E1
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branches as the sister group of B. thuringiensis BGSC 4AW1 (Suppl. Fig. S3D).
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Hypothesis
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B. cereus BGSC 6E1 is the recombinant and have acquired the Mph allele of B. thuringiensis
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BGSC 4AW1 (the minor parent). In this case, the major parent would be a relative of B. cereus
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BGSC 6E1, root of the Ba++ group.
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RDP analysis
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Included isolates: B. cereus BGSC 6E1, B. thuringiensis BGSC 4AW1, all other members of the
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Ba++ group, B. cereus VD156 (outgroup). Analysis performed on the 16 Kbp region with the
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polymorphic Mph region included.
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Five methods provided significant support for a ~1-2 Kbp recombination event that occurred in
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B. cereus BGSC 6E1, with B. thuringiensis BGSC 4AW1 as the minor parent (Suppl. Table S2).
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The recombined section spans over 1 Kbp of the polymorphic Mph region.
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