Locus Size (bp) Alleles Variable sites π p-distance max. (%) dN/dS ratio         n %       Wolbachia (n=64) wsp 525 13 155 29.52 0.1030 20.08 0.60   ftsZ 507 14 20 3.94 0.0126 2.37 0.07   groEL

491 11 18 3.67 0.0087 1.83 0.29   trmD 453 18 34 7.51 0.0176 4.42 0.23 Cardinium (n=15) CLO 407 6 15 3.69 0.0151 2.22 –   gyrB 631 8 127 20.13 0.0839 14.9 0.06 π = nucleotide diversity Forty-four out of the 64 strains were grouped into five clonal complexes (I-V; Figure 2 and Table 2). All other strains differed at more than one locus from the strains in these complexes. A total of 17 alleles deviated from the alleles from the founding genotypes within the clonal complexes (Table 2). A significant higher number of these variant alleles were found for trmD compared to the other loci (Table 2; Chi-square test; p=0.003), which is consistent TGF-beta/Smad inhibitor with the observation that AZD6738 molecular weight this locus contains the most alleles. Table 2 Clonal complexes found forWolbachia learn more complex I II III IV V STa 4 9 5 2 1 11 6 30 29 28 27 16 15 14 13 36 10 12 24 25 33 34 wsp 1 – - -

– - 5 18 12 – - – 5 3 8 – - – - 4 16 12 – 6 – ftsZ 2 – - – 1 1 – - 10 – - – 3 – - – - – - 10 – 14 – groEl 8 – - 4 4 4 4 – - 8 – - – 8 – 4 4 – - – - 12 11 2 3 – trmD 1 3 1* 10 15 – - 6 7 6 7 1 14 9 6 7 2 1* 1 – - 5 2* 17 9 15 8 15 8 8 – 9 11 1* Speciesb BK-B BK-B BK-B BK-B BK-B BK-D BK-D BK-B BspI BK-D BK-B BK-D BK-D BK-D BR BR BspI BR BK-D BK-D BS BS Freq.c 2 1 1 12 1 1 1 Ixazomib research buy 1 1 1 1 2 1 1 1 1 1 1 3 1 8 1 a Allelic variants within each clonal complex are depicted, listed per sequence type (ST). Likely recombinational changes are depicted in plain text, and putative mutations are shown in bold. Disputable cases are highlighted in italics (possible recombinational changes). For each allelic variant the allele number is given, with in superscript the number of polymorphic sites between the allelic variant and the typical

allele of the clonal group. * indicates non-unique mutations (in cases where one or two mutations were found). b Host species name in which each ST was detected is indicated (for abbreviations see legend Figure 2). c The frequency of each sequence type is listed. Recombination between Wolbachia strains We investigated intergenic recombination by analysis of allelic variation within the clonal complexes. This approach reveals whether variant alleles arose by point mutation or by recombination. Of the 17 variant alleles, four differed from the typical allele in the clonal complex by a single nucleotide change (Table 2). Three of these single nucleotide changes, however, were non-unique. Two other alleles differed by two nucleotide changes, and could be either derived by point mutations or recombination (the chance of two independent mutations both occurring in one out of four genes is 0.25).