Fig 1.
Bb diversity in nymphal ticks is derived from two sources.
(a) Larval ticks may be infected with a single Bb strain that may undergo in situ evolution over the course of the tick’s ten-month infection, a single infection. (b) Alternatively, larval ticks may be infected with a diverse infecting inoculum from the vertebrate host and the mixed-strain Bb population may further undergo in situ evolution over the tick’s 10-month infection. De novo mutations acquired over the ticks’ infection are represented by x’s.
Fig 2.
Within-tick Bb diversity across ticks.
(a) Deep sequencing reveals within-tick Bb variation. We generate a within-tick consensus Bb sequence for each host, map sequence reads to the consensus sequence, and identify iSNVs (indicated in grey). (b) Number of Bb iSNVs across individual ticks. Histogram bars represent the count of individual ticks harboring total Bb diversity falling within the iSNV bin on the x-axis. (c) The normalized number of Bb iSNVs per site with > 40 X coverage (60% iSNV calling power for minor alleles comprising 10% of the population, S1 Text). No significant differences exist in iSNV rates between chromosome and plasmids (Mann-Whitney test, p-value > 0.1). (d) Genetic distance for the Bb population infecting each tick. Genetic distance is calculated as the sum of the minor allele frequencies across the Bb genome. The red dotted line indicates the estimated threshold above which ticks are classified as harboring “mixed” infections.
Fig 3.
Within-tick Bb diversity in single infected ticks.
(a) The minor allele frequency (MAF) spectrum of a representative “singly” infected tick sample. The Bb population infecting Bbcap22 (600 X coverage) harbors few variants with MAF < 5%, likely accrued via in situ evolution. Histogram bars represent the count of genomic sites with a minor allele frequency that falls within the MAF frequency bin on the x-axis. Note the differing y-axes for (a) and (c, d, and e). (b) MAF spectrum of a “multiply” infected tick sample, Bbcap5 (109 X coverage). The Bb population infecting Bbcap5 harbors a high number of intermediate frequency minor alleles, a level of diversity likely preexisting within a diverse inoculum. (c) MAF spectrum of a “multiply” infected tick sample, Bbcap17 (221 X coverage). (d) MAF spectrum of a “multiply” infected tick sample, Bbcap31 (309 X coverage).
Fig 4.
Detecting selection across the Bb genome.
(a) Within a single tick (Bbcap17), dN plotted against dS for each of the 876 genes on the Bb chromosome and plasmids cp26 and lp54. Each point represents a single gene. The line represents the neutral expectation dN = dS. Red points falling in the upper left half of the plot have a signal of positive selection while blue points falling in the lower right have a signal of purifying selection. Grey points represent genes with nonsynonymous variants, but no synonymous variants, so dN/dS could not be determined. (b) Overlap between genes under positive selection (dN/dS >1) in each multiply infected tick (the 10 ticks with greatest within-tick Bb diversity are shown for clarity, resulting in 9 pairwise comparisons for each tick). For each pair of ticks, the odds ratio representing the strength of association between the two gene sets under positive selection is colored according to the color ramp: dark blue indicates that gene sets are strongly associated. P-values from a Fisher’s exact test of association between gene sets are super-imposed on each cell.