Tissue heterogeneity is associated with phenotypic but not genomic diversity in Wolbachia endosymbionts

The mechanisms underlying within-host diversification in parasite populations are poorly understood. Yet, phenotypic and genotypic variation in parasites can shape their evolutionary trajectories and have important epidemiological consequences. Our aim was to determine whether the constraints associated with infecting different host tissues lead to the emergence and coexistence of multiple parasite sub-populations with distinct phenotypes. We tested this hypothesis using the most widespread bacterial endosymbiont, Wolbachia. We injected Wolbachia bacteria isolated from three tissues of the common pill-bug (Armadillidium vulgare) into uninfected individuals and monitored the growth rate and virulence of each bacterial sub-population in the new hosts. Our results highlight that within-host tissue heterogeneity leads to diverse Wolbachia phenotypes. High-depth genome re-resequencing of Wolbachia sub-populations revealed that this polymorphism was not due to genomic variation but was more likely a result of phenotypic plasticity. Indeed, we found no recurrent tissue-specific genomic variation among infected individuals. Our single nucleotide polymorphism (SNP) filtration pipeline, developed to ensure SNP validity, detected only one substitution. This Wolbachia variant, observed in one female, was present in all three bacterial sub-populations, with frequencies ranging from 24% to 58% depending on the tissue. Overall, our results support the stability of the Wolbachia genome with respect to the rarity of point mutations, in agreement with reports from other symbiotic systems. From a methodological perspective, our study highlights the need for considerable caution when detecting variants in endosymbiont populations, as our conservative approach led us to exclude more than 99.5% of the initially called variants.