Laboratory evolution drives continued genome degradation in the facultative tsetse symbiont Sodalis glossinidius

Bacterial symbionts of insects undergo dramatic genome reduction during their evolutionary transition from free-living to host-dependent lifestyles, but the dynamics of genome degradation remain poorly understood due to the difficulty of observing these processes in real-time. Sodalis glossinidius , a facultative bacterial endosymbiont of tsetse flies, provides an exceptional opportunity to study this transition experimentally: Unlike highly specialised obligate symbionts, S. glossinidius can be cultured in vitro and retains a large genome (4 Mbp) with extensive pseudogene content (49%, vs. ~ 1% in free-living bacteria), suggesting a recent evolutionary transition. Here, we present a comparative genomic analysis of S. glossinidius strains isolated from laboratory colony-derived Glossina morsitans morsitans , comparing one strain after ten years of serial passaging in laboratory culture (SgGmmC1*) to a counterpart isolated at the same time from the same colony (SgGmmB4). Hybrid genome assembly using Oxford Nanopore and Illumina technologies produced a high-quality 4.29 Mbp genome comprising one circular chromosome and four plasmids. Comparative analysis revealed a significant deletion (16,493 bp) containing 31 genes, including thiM (involved in thiamine biosynthesis) and genes encoding sulfur transporters. Additionally, we identified multiple small-scale chromosomal mutations (8 deletions, 39 insertions, 11 SNPs) resulting in frameshifts in genes including a hemolysin precursor ( shlA ). Our findings demonstrate that, under stable laboratory conditions without the selective pressures of the host environment, S. glossinidius continues to undergo genome degradation. The loss of thiM supports previous hypotheses of complementary metabolic pathways between S. glossinidius and the primary symbiont Wigglesworthia glossinidia for thiamine biosynthesis. This study provides insights into the evolutionary trajectory of facultative symbionts and has implications for studying the patterns of genome evolution in bacterial symbionts adapting to novel ecological niches, as well as paratransgenic approaches using S. glossinidius for trypanosome control.