Parallel adaptation and cryptic global expansion of Mycobacterium tuberculosis Lineage 3

The "modern" lineages of Mycobacterium tuberculosis (Lineages 2, 3, and 4) drive the global tuberculosis pandemic, yet the evolutionary dynamics of Lineage 3 (L3) remain poorly defined. Although historically stereotyped as geographically restricted and evolutionarily marginal, our analysis of 7,679 genomes reveals that L3 is far more widespread than previously appreciated. It circulates across five continents, diversifying along a trajectory independent of the canonical adaptive steps that facilitated the rise of Lineages 2 and 4. We resolve the dominant L3.1.1 clade—accounting for > 95% of all isolates—into six actively circulating sub-lineages (L3.1.1.i1–i6). These groups exhibit contrasting demographic histories, with three notably undergoing recent exponential expansion. Remarkably, we detect strong parallel positive selection on an identical set of twelve genes across all six sub-lineages, indicating a clear pattern of convergent adaptation intrinsic to the lineage rather than shaped by regional environments. While nine of these genes are associated with drug resistance, three ( Rv2082 , phoR , Rv3401 ) likely reflect adaptation to host or environmental pressures. Despite L3 exhibiting a high overall burden of drug resistance (~ 30%), this resistance rarely enhances transmission in most subpopulations. The L3.1.1.i6, however, represents a striking exception: nearly half of its isolates are resistant, and compensatory evolution has restored high transmissibility even among pre-XDR strains. This mirrors the convergence toward the high-transmission, high-resistance phenotype characteristic of Lineage 2. Collectively, these findings demonstrate that L3 is not a weakened derivative of other modern lineages but a globally dispersed pathogen advancing along a distinct adaptive path. The ongoing expansion of L3, particularly the emergence of the resistance-driven L3.1.1.i6, underscores the urgent need to reassess its epidemiological significance in the contemporary tuberculosis landscape.