Pangenome analysis of <i>Lactobacillus mulieris</i> strains reveals distinct subspecies clusters with defined ecological adaptations

<i>Lactobacillus mulieris</i> is a recently described species, reportedly isolated from human urine, vagina, and gut. Previous genomic studies of <i>L. mulieris</i> highlighted significant genetic diversity among its strains. To gain a deeper understanding of this genomic diversity, we conducted a comprehensive genomic comparison of 70 <i>L</i>. <i>mulieris</i> strains from diverse sources. Phylogenomic and genome relatedness analysis identified three distinct clades, each representing a potential subspecies cluster. Pangenome analysis revealed distinct gene clusters shaping the functional characteristics and unique ecological adaptations of each clade. Clade 1 demonstrated a generalist lifestyle, with strains isolated from diverse sources and enriched in serine/threonine protein kinases, suggesting adaptive versatility. Clade 2, predominantly composed of urinary isolates, displayed enrichment in genes facilitating nutrient acquisition and osmotic regulation, enabling survival in the nutrient-limited and high osmolarity conditions of the urinary tract. Clade 3, exclusively composed of vaginal isolates, exhibited significant enrichment in genes supporting glycogen metabolism, carbohydrate transport, and capsular polysaccharide biosynthesis-features indicative of adaptation to the vaginal environment. Collectively, our findings provide essential genomic insights into the ecological specialization of <i>L. mulieris</i>, shedding light on their genetic variability and adaptive traits within their respective ecological niches.IMPORTANCERecognizing the genomic diversity within <i>Lactobacillus mulieris</i> is essential for understanding its ecological specialization and adaptation strategies across distinct human-associated environments. By identifying three distinct clades with unique functional traits, our study highlights the critical role of niche-specific genetic adaptations in microbial survival. The presence of specialized gene functions within each clade underscores how evolutionary pressures shape bacterial resilience in different environments. Despite their coexistence in overlapping environments, these clades exhibit distinct genomic profiles that may influence their colonization potential and interactions with the host and within the host-associated microbiota. Our findings emphasize the need for a classification framework that accounts for these genetic and functional differences and the necessity for further investigation to understand their distinct roles and impact on human health.