Isolation and characterization of mollicute symbionts from a fungus-growing ant reveals genome reduction and host specialization

Two mollicute species belonging to the Mesoplasma and Spiroplasma genera have been detected in several species of fungus-growing ants using molecular methods. However, their ecological roles remain largely only inferred from metagenomic data. To better understand their diversity and specialization, we cultured both of these Mesoplasma and Spiroplasma symbionts from the fungus-growing ant Trachymyrmex septentrionalis , providing the first isolated mollicutes from any fungus-growing ant species. The genomes of our isolates and related metagenome-assembled genomes (MAGs) from T. septentrionalis fungus gardens comprise two unique phylogenetic lineages compared to previously described Mesoplasma and Spiroplasma species, and from related MAGs previously sequenced from the leaf-cutting ant Acromyrmex echinatior . This suggests that the T. septentrionalis symbionts comprise undescribed species with distinct host specificities. Mesoplasma genomes and MAGs also demonstrate regional specificity with their T. septentrionalis ant hosts. Both Mesoplasma and Spiroplasma strains from T. septentrionalis can catabolize glucose and fructose; both sugars are common in the ant’s diet. Similarly, both these Mesoplasma and Spiroplasma can catabolize arginine but only Mesoplasma can catabolize N -acetylglucosamine, which could both produce ammonia for the ants or fungus garden. Based on our genomic and phenotypic analyses, we describe these T. septentrionalis symbionts as Mesoplasma whartonense sp. nov. and Spiroplasma attinicola sp. nov. , providing insight into their genomic and phenotypic diversity, and cultures to facilitate future studies of these common but poorly understood members of the fungus-growing ant symbiosis.