Abstract
Many bacterial taxa evolved facultative symbiotic associations with insects and spread through host populations by horizontal and maternal transmission. Co-infection at the individual host level may facilitate or constrain the spread of facultative symbionts. Due to insufficiently detailed genotyping, co-infections of maternally transmitted symbionts often remain hidden, limiting our understanding of (co-)infection dynamics. Spiroplasma bacteria exhibit multiple independent origins of symbiosis with insects and have poorly understood patterns of transmission and co-infection. Here, we examined these traits of Spiroplasma symbiosis using Myrmica ants, a system known for high frequencies of single Spiroplasma infections. Through exhaustive genotyping of 75 colonies across seven Myrmica species, we uncovered multiple cryptic co-infections involving two distinct Spiroplasma clades that display significantly different infection frequencies in workers. Within Myrmica ruginodis, infection heterogeneity was contingent on ant caste and was lower in workers. Remarkably, the sMyr Spiroplasma variant infected four Myrmica species and was widespread in queens and workers. We provide phylogenomic and functional genomic support for an exceptionally stable symbiosis with maternally acquired sMyr, with a predicted infection persistence of seven million years in the Myrmica scabrinodis species group. Our findings reveal that Spiroplasma can display complex infection heterogeneity and evolve an evolutionary stable maternally acquired infection within insect hosts.