AbstractLepidoptera (butterflies and moths) are diverse and ecologically important, yet we know little about how they interact with microbes as adults. Due to metamorphosis, the form and function of their adult-stage microbiomes might be very different from microbiomes in the larval stage (caterpillars). We studied adult-stage microbiomes ofHeliconiusand closely related passion-vine butterflies (Heliconiini), which are an important model system in evolutionary biology. To characterize the structure and dynamics of heliconiine microbiomes, we used field collections of wild butterflies, 16S rRNA gene sequencing, quantitative PCR, and shotgun metagenomics. We found thatHeliconiusharbor simple and abundant bacterial communities that are moderately consistent among conspecific individuals and over time. Heliconiine microbiomes also exhibited a strong signal of host phylogeny, with a major distinction betweenHeliconiusand other butterflies. These patterns were largely driven by differing relative abundances of bacterial phylotypes shared among host species and genera, as opposed to the presence or absence of host-specific phylotypes. We suggest that phylogenetic structure in heliconiine microbiomes arises from conserved host traits that differentially filter microbes from the environment. While the relative importance of different traits remains unclear, our data indicate that pollen-feeding (unique toHeliconius) is not a primary driver. Using shotgun metagenomics, we also discovered trypanosomatids and microsporidia to be prevalent in butterfly guts, raising the possibility of antagonistic interactions between eukaryotic parasites and co-localized gut bacteria. Our discovery of characteristic and phylogenetically structured microbiomes provides a foundation for tests of adult-stage microbiome function, a poorly understood aspect of lepidopteran biology.ImportanceMany insects host microbiomes with important ecological functions. However, the prevalence of this phenomenon is unclear, because in many insect taxa microbiomes have only been studied in part of the life cycle, if at all. A prominent example is the butterflies and moths, in which the composition and functional role of adult-stage microbiomes are largely unknown. We comprehensively characterized microbiomes in adult passion-vine butterflies. Butterfly-associated bacterial communities are generally abundant in guts, consistent within populations, and composed of taxa widely shared among hosts. More closely related butterflies harbor more similar microbiomes, with the most dramatic shift in microbiome composition occurring in tandem with a suite of ecological and life history traits unique to the genusHeliconius. Butterflies are also frequently infected with previously undescribed eukaryotic parasites, which may interact with bacteria in important ways. These findings advance our understanding of butterfly biology and of insect-microbe interactions generally.
