Characterization of the Biogeography and Temporal Dynamics of the \u3ci\u3eTrachymyrmex septentrionalis\u3c/i\u3e Fungus Garden Microbiome

Like all fungus-growing ants, Trachymyrmex septentrionalis engages in an obligate mutualism with a Basidiomycete fungus that it raises as its primary nutrition source. Therefore, the success of the symbiosis is dependent upon the health of the fungus garden containing this fungus, which may be affected by the composition of its microbiome. Most of what is known about fungus garden microbiomes comes from studies of the Neotropical fungus growing ants, especially the most evolutionarily derived and economically impactful “leaf-cutting” species. Although fungus-farming ants inhabit a vast range, little is known about how dispersal of microbes from their biogeographically and temporally distinct habitats affect the composition and function of fungus garden microbiomes, which ultimately may affect symbiotic fitness. Therefore, this dissertation will infer the effects of dispersal from environmental microbiomes on the composition of the Trachymyrmex septentrionalis fungus garden microbiome across these ants’ geographic range and active seasons, and test the potential consequences of these dispersal events on symbiotic fitness. The extensive fieldwork required to conduct these types of studies inevitably creates delays between sample collection and processing. Therefore, a robust sample preservation strategy is needed to maintain the composition of the microbial community during these delays. This work validates a preservative buffer that is non-hazardous, cost-effective, and preserved microbial composition with high fidelity, making it broadly applicable for microbial community ecology studies. To investigate the effects of environmental dispersal on T. septentrionalis fungus garden microbiomes across their geographic range, we analyzed 16S rRNA gene sequence data from \u3e 100 colonies that we collected from six states, and 90 metagenomes from five of those states. We found that fungus garden microbiome composition and its encoded functional genes both exhibited biogeographic signatures. Microbiome composition was also distinct from the composition of the soil microbiomes that were collected adjacent to the nest chambers from the same ant colonies. Additionally, T. septentrionalis fungus garden microbiomes had a minimally conserved and low-abundance core microbial community. Together, these results suggest that the biogeographic signature that we detected is due to the microbes present on ant forage, which is determined by local ecological conditions, and that these bacteria may not have specific relationships with their ant hosts. Because environmental microbiome composition may also vary seasonally, we analyzed the 16S rRNA sequences of T. septentrionalis fungus gardens from two independent populations during each month of the ants’ active season. We found that fungus garden microbiome composition varied in a site-specific manner, and that alpha-diversity decreased as seasons changed. In a separate experiment, we found that a single microbial dispersal event did not change fungus garden fitness over a short time span. These results demonstrated that T. septentrionalis fungus garden microbiomes change seasonally, these temporal compositional changes are related to geography, and that dispersal of environmental microbiomes may not affect host fitness. Most multicellular organisms host distinct microbial communities, or microbiomes, at sites in and on their bodies. The composition of these host-associated microbiomes has been linked to both beneficial and detrimental consequences for the host, and much research has been dedicated to understanding how host-mediated selection shapes their microbiome. However, the contributions to microbiome composition and potential fitness consequences of dispersal from environmental microbiomes into a host-associated microbiome remain less well understood. Here we show that the dispersal of environmental microbes into the T. septentrionalis fungus garden microbiome dramatically affects its composition both spatially and temporally, although this may not affect symbiotic fitness. Future studies using other fungus-growing ant species may benefit from considering the impact of environmental microbes on fungus garden microbiomes. More broadly, this work demonstrates that dispersal from environmental microbiomes should be considered as part of research into host-associated microbiome composition and function, and its effects on host fitness