Cephalotes are herbivorous ants (>115 species) feeding on low-nitrogen food sources, and they rely on gut symbionts to supplement their diet by recycling nitrogen food waste into amino acids. These conserved gut symbionts, which encompass five bacterial orders, have been studied previously for their primary nitrogen metabolism; however, little is known about their ability to biosynthesize specialized metabolites which can play a role in bacterial interactions between communities living in close proximity in the gut. To evaluate the biosynthetic potential of their gut symbionts, we mine 14 cultured isolate genomes and gut metagenomes across 17 Cephalotes species to explore the biodiversity of biosynthetic gene clusters (BGCs) producing specialized metabolites. The diversity of BGCs across Cephalotes phylogeny was analyzed using sequence similarity networking and BGC phylogenetic reconstruction. Our results reveal that the conserved gut symbionts involved in the nutritional symbiosis possess 80% of all the 233 BGCs retrieved in this work. Furthermore, the phylogenetic analysis of BGCs reveals different patterns of distribution, suggesting different mechanisms of conservation. A siderophore BGC shows high similarity in a single symbiont across different ant host species, whereas a BGC encoding the production of non-ribosomal peptides (NRPs) found different symbionts within a single host species. Additionally, BGCs were abundant in four of the five bacterial orders of conserved symbionts co-occurring in the hindgut. However, one major symbiont localized alone in the midgut lack BGCs. Because the spatial isolation prevents direct interaction with other symbionts, this result supports the idea that BGCs are maintained in bacteria living in close proximity but are dispensable for an alone-living symbiont. These findings together pave the way for studying the mechanisms of BGC conservation and evolution in gut bacterial genomes associated with Cephalotes. This work also provides a genetic background for further study, aiming to characterize bacterial specialized metabolites and to understand their functional role in multipartite mutualisms between conserved gut symbionts and Cephalotes turtle ants.

In insect-microbe nutritional symbioses the symbiont supplements the low nutrient diet of the host by producing amino acids and vitamins, and degrading lignin or polysaccharides. In multipartite mutualisms composed of multiple symbionts from different taxonomical orders, it has been suggested that in addition to the genes involved in the nutritional symbiosis the symbionts maintain genes responsible for the production of metabolites putatively playing a role in the maintenance and interaction of the bacterial communities living in close proximity. To test this hypothesis we investigated the diversity of biosynthetic gene clusters (BGCs) in the genomes and metagenomes of obligate gut symbionts associated with the herbivorous turtle ants (genus: Cephalotes). We studied 17 Cephalotes species collected across several geographical areas to reveal that (i) mining bacterial metagenomes and genomes provides complementary results demonstrating the robustness of this approach with metagenomic data, (ii) symbiotic gut bacteria have a high diversity of BGCs which is correlated with host geography but not host phylogeny, (iii) the majority of the BGCs comes from the bacteria involved in the nutritional symbiosis supporting conserved metabolic functions for colonization, communication and competition in the gut environment, (iv) phylogenetic analysis of arylpolyene, polyketide (PK), and siderophore shows high similarity between BGCs of a single symbiont across different ant host species, while non-ribosomal peptide (NRP) shows high similarity between BGCs from different bacterial orders within a single host species suggesting multiple mechanisms for genome evolution of these obligate mutualistic gut bacteria.