Symbiosis with microorganisms is a globally ubiquitous and taxonomically widespread evolutionaryinnovation. Such symbiosis frequently takes the form of mutualisms where both partners gain a net benefit from the interactions. Nutritional mutualisms are a common form of symbiosis, often allowing host and symbiont to explore new niches by expanding the range of available nutrients the host can rely on. Microbial mutualists can also be major contributors of anti-pathogen, parasite and predator defence. Such functions are often mediated through the production of secondary metabolites, for which the microbe can be coopted for.The fungus-farming termites subfamily (Macrotermitinae, Termitidae: Blattodea) engage in a tripartite obligate mutualism with a single genus of basidiomycete fungus, Termitomyces (Agaricales: Lyophyllaceae) and complex bacterial communities within the guts and externally maintained fungus gardens (combs). The termites provide constant plant biomass for the fungus that reside inside the dense nests with strictly controlled microenvironments. Lignocellulosic rich plant biomass is exceptionally recalcitrant presenting a significant barrier to digestion, yet the fungus-farming termite symbiosis efficiently utilise it. Termitomyces, along with bacterial comb communities, fully degrades the plant biomass and Termitomyces acts as a nutritious food source for the termites. In return, the fungus benefits from a constant supply of growth substrate and optimal growth conditions. Despite the fungal crop being maintained in monoculture and the termites foraging in pathogen rich environments, this symbiosis demonstrates a robustness towards disease and infection. Many factors are thought to contribute to this effective defence of which secondary metabolite production by bacterial mutualists and Termitomyces themselves are thought to be one.

This thesis explores the contributions of microbial symbionts to the fungus farming termite symbiosis through both their contributions to nutrient acquisition and antagonist suppression by exploring the genetic potential of metabolic enzyme and secondary metabolite production. In chapter 1 we detail the current state of the field regarding microbial contributions to both nutrient acquisition and colony defence within fungus farming termite symbiosis, and how these contributions allow the symbiosis to be extremely successful decomposers along with other derived ecosystem services. Chapter 2 explores the specific contributions of the prolific bioactive secondary metabolite producing phylum Actinobacteria which is commonly found in the symbiosis.We characterise the extensive carbohydrate active enzyme (CAZyme) potential and secondary metabolite production potential and how this does not differ from close free-living relatives showing no termite associated genomic adaptations in these two metrics. In chapter 3 we utilised amplicon sequencing to expand the categorisation of secondary metabolite production potential of termite guts and fungus comb. Wecharacterised the taxonomic and non-ribosomal peptide (NRP) compositions of seven species covering the breath of the Macrotermitinae phylogeny. We show both are directed by termite species and sample type with minimal shared features yet structural similarity across species and species specific NRP cores. Next, we shift focus to Termitomyces and characterise the fungal symbionts biosynthetic potential across the phylogeny of the genus. We demonstrate the vast potential for novel compound discovery and termite host species specific biosynthetic potential with patterns of positive selection. In chapter 5 we show how acquisition of the fungal cultivar partially directs the CAZyme and nitrogen cycling potential of the termite gut microbiomes but that the majority of that potential is seeming present from colony inception, likely due to vertical transmission.Chapter 6 looks at the extended phenotype of the termite nest as a layer of defence by showing that elevated levels of CO 2 protect against generalist but not specialist antagonists. Chapter 7 details a bioinformatics tool that examines the ability of an amplicon primer set to differentiate between species of any domain of life.

Collectively, this thesis demonstrates the vast potential of microbial symbionts to contribute to both nutrient acquisition and colony defence of the fungus-farming termite symbiosis and that the harsh internalmicroclimate likely contributes to the latter. Furthermore, we show that both Termitomyces and the bacterial symbionts harbour vast potential for novel compound discovery that should be further explored to understand its ecological relevance.