On the ecology and evolution of microorganisms associated with fungus-growing termites

Organisms living in symbiosis fascinate us with their adaptations to live in extreme proximity to, or even inside, a partner that may be from a completely different Class, Phylum or Kingdom. Combinations of organisms that live in mutualistic symbiosis seem very exceptional, but when studying any organism more closely one may find involvement in mutualistic symbiosis to be the rule rather than an exception. For example, most of the animals have microorganisms in their guts that help digestion, and many plants have fungi around their roots that aid in uptake of nutrients from the soil. Having complementary traits and reciprocally benefitting each other, cooperating organisms may evolve into extremely successful species. CHAPTER 1 introduces the topic of this thesis: fungus-growing termites. Fungus-growing termites play a dominant role as ecosystem engineers in sub-Saharan Africa and South Asia. They change soil properties by their building and foraging activities, and are major players in decomposition of wood and dead vegetation. Though they are often regarded as a pest, termites can be very useful for people. Besides eating the termites and mushrooms that emerge from the termite mound, people use termite soil-engineering to improve the fertility of agricultural fields. The termite and fungus live in obligate mutualistic symbiosis. Termites (Blattodea: Termitidae, subfamily Macrotermitinae) provide the fungus Termitomyces (Basidiomycota: Agaricales: Lyophyllaceae) with fragmented dead plant material and create a controlled environment perfect for the fungus, whereas Termitomyces decomposes the low-quality matter into a nutritious food source and produces mushroom primordia both of which are eaten by the termites. The symbiosis exists in a world where other organisms are awaiting their chance to exploit the richness of the termite nests. Hence, one could expect to find other organisms in the nest, next to termites and Termitomyces. There is at least one fungus associated with fungus-growing termites that emerges very prominently after termites are no longer active: species of Xylaria (Ascomycota: Xylariales: Xylariaceae, subgenus Pseudoxylaria) are frequently overgrowing the fungus gardens of dead termite nests. What is the status of Pseudoxylaria in the fungus-growing termite symbiosis, does it play a role? How are the fungus-growing termite gardens kept free of weeds, parasites and pathogens? These questions form the foundation of this thesis on the ecology and evolution of microorganisms associated with fungus-growing termites, with particular focus on the role and interactions with associated Pseudoxylaria. CHAPTER 2 investigates the specificity of Pseudoxylaria for fungus-growing termites. I hypothesize that specificity or selectivity for fungus-growing termites would mean that Pseudoxylaria is not present coincidentally as opportunist, but truly associated with fungus-growing termite symbiosis. Hundred and eight South-African fungus-growing termite nests were sampled for Pseudoxylaria, and it was found in most of the nests. Partial rDNA sequences of the obtained isolates were compared with those of Xylaria from the environment and isolates from other parts of the world. I found 16 different molecular types (‘species’) of Pseudoxylaria. They formed a separate group, showing that Pseudoxylaria specifically occurs in fungus-growing termite nests indeed. No specificity for the termite genus or species was found, implying that Pseudoxylaria may have specialised on the fungus garden substrate, rather than on the termite host or the mutualistic fungus Termitomyces. CHAPTER 3 focuses on the role of Pseudoxylaria in the fungus-growing termite nest. Pseudoxylaria is inconspicuous in healthy termite nests and usually only occurs when termites are no longer present in the nest, or when pieces of fungus garden are incubated without termites in the lab. Therefore, it seems to be suppressed and an unwelcome nest inhabitant. I postulate that Pseudoxylaria is a benign stowaway that practices a sit-and-wait strategy to survive in the termite nest. First, Pseudoxylaria and Termitomyces were grown independently on different carbon sources; to test if they have a complementary diet preference, degrading complementary substrate components as had been suggested previously. The carbon source use of both fungi overlapped, implying that Pseudoxylaria is not a beneficial or benign symbiont. Second, the role of Pseudoxylaria in termite nests was inferred from interactions between mycelia of Pseudoxylaria, Termitomyces, and their free-living relatives. Both fungi were grown on the same plate, and also combinations with each other’s free-living relatives were tested. This revealed that Pseudoxylaria is not parasitizing Termitomyces. Furthermore, Pseudoxylaria grew relatively less than its free-living relatives when combined with Termitomyces. This result suggests that the symbiotic lifestyle adopted by Pseudoxylaria went together with adaptations that changed the interaction between both fungi, consistent with Pseudoxylaria being a stowaway. CHAPTER 4 tests the hypothesis that termite workers play a crucial role in maintaining the fungus garden hygiene. The occurrence of microorganisms other than Termitomyces was monitored for pieces of fungus garden that were incubated with, without, or temporarily without termite workers. The effect that workers had on the fungus-comb hygiene, as well as observations on worker cleaning behaviour and their response to mycelium tissue of Pseudoxylaria and Termitomyces, show that termites play an important role in maintaining the fungus-garden hygiene indeed. CHAPTER 5 explores the potential of Actinobacteria for a mutualistic role as defensive symbiont against Pseudoxylaria in the fungus-growing termite nest. Actinobacteria play a mutualistic role as defensive symbionts in many biological systems. It was unclear by which mechanism the termites suppress Pseudoxylaria. Thirty fungus-growing termite colonies from two geographically distant sites were sampled for Actinobacteria. Resulting isolates were characterized based on morphology and 16S rRNA sequences. Next, the obtained Actinobacteria were tested for their antibiotic effect on both Pseudoxylaria and Termitomyces. This chapter describes the first discovery of an assembly of Actinobacteria occurring in fungus-growing termite nests. Actinobacteria were found throughout all sampled nests and materials, and in the phylogenetic tree their 16S rRNA sequences were interspersed with those of Actinobacteria from origins other than fungus-growing termites. The bioassays showed that many Actinobacteria inhibited both the substrate competitor Pseudoxylaria and the termite cultivar Termitomyces. The lack of specificity of the Actinobacteria for fungus-growing termites, and lack of specific defence against Pseudoxylaria, make it unlikely that Actinobacteria play a role as defensive symbionts in fungus-growing termites. Final CHAPTER 6 reflects on the previous chapters, focussing on underlying mechanisms. What caused fungus-growing termites to survive for thirty million years already, and what makes them so successful that they dominate semi-arid ecosystems in sub-Saharan Africa and South Asia? How are conflicts of interest between symbiotic partners resolved? How does cooperation between termites and Termitomyces remain stable over evolutionary time scales? The roles of termites, Termitomyces, Pseudoxylaria, and other organisms in the fungus-growing termite nest are discussed more elaborately. In addition, the question to what extent certain aspects determine whether an organism behaves parasitically or mutualistically, and the question whether symbiont role affects the level of specificity between symbiotic partners, are examined. An analogy is drawn with human agriculture and directions for future research are given. The chapter ends with main conclusions of this thesis. Fungus-growing termites are so successful in maintaining a Termitomyces monoculture that the means by which they accomplish this may be further studied for human agricultural interests. Pseudoxylaria species occur specifically in fungus-growing termite nests, where they are suppressed by termites while awaiting an opportunity to overgrow the fungus garden.