The termite-fungus symbiosis is an ancient stable mutualism of two partners that reproduce and disperse independently. With the founding of each termite colony the symbiotic association must be ...re-established with a new fungus partner. Complementarity in the ability to break down plant substrate may help to stabilize this symbiosis despite horizontal symbiont transmission. An alternative, non-exclusive, hypothesis is that a reduced rate of evolution may contribute to stabilize the symbiosis, the so-called Red King Effect.
To explore this concept, we produced the first linkage map of a species of Termitomyces, using genotyping by sequencing (GBS) of 88 homokaryotic offspring. We constructed a highly contiguous genome assembly using PacBio data and a de-novo evidence-based annotation. This improved genome assembly and linkage map allowed for examination of the recombination landscape and its potential effect on the mutualistic lifestyle.
Our linkage map resulted in a genome-wide recombination rate of 22 cM/Mb, lower than that of other related fungi. However, the total map length of 1370 cM was similar to that of other related fungi.
The apparently decreased rate of recombination is primarily due to genome expansion of islands of gene-poor repetitive sequences. This study highlights the importance of inclusion of genomic context in cross-species comparisons of recombination rate.
Fungus-growing termites rely on mutualistic fungi of the genus
and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has ...evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced
Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites.
Fungus-growing termites have a substantial ecological footprint in the Old World (sub)tropics due to their ability to decompose dead plant material. Through the establishment of an elaborate plant biomass inoculation strategy and through fungal and bacterial enzyme contributions, this farming symbiosis has become an efficient and versatile aerobic bioreactor for plant substrate conversion. Since little is known about what enzymes are expressed and where they are active at different stages of the decomposition process, we used enzyme assays, transcriptomics, and plant content measurements to shed light on how this decomposition of plant substrate is so effectively accomplished.
Although mutualistic symbioses per definition are beneficial for interacting species, conflict may arise if partners reproduce independently. We address how this reproductive conflict is regulated in ...the obligate mutualistic symbiosis between fungus-growing termites and
Termitomyces
fungi. Even though the termites and their fungal symbiont disperse independently to establish new colonies, dispersal is correlated in time. The fungal symbiont typically forms mushrooms a few weeks after the colony has produced dispersing alates. It is thought that this timing is due to a trade-off between alate and worker production; alate production reduces resources available for worker production. As workers consume the fungus, reduced numbers of workers will allow mushrooms to ‘escape’ from the host colony. Here, we test a specific version of this hypothesis: the typical asexual structures found in all species of
Termitomyces
—nodules—are immature stages of mushrooms that are normally harvested by the termites at a primordial stage. We refute this hypothesis by showing that nodules and mushroom primordia are macro- and microscopically different structures and by showing that in the absence of workers, primordia do, and nodules do not grow out into mushrooms. It remains to be tested whether termite control of primordia formation or of primordia outgrowth mitigates the reproductive conflict.
Fungus-growing termites are associated with genus-specific fungal symbionts, which they acquire via horizontal transmission. Selection of specific symbionts may be explained by the provisioning of ...specific, optimal cultivar growth substrates by termite farmers. We tested whether differences in in vitro performance of Termitomyces cultivars from nests of three termite species on various substrates are correlated with the interaction specificity of their hosts. We performed single-factor growth assays (varying carbon sources), and a two-factor geometric framework experiment (simultaneously varying carbohydrate and protein availability). Although we did not find qualitative differences between Termitomyces strains in carbon-source use, there were quantitative differences, which we analysed using principal component analysis. This showed that growth of Termitomyces on different carbon sources was correlated with termite host genus, rather than host species, while growth on different ratios and concentrations of protein and carbohydrate was correlated with termite host species. Our findings corroborate the interaction specificity between fungus-growing termites and Termitomyces cultivars and indicate that specificity between termite hosts and fungi is reflected both nutritionally and physiologically. However, it remains to be demonstrated whether those differences contribute to selection of specific fungal cultivars by termites at the onset of colony foundation.
Life is organised in a hierarchical fashion; smaller replicating entities cooperate to make more complex organisational forms. For example, DNA is organised in genes, genes are organised on ...chromosomes, chromosomes are organised in nuclei, nuclei and organelles are organised in cells and cells can be organised in multicellular organisms. One form of higher-level organisation is an obligate symbiotic mutualism; two different species that become mutually dependent on each other. From an evolutionary perspective there is a tension between lower-level selection and higher-level organisation; natural selection at the lower level can oppose the higher-level organisation, if reproductive interests between the two levels are not aligned. In this thesis I explored this tension and the stabilising mechanisms that align the interests of different organisational levels at different levels of selection in the termite-fungus symbiosis.The symbiosis between fungus-growing termites (Macrotermitinae) and Termitomyces fungi (Basidiomycota) evolved on time, approximately 30 million years ago, without subsequent reversals to non-symbiotic states. The symbiosis has often been described as a farming system in which the termite farmers cultivate their domesticated fungus. Over time both the termites and their fungi have become mutually and obligately dependent on each other, even though in most cases the termites and fungi have retained independent reproduction and dispersal. Independent reproduction implies that the reproductive interests of the termites and their symbionts are not completely aligned, leaving room for conflict between the partners. Since the symbiosis has remained stable over evolutionary time, it is likely that there are mechanisms that have stabilised this level of organisation.One of the major questions in the termite-fungus symbiosis is how sexual reproduction in the partners is correlated in time. Even though the termites and their fungal symbionts reproduce and disperse independently to establish new colonies, the fungal symbiont typically forms mushrooms a few weeks after the colony has produced reproductive termites. It has been hypothesised that this timing of mushroom formation is due to a trade-off between alate and worker production by the queen of the termite colony. Under the assumption of a maximal rate of termite reproduction, investment in the production of alates leads to a reduction in the production of workers. Because workers consume the fungus, reduced numbers of workers will allow mushrooms to ‘escape’ from the host colony. In chapter 2 we tested a specific version of this hypothesis, viz. that the typical asexual structures found in all species of Termitomyces – nodules – are immature stages of mushrooms that are normally harvested in a primordial stage, except when there are too few workers. We refuted this version of the hypothesis by showing that nodules and mushrooms are completely different structures from the earliest developmental stages that we could sample. While our results indicate that a reduced number of workers is a necessary condition for the production of mushrooms, they also show that it is not a sufficient condition, and that other factors are also necessary to trigger the formation of mushrooms. In chapter 6 I discussed a possible mechanism that may trigger mushroom formation in Termitomyces fungi.Due to the independent reproduction and dispersal of the termites and their fungi, the interaction between host and symbiont needs to be re-established at the start of each termite colony. It is known that there is a certain interaction specificity between termites and Termitomyces fungi, but unknown what factors contribute to the observed combinations of termite and fungus. It has been hypothesised that substrate provisioning by termite farmers could explain the observed interaction specificity. In chapter 3 we explored whether differences in nutrient requirement between fungi from different termite species can be found. We tested if differences in in vitro performance of Termitomyces cultivars from nests of three termite species on various substrates are correlated with the interaction specificity of their hosts. We showed that there were quantitative differences between biomass formation on different carbon sources and in a two-factor geometric framework experiment (simultaneously varying carbohydrate and protein availability), which indicates that substrate provisioning may contribute to selection of an adapted symbiont. However, future research needs to show whether those differences indeed contribute to selection of specific fungal cultivars by termites at the founding of a colony.In the termite-fungus symbiosis, horizontal symbiont transmission is also associated with sexual reproduction of the fungus. The dispersing fungal spores are sexual spores produced in the mushrooms. It has been shown that for inhabitant symbionts, like Termitomyces, those that undergo little genetic change should be selected as they live in a stable biotic environment to which they have become adapted. Following from this observation, there should have been selection for a low recombination rate in Termitomyces fungi. In chapter 4 we constructed a new, more contiguous reference assembly of the Termitomyces symbiont of M. natalensis that allows for the study of recombinational landscapes. Also, we isolated a full-sibling mapping population of this Termitomyces species and used these to create the first linkage map of a Termitomyces fungus using a Genotyping-by-Sequencing approach. Finally, we performed an initial study into the recombination landscape of this Termitomyces species and showed that its recombination rate varies substantially across the genome. To be able to answer whether Termitomyces fungi indeed have evolved a low recombination rate, the recombination landscapes of more Termitomyces species as well as those of its close, free-living relatives should be studied.In chapter 5 we zoomed in on the basidiomycete life cycle and explored how the peculiarities of basidiomycete life cycle open possibilities for lower-level selection that conflicts with the higher-level organisation (the fungal mycelium). The first difference between basidiomycetes and the vast majority of sexual life cycles is that after gamete fusion, the nuclei remain separate for almost the whole life cycle. The second difference is that the nuclei of two fusing gametes can move through the whole body – the whole mycelium - of their mating partner. We show that by remaining separate, the fates of these two separate nuclei are not fully aligned, which means that selection can act on the individual nuclei at the cost of the dikaryon. Also, we show that these life cycle peculiarities could enhance the conflict of interest between nuclei and mitochondria, possibly leading to reduced fitness of the dikaryon. In chapter 6 I elaborated on this chapter by discussing how the Termitomyces life cycle deviates from the ‘standard’ basidiomycete life cycle. Most Termitomyces species that were studied did not show nuclear migration, and do not have the typical clamp connections to ensure that only two nuclei (one of each mate) are present in each mycelial cell. I argue that absence of nuclear migration may reduce nuclear competition at the cost of the heterokaryon, but may enhance the conflict between nuclei and mitochondria through competition between the different parental mitochondria.Although the stability of the termite-fungus symbiosis has attracted the interest of many evolutionary biologists, the interest in the termite-fungus symbiosis is not for fundamental questions only. All mushrooms of Termitomyces fungi are edible and considered delicacies in the areas where they are found. The work described in this thesis concerning Termitomyces will also aid the search for Termitomyces mushroom cultivation methods. The work in chapter 2 brings us closer to find the factors that promote mushroom formation. The work in chapter 3 will aid the optimisation of Termitomyces growth substrate. Finally, the work in chapter 4 could in the future help for breeding and analysing desirable traits for the cultivation of Termitomyces mushrooms, so that in future we will be able to re-domesticate the fungus that was domesticated by termites 30 million years ago.
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