Evolutionary Studies of Guyanagaster Reveal a Novel Tripartite Mutualism in the Neotropics
Guyanagaster necrorhizus gen. et sp. nov., believed to be endemic to tropical rainforests of western Guyana, was described in 2010. Guyanagaster is related to mushroom-forming, root-rotting species of Armillaria, but phylogenetic analyses were inconclusive as to whether it evolved within Armillaria or as sister to it. This species is a gasteroid fungus, lacking the ability to forcibly discharge its basidiospores. Dispersal of gasteroid fungi depends on the evolution of a passive means for basidiospore dispersal that engages exogenous forces, notably mechanical mechanisms and animal dispersal. Sporocarps of Guyanagaster are subhypogeous, entirely enclosed within a thick, durable and persistent peridium; the spores are enmeshed within a tough, decay-resistant gleba that does not become powdery at maturity. The gleba undergoes color changes as the spores mature, from white to pink to dark orange. Guyanagaster has a novel suite of characteristics that do not make it amenable to the dispersal mechanisms of any other known gasteroid fungus. The phylogenetic relationship of Guyanagaster and Armillaria was resolved by a comprehensive phylogenetic reconstruction using sequence data of more than half of the approximately 35 known Armillaria species worldwide, including Guyanagster specimens from two collecting sites. A molecular-clock analysis was used to date the origin of this clade and lineages within it. Guyanagaster represents the earliest diverging lineage from the armillarioid clade. This lineage evolved during the Eocene (56 to 33.9 million years ago), when the global climate was tropical and humid. Annulate, mushroom-forming Armillaria species evolved once the climate transitioned to cool and temperate during the late Eocene. No mushroom-forming taxa were resolved within the Guyanagaster lineage, suggesting that species in this genus are specialized to their tropical rainforest habitat, which may have buffered them from extinction. Additionally, a new species of Guyanagaster, Guyanagster lucianii, was described from a collecting location approximately 125 km east of the holotype site of G. necrorhizus. Repeated field observations of termites consuming the mature G. necrorhizus gleba led to the hypothesis that these insects act as the dispersal agent for this fungus. To test this hypothesis, population genetics analyses were conducted based upon microsatellite genotyping of 198 collections. Conservative estimates from population assignment programs suggest that dispersal does not occur beyond distances greater than five km. Further spatial genetic structure analyses confirm that G. necrorhizus sporocarps within 400 m are more closely related than would be expected by chance, suggesting that 400 m is the likely maximum dispersal distance of G. necrorhizus spores. This distance is consistent with previous studies analyzing the foraging areas of termites. Examination of termites that had fed on G. necrorhizus gleba determined that numerous spores had adhered to their exoskeletons. Further examination of consumed spores dissected from termite guts and spores adhering to their exoskeletons showed that most of the eaten spores were degraded, while spores on the exoskeleton were all nearly intact. This suggests that the termites derive a nutritional benefit from consuming the gleba, but that the spores adhering to the termite exoskeletons are likely the dispersal diaspores of Guyanagaster. Guyanagaster harbors a community of bacteria within its sporocarp. This community was characterized for 52 G. necrorhizus specimens at different maturity stages. Averaged together, 99% of the bacterial community are from the Enterobacteriaceae. Species in this genus are important nitrogen fixers in soil and insect intestines, as well as in leaf-cutter-ant nests. To determine if nitrogen fixation occurs in G. necrorhizus sporocarps, we employed the acetylene reduction assay, which tests for an active nitrogenase enzyme (the enzyme responsible for nitrogen fixation). Significant levels of nitrogen fixation were detected in the mature (i.e., dark orange gleba), intact G. necrorhizus sporocarps. Analysis of the nitrogen content of G. necrorhizus sporocarps at different maturities confirmed that it was significantly greater in the mature specimens. Termite feeding has only been observed on these mature G. necrorhizus sporocarps, suggesting that wood-feeding termites with nitrogen-deficient diets are rewarded for dispersing this fungus by an increase in a necessary dietary nutrient. Toxic superoxide anions are a byproduct of the nitrogen fixation process, which can deactivate the nitrogenase enzyme. Understanding how G. necrorhizus regulates this environment to produce one that promotes nitrogenase activity is crucial. The genome of G. necrorhizus was sequenced, along with global proteome profiles for two dried G. necrorhizus basidiomes—one immature (white gleba) and one mature (dark-orange gleba). Analysis of the genome of G. necrorhizus indicated that the copper-zinc superoxide dismutase gene was duplicated. The protein product of this gene was more abundant in the mature G. necrorhizus sporocarp compared to the immature one. Superoxide dismutases are crucial to the proper functioning of the Rhizobium nodules associated with legumes because of their ability to scavenge superoxide anions with remarkable efficiency. The finding of this protein product in G. necrorhizus gleba at the time point when nitrogen fixation is occurring at its highest rate suggests that it plays a role in protecting the nitrogenase enzyme. Taken together, these results suggest that G. necrorhizus is highly specialized for termite dispersal. When comparing this tripartite mutualism to examples of fungal-farming insects, a remarkable convergence was observed in the roles played by each member. Although this system does not meet all of the requirements, it could be in the early evolutionary stages of a full transition to insect agriculture.
Aime, Purdue University.
Biology|Ecology|Evolution and Development
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