Nitrogen (N) fixation is a driving force for the formation of symbiotic associations between N₂-fixing bacteria and eukaryotes.¹ Limited examples of these associations are known in fungi, and none with the sexual structures of species outside of lichens.^2-6  The basidiomycete, Guyanagaster necrorhizus, is a sequestrate fungus endemic to the Guiana Shield.⁷ Much like the root rot-causing species in its sister genera, Armillaria and Desarmillaria, G. necrorhizus sporocarps fruit from the roots of decaying trees (Figures 1A, 1B and 1C),⁸ and genome sequencing is consistent with observations that G. necrorhizus is a white-rotting decomposer. This species also represents the first documented instance of an arthropod-dispersed sequestrate fungus. Numerous species of distantly related wood-feeding termites, which scavenge for N-rich food, feed on mature G. necrorhizus gleba, the interior spore-bearing tissue. During feeding, mature spores adhere to termites for subsequent dispersal.⁹ Using chemical assays, isotope analysis and high-throughput sequencing, we show that the sporocarps harbor actively N₂-fixing Enterobacteriaceae species and that the N content within fungal tissue increases with maturation. Untargeted proteomic profiling suggests that ATP generation in the gleba is accomplished via fermentation. The use of fermentation—an anaerobic process—indicates that the sporocarp environment is anoxic, likely an adaptation to protect the oxygen-sensitive nitrogenase enzyme. Sporocarps also have a thick outer covering, possibly to limit oxygen diffusion. The enriched N₂ content within mature sporocarps may offer a dietary inducement for termites in exchange for spore dispersal to woody substrates. These results show that the flexible metabolic capacity of fungi may facilitate N₂-fixing associations, as well as higher-level organismal associations.

This dataset is composed of multiple different data types, including: 1) vouchered specimens of the fungus, Guyanagaster necrorhizus, 2) nitrogen elemental analysis of G. necrorhizus gleba, 3) ethylene production of G. necrorhizus gleba (for the acetylene reduction assay), 4) bacterial community data of G. necrorhizus sporocarps, 5) plant cell wall degrading gene copy number in the genome of G. necrorhizus and other closely related species, and 6) proteomics data from G. necrorhizus gleba. Data from 1, 2, 3, 5 and 6 are raw data. Data from the microbiome dataset includes the count table and taxonomy table output from DADA2.

Data_S1.csv: master file sheet that includes all of the G. necrorhizus sporocarps that were used in this study, and the year collected.

Data_S2.csv: This file contains the % N and stable N15 values of G. necrorhizus gleba tissue and the maturity group of the corresponding sporocarp.

Data_S3.csv: Contains the ethylene measurements from gleba tissue, and the corresponding maturity group and replicate. The ethylene value was normalized to per hour and per gram weight. The ethylene value is a proxy for nitrogenase activity.

Data_S4.csv: Contains the gene copy number for a group of plant cell wall degrading enzymes. Besides G. necrorhizus, there are five Armillaria species, and five additional closely related species. These data were derived from the CAZyme annotation pipeline in MycoCosm.

Data_S5.csv: Contains the count data and taxonomy output from DADA2 for all samples (negative controls were removed).

Spectral_Counts_Gn.csv: Contains the soluble spectral counts for all of the G. necrorhizus protein hits for three biological replicates at MG I and MG IV. NA indicates values where the peptide and/or spectral count was equal to 0.

Data_S6.csv: Contains the fold change of the proteins hits between MG I and MG IV, and the p-value of the t-tests between the two treatments. While certain values were imputed (in Perseus) when missing in only several of the samples, NA indicates missing values that could not be imputed because the proteins were only present in one or two samples.

ASVs_Gn_20200714.fa: A fasta file containing all of the ASVs after processing with DADA2.

ASV3_Enterobacteriaceae.fa: alignment of the 16S fragment of ASV3 and other Enterobacteriaceae species.