Three fungal genomes decoded in one run reveal secrets of a famous symbiosis

In a breakthrough that could reshape how scientists study fungal partnerships, researchers have sequenced three complete genomes in a single experiment from the naturally interacting community of the snow fungus, Tremella fuciformis, and its long-term companion Annulohypoxylon stygium. The achievement, published in IMA Fungus, goes far beyond the traditional practice of decoding one species at a time and offers the first holistic view of an entire fungal interaction community at the chromosome level.

Tremella fuciformis is no obscure laboratory organism. Known widely as the snow fungus or white jelly mushroom, it produces soft, translucent, chrysanthemum-shaped fruiting bodies prized in Chinese cuisine and traditional medicine for their rich polysaccharides. In the wild, however, this basidiomycete fungus almost never appears alone. Its fruiting bodies are consistently accompanied by Annulohypoxylon stygium, a filamentous ascomycete renowned for its ability to decompose lignin and cellulose in decaying broadleaf wood. Scientists have long suspected that Annulohypoxylon provides nutritional support for Tremella, but the precise nature of their partnership has remained largely cryptic, hidden within the woody substrates where both fungi coexist.

Traditional genome sequencing yields genetic information for only one species per run, and metagenomic approaches designed for complex microbial communities struggle to assemble complete genomes when species composition is unknown or when low-abundance members interfere with assembly. The research team, led by investigators working with wild strains isolated from natural habitats in Yunnan Province, China, took a different route. They treated the mixed mycelia of Tremella fuciformis strain YN01 and Annulohypoxylon stygium strain YNAS01 as an integrated unit and subjected them to PacBio circular consensus sequencing alongside chromosome conformation capture, or Hi-C, sequencing in a single experiment.

The technical pipeline was elegant in its simplicity. A preliminary assembly of all sequencing reads produced mixed contigs, which were then aligned against published reference genomes to classify each contig by species. These reference-guided clusters allowed the team to partition roughly 1.1 million high-fidelity reads into species-specific sets: 38.85 percent belonged to Tremella and 61.15 percent to Annulohypoxylon. Independent assembly of each read set ultimately yielded two complete haploid genomes for the heterokaryotic Tremella, designated hapA and hapB, and one complete genome for Annulohypoxylon, all validated across four dimensions including completeness, base accuracy, chromosome typing, and nuclear phasing.

The resulting assemblies are remarkable for their quality. The hapA genome of Tremella spans 26.75 megabases across 11 telomere-to-telomere chromosomes, while hapB spans 26.30 megabases with similarly symmetric chromosome sets. Annulohypoxylon stygium yielded a 38.83-megabase genome organized into 10 complete chromosomes plus a circular mitochondrial sequence. Base-level accuracy, assessed with the Inspector tool, exceeded 99.9999 percent for all three genomes, with erroneous bases reduced to just 4, 25, and 47 respectively. Hi-C interaction maps confirmed continuous chromosomal architecture without structural breaks, and the NUCLEARPHASER program successfully resolved the 22 Tremella chromosomes into two physically independent haplotypes corresponding precisely to the hapA and hapB karyotypes.

Quantifying the cellular composition of the community revealed a viable cell ratio of Tremella to Annulohypoxylon of approximately 1 to 1.09, estimated from the ratio of sequencing yield to genome size. This near-even balance suggests that both fungi maintain substantial populations within the interaction community, making the system an ideal experimental model for simultaneous genome assembly. The team also identified the mating-type loci in Tremella, locating the homeodomain locus on chromosome 6 and the pheromone-receptor locus on chromosome 10 of both haplotypes, confirming that the species is tetrapolar, a finding consistent with fungal sexual reproduction patterns.

Perhaps the most striking discovery concerns the ribosomal DNA regions within the two Tremella nuclei. The rDNA array of hapA contains 46 tandem repeats of a 7,969-base-pair unit, whereas hapB harbors 34 repeats of a 9,028-base-pair unit, with three insertions accounting for its greater length. When the team amplified the internal transcribed spacer, or ITS, region using universal fungal primers, both haplotypes produced identical 474-base-pair fragments, yet five single-nucleotide differences were detected between them, accounting for 1.1 percent sequence divergence. This finding carries significant implications for fungal identification, suggesting that the widely used ITS barcode may inadequately reflect the genetic complexity of heterokaryotic fungi when applied as a single-locus marker.

Investigating whether long-term symbiosis had enabled genetic exchange between the two species, the team identified segments with sequence identity above 80 percent, but these were confined to universally conserved ribosomal sequences rather than evidence of horizontal transfer. Screening of high-fidelity reads revealed 89 putative interspecies chimeric reads, but polymerase chain reaction validation across predicted breakpoints failed to confirm any of them, indicating they were technical false positives likely arising from sequencing errors or library construction artifacts. Hi-C collinearity heatmaps showed no significant interaction hotspots between the two genomes, providing robust evidence that Tremella and Annulohypoxylon maintain strict genomic isolation despite their ecological partnership.

Within the Tremella heterokaryon itself, the team documented 380,009 single nucleotide polymorphisms and insertions-deletions between the two nuclei, corresponding to an average density of one variant per 190 base pairs, with pronounced variation across chromosomes. Structural variations totaled 1,146 events ranging from 52 to 75,125 base pairs, representing nearly 19 percent of the hapA genome, and over 80 percent of these were associated with transposable elements, predominantly long terminal repeat retrotransposons. Two chromosomes, Chr09A and Chr11A, were classified as accessory chromosomes based on their reduced gene density, elevated repetitive sequence content, and absence of conserved Basidiomycota genes, features consistent with a rapidly evolving accessome involved in adaptive evolution.

Beyond the biological insights, the study establishes a practical framework for future research. The team constructed a comprehensive genome-wide molecular marker set covering over 231,000 effective windows for Tremella and more than 351,000 for Annulohypoxylon, validated with independent resequencing datasets at 99.9999 percent consistency. These markers enable precise strain identification, determination of interaction compatibility between partners, and screening of compatible strain pairs for breeding programs without costly de novo genome assembly. Analysis of 16 Tremella strains revealed three distinct pairing groups based on specificity with Annulohypoxylon, with successful pairing occurring within but not between groups. This molecular toolkit promises to improve cultivation efficiency for a fungus of significant culinary and medicinal value while providing a methodological reference for studying interaction communities in other fungal systems.

Subject of Research: Holistic genome assembly and analysis of the Tremella fuciformis interaction community uncovers intergenomic insights beyond dual genomes

Article Title: Holistic genome assembly and analysis of the Tremella fuciformis interaction community uncovers intergenomic insights beyond dual genomes

Article References: Lin, F., Chen, H., Ye, J., Xu, H., Lin, H., Cen, L., Luo, D., Chen, X., Hu, H., Wang, Z., Deng, Y., & Deng, L. (2026). Holistic genome assembly and analysis of the Tremella fuciformis interaction community uncovers intergenomic insights beyond dual genomes. IMA Fungus, 17, Article e185345. https://doi.org/10.3897/imafungus.17.185345

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.185345

Keywords: genomics, mycology, symbiosis, Tremella fuciformis, genome assembly, fungi, Holistic, genome, assembly, analysis, Tremella, fuciformis