Amanita is the largest genus in the family Amanitaceae, encompassing roughly 650 species and divided taxonomically into three subgenera and eleven sections. Different sections produce strikingly different toxins. The notorious death cap, Amanita phalloides, and its relatives in section Phalloideae cause fatal liver damage through amatoxins such as alpha-amanitin. Species in sections Amidella and Roanokenses, including A. proxima and A. smithiana, inflict kidney injury through aminohexadienoic acid. Section Amanita, by contrast, is the home of the neurotoxic isoxazoles: ibotenic acid, which mimics the neurotransmitter glutamic acid and stimulates NMDA receptors, and muscimol, its decarboxylation product, which mimics gamma-aminobutyric acid and activates GABA receptors. Together these compounds produce the central nervous system excitation, confusion, and hallucinations characteristic of fly agaric poisoning, and they have been implicated in severe intoxications and even fatalities across Asia, Europe, and North America.

Historically, the psychoactive reputation of A. muscaria was attributed to muscarine, the alkaloid first isolated from the species in 1869, which binds acetylcholine receptors and mimics parasympathetic nervous system stimulation. But muscarine is present in fly agaric at only around 0.0003 percent by weight, far too little to cause intoxication, while in some mushrooms of the families Clitocybaceae and Inocybaceae concentrations can reach 1.6 percent. Ibotenic acid and muscimol, isolated in the 1960s, were eventually recognized as the true active agents. Until recently, however, the biosynthetic machinery behind these isoxazoles remained obscure. That changed when Obermaier and Müller elucidated two alternative pathways in A. muscaria that convert glutamine or glutamate into ibotenic acid and muscimol using seven core enzymes, encoded by the genes iboA, iboC, iboD, iboF, iboG1, iboG2, and iboH, arranged contiguously in a biosynthetic gene cluster.

Biosynthetic gene clusters of this kind, which in fungi also underlie the production of compounds as consequential as penicillin, lovastatin, cyclosporin, psilocybin, and the aflatoxins, are ideal evolutionary markers because their presence or absence can be traced directly in genome data. The research team, led by Chinese mycologists working with specimens archived in herbaria at Hunan Normal University and the Kunming Institute of Botany, collected 101 Amanita fruiting bodies between 2006 and 2022 across Central, Eastern, Northeastern, Southern, and Southwestern China. Ninety-nine of these represented 29 species of section Amanita, including 26 described species and three newly encountered unnamed species, providing sampling that spans essentially every major lineage of the section known worldwide.

Reconstructing the evolutionary framework demanded multiple tiers of analysis. The team first built barcode trees from the standard fungal DNA markers ITS and LSU, covering 75 species of section Amanita globally, and then a five-locus tree adding the nuclear genes RPB2, TEF1, and TUB2 for the 29 Chinese species. These analyses resolved three major clades within the section, but several internal relationships remained statistically shaky. To break through this ambiguity, the researchers sequenced 25 whole genomes on an Illumina NovaSeq platform, assembled them de novo, identified 467 shared single-copy genes, and concatenated nearly 293,000 aligned amino acid positions into a phylogenomic matrix. The resulting species tree achieved 100 percent bootstrap support at essentially every node, definitively resolving the previously tangled subclades of the species-rich Clade A and cementing the framework onto which toxin data could be mapped.

With the tree in hand, the team searched the 25 genomes for homologs of the seven ibo genes using the known sequences from A. muscaria as queries, retrieving 134 genes in total. The results were strikingly clean: ibo genes were present in 21 species, all belonging to a single strongly supported monophyletic clade, Clade A, and entirely absent from species in the other two clades of the section. In 17 species all seven genes were recovered, while in four species a subset was missing, most plausibly because DNA degradation in dried museum specimens fragmented the genome assemblies rather than because the genes are truly gone; one such species, A. griseopantherina, is known to produce muscimol, which would be impossible without a complete cluster. Amino acid identities of the ibo proteins ranged from about 70 to 97 percent relative to fly agaric, with most exceeding 80 percent, consistent with a shared ancestral origin.

Muscarine told a complementary story. Using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry, the team screened all 101 dried samples, detecting muscarine in 22 samples representing eight species: A. concentrica, A. flavopantherina, A. ibotengutake, A. muscaria, A. orientigemmata, A. pseudosychnopyramis, A. parvisychnopyramis, and A. subfrostiana. These eight form a small, fully supported monophyletic subclade nested within Clade A, and muscarine was found nowhere else in the section. Beyond the two species previously known to contain the alkaloid, six species are documented here for the first time. The concentrations, expressed on a dry-weight basis, ranged from just over 1 to a remarkable 6,428.57 milligrams per kilogram in A. concentrica, a level comparable to the most muscarine-laden members of the Clitocybaceae and far above what has ever been reported in fly agaric itself.

Molecular dating using a Bayesian relaxed-clock approach added the temporal dimension. Calibrating against the Cretaceous split between Amanitaceae and Pluteaceae, estimated at around 113 million years ago, the analysis placed the origin of the genus Amanita at roughly 96 million years ago and of section Amanita at about 45 million years ago. The ibotenic acid gene cluster was acquired approximately 28 million years ago, with muscarine biosynthesis emerging later, around 15 million years ago, in the ancestor of the small subclade. Crucially, the monophyletic distributions with no detectable losses indicate that each toxin system evolved once and was faithfully retained as its bearer species diversified. Gene-by-gene trees showed partial topological conflicts with the species tree, suggesting recombination, duplication, or loss events within the cluster, a pattern that echoes conflicts reported for the amanitin biosynthetic genes in section Phalloideae and contrasts with the tidy congruence observed for the psilocybin cluster in Psilocybe.

The practical implications are sobering. Because muscarine’s estimated lethal dose for humans falls somewhere between 40 and 495 milligrams depending on the source consulted, a kilogram of fresh A. concentrica could in principle deliver a fatal dose of muscarine alone, without any contribution from ibotenic acid or muscimol. In Chinese fly agaric samples, muscarine reached 84 to 474 milligrams per kilogram, orders of magnitude above earlier assumptions that had led clinicians to dismiss the alkaloid in this species. Patients poisoned by members of the doubly armed subclade may therefore present overlapping central and parasympathetic symptoms, and the study’s integrated genetic and chemical map now gives healthcare providers a rational basis for anticipating which toxin profiles to expect from which mushrooms. It also parallels the pattern seen in Clitocybaceae, where muscarine likewise arose once around 20 million years ago and persisted, while in Inocybaceae the trait has evolved and been lost repeatedly. As genome-scale phylogenetics continues to replace single-locus barcoding, the hidden evolutionary architecture of fungal toxins, once murky, is coming sharply into focus, and with it a far better understanding of one of nature’s most consequential chemical arsenals.

Subject of Research: Evolutionary origins and distribution of neurotoxin biosynthetic gene clusters in Amanita section Amanita

Article Title: The distribution and evolution of muscarine and the ibotenic acid biosynthetic gene cluster within the genus Amanita section Amanita revealed by phylogenomics

Article References: Su, Y.-T., He, Z.-M., Yang, Y.-Z., Xu, F., Lai, M.-M., Yang, Z. L., Zhang, P., & Chen, Z.-H. (2026). The distribution and evolution of muscarine and the ibotenic acid biosynthetic gene cluster within the genus Amanita section Amanita revealed by phylogenomics. IMA Fungus, 17, Article e175874. https://doi.org/10.3897/imafungus.17.175874

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.175874

Keywords: Amanita, mushroom toxins, ibotenic acid, muscimol, muscarine, biosynthetic gene cluster, phylogenomics, mycology, molecular dating, fly agaric, mushroom poisoning, secondary metabolites