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

## TL;DR

This study explores the evolution and distribution of toxins in the Amanita mushroom genus using genetic and biochemical analyses.

## Contribution

The study reveals the independent origins and monophyletic distribution of two neurotoxic gene clusters in Amanita mushrooms.

## Key findings

- IboBGC genes are found in 21 species forming a monophyletic clade within Amanita section Amanita.
- Muscarine is detected in eight species within a nested subclade of the iboBGC clade.
- The iboBGC and muscarine originated independently approximately 28 Mya and 15 Mya, respectively.

## Abstract

Numerous severe cases of neurotoxic mushroom poisoning worldwide are caused by ibotenic acid and muscimol produced by specific species belonging to section Amanita of the genus Amanita. Recent studies have demonstrated that both toxins are produced through the ibotenic acid biosynthetic gene cluster (iboBGC) in these species. In addition to these two toxins, section Amanita is also thought to include several species producing another neurotoxic compound, muscarine. However, the taxonomic distribution and evolutionary history of these toxins within the section remain poorly understood. In this study, phylogenetic analyses based on nucleotide sequences of two loci (ITS and LSU) and five loci (ITS, LSU, RPB2, TEF1, and TUB2), together with a phylogenomic analysis using 467 single-copy genes, were conducted to reconstruct the phylogenetic framework of section Amanita. BEAST analysis was used to estimate divergence times within the section. Gene identification of the iboBGC was conducted using 25 Amanita genomes, followed by phylogenetic analyses of each ibo gene. Biochemical analysis of muscarine was performed on 31 representative species. Based on these analyses, ibo genes were detected in 21 species forming a major monophyletic clade within the section Amanita, whereas muscarine was detected in eight species that constituted a small subclade nested within this clade. Finally, our phylogenetic, phylogenomic, chemotaxonomic, and molecular dating results indicate a monophyletic distribution of the iboBGC and muscarine within the section Amanita, with independent origins approximately 28 million years ago (Mya) and 15 Mya, respectively, and no evidence of subsequent losses.