Genomic Insights into the Phylogenetics, Ecology, and Reproductive Biology of Lorchel Mushrooms (Gyromitra spp.) and the Biosynthesis and Evolution of the Gyromitrin Mycotoxin

Abstract

Gyromitrin is a mycotoxin produced by the fungus Gyromitra esculenta and related species, which are collectively called false morels or lorchels. Despite the health risks of gyromitrin exposure, G. esculenta mushrooms are consumed as a delicacy in some parts of the world after detoxification. While the chemical structure of gyromitrin has been known for decades, the distribution, evolutionary history, and genetics of gyromitrin are poorly studied or completely unknown. My aims in this thesis were to identify the gyromitrin biosynthesis genes and elucidate their evolutionary history while augmenting our knowledge of lorchel biology and taxonomy. To achieve these goals, I first developed a new analytical protocol for detecting gyromitrin in lorchel mushrooms. I then amassed a robust sampling of Discinaceae specimens and sequenced rDNA barcodes to build a multi-locus phylogenetic tree for a preliminary investigation of gyromitrin evolution. I discovered that gyromitrin has a more limited distribution in Discinaceae than expected based on anecdotal poisoning data. The position of these taxa in the multi-locus phylogenetic tree suggests gyromitrin has a discontinuous distribution, but low support among basal branches makes this conclusion tentative. Next, I scaled up my investigations and sequenced dozens of whole genomes, including from specimens up to 60 years old. From these I inferred the first-ever phylogenomic tree for the lorchel family, which allowed me to revise genus- and subgenus-level taxonomy in Discinaceae. These genomes provided a unique opportunity to explore not only the evolutionary history of Discinaceae but also fundamental aspects of lorchel biology, such as their reproduction and ecology. Through phylogenomic analyses, I resolved the deep-branching relationships in the family and found that gyromitrin does indeed have a discontinuous distribution. This distribution is suggestive of horizontal gene transfer, although I could not rule out numerous gene loss events or widespread transcriptional silencing of the gyromitrin genes. Taxonomic revisions based on the phylogenomic tree resulted in the recognition of a single genus, Gyromitra, composed of 10 subgenera. I demonstrated that Gyromitra species are predominantly heterothallic (obligately outcrossing) and that all clades possess genomic signatures of saprotrophism except for the truffloid taxa in subg. Hydnotrya, which are ectomycorrhizal. Lastly, I utilized the phenotypic data and genomes from these investigations to search for candidate gyromitrin biosynthesis genes with bioinformatics analyses. I identified three candidate gyromitrin biosynthesis gene clusters. My top candidate gene cluster was one that included four to five genes with predicted functions compatible with gyromitrin production and prominent phylogenetic incongruence. These gene trees showed the gyromitrin-producing species as more closely related than expected, suggesting horizontal gene transfer may have occurred between the ancestors of the gyromitrin-producing clades. The candidate gyromitrin gene clusters identified here serve as exceptional targets for future validation studies using heterologous expression and gene knockouts. This work showcases a comparative genomics approach for the identification of unknown and noncanonical biosynthesis gene clusters that could be applied in the identification of other secondary metabolite gene clusters.