The project was based on 3 work packages:
In WP1, I assembled a large collection of Mycena species prioritizing specimens originating from non-temperate regions, to increase the geographical and ecological breadth of current sampling. We sampled mainly Fungarium specimens (207 specimens from the Naturalis Biodiversity Center collection, 167 specimens from the Royal Botanic Gardens, Kew, and 8 from specimens Ghent University). We included 10 recently dried specimens (from 2021) for whole-genome sequencing. Sampling remains until now an ongoing process and our dataset is constantly updated.
Genomic DNA (gDNA) extraction was carried from all samples with an optimized CTAB (Cetyl trimethylammonium bromide) method, to maximise the quality and quantity of the gDNA to be used in different sequencing applications (traditional barcoding, whole-genome sequencing, target enrichment by hybridization capture). All specimens were barcoded prior to genome-scale applications using two sets of primers targeting the large subunit region (LSU) of the ribosomal DNA sequence (rDNA). Considering that Fungarium gDNA from historical specimens can be highly degraded, we targeted a “short region” (~300 base pairs) and a “long region” (~600 base pairs). Draft assemblies and annotations of 10 new Mycena species (including bioluminescent and non-bioluminescent species) were generated and used in the subsequent work packages.
Our main conclusions from this package are:
• The diversity of the Mycena group goes far beyond the current known diversity.
• Fungarium collections and the use of voucher specimens integrated with DNA analysis is the most accurate strategy to refine the genus classification.
• Age of the specimen only partially correlates with DNA extraction, PCR and barcoding success and does not represent a reliable predictor. Despite the highly fragmented nature of genomic DNA of historical specimens, it is always valuable to attempt sequencing.
• Mycena genomes are highly variable in terms of size, repeat content and heterozygosity. The within genus diversity can exceed 20% amino acid identity.
In WP2 I developed one of the first “Museomics” approach to generate genome-scale data from fungal museum specimens. A first phase of this work package involved the design of a new, custom set of probes (biotinylated oligonucleotides) called bait to hybridize target regions of interest from the selected taxa prior to sequencing. The bait was design using whole-genome sequencing data from 40 Mycena species (10 newly generated genomes and 30 genomes available on public databases) and 8 outgroup species including Mycena look-alike species. Both phylogenomic markers (347 genes) and genes from the bioluminescence gene cluster (3 genes) were included in the design.
A second phase involved the actual library preparation, enrichment, target capture and sequencing of the DNA. Analysis of the sequencing data enable the generation the first genome-scale phylogenetic hypothesis of this genus that includes >200 species.
Our main conclusions from this package are:
• Target-capture success is higher than barcoding success and should become a routine method to generate phylogenetic information from historical collection in fungi as in plants.
• Current database of universal, single-copy orthologous phylogenomic markers for fungi are not representative for Mycenas. The bait design is a crucial aspect of the target capture methodology that should deserve more attention.
• Key genes of the fungal bioluminescent cluster can be found is species with unknown bioluminescent status.
In WP3 we attempted to generate the first chromosomal-level assemblies of 4 Mycena species, including one of the largest fungal genomes reported so far, using a combination of two methodologies, PacBio high fidelity (HiFi) and chromatin conformation capture (Hi-C) sequencing. Three of the selected species represent a potential species complex, a group of closely related organisms with unclear species boundaries and high genetic similarities. The 3 species are currently indistinguishable using DNA barcoding but show striking differences in genome structure based on preliminary assemblies. A fourth outgroup species was also included; genetically distant from the ingroup, it shares several morphological and ecological features with it. The generation of Hi-C data was successful. The PacBio sequencing failed, probably to the presence of inhibitors in the DNA extractions. In a follow-up project we are exploring alternative pipelines for the generation of high-quality gDNA compatible with the PacBio workflow.
Our main conclusions from this package are:
• Culturing of non-common Mycena species and genomic DNA extraction represented the bottleneck of generating chromosomal-level assemblies of non-conventional mushroom-forming species.
• Heterozygosity and phasing are crucial aspects in the generation of high-quality assemblies and some to the available assemblies might be inflated by un-collapsed heterozygosity.
• Mycena genus as high potential to study genome architecture evolution in fungi.
