Periodic Reporting for period 1 - GLiMMer (Genomics of Luminescence in Mycena Mushrooms)
Reporting period: 2022-08-01 to 2024-07-31
Bioluminescence, the emission of light by living organisms, is a fascinating phenomenon that evolved independently multiple times across the tree of life. It has been associated to a variety of ecological functions such as courtships, communication, luring prey and repulsing predators. In Fungi, bioluminescence has a single, de novo origin, and the majority of known bioluminescent fungal species (68/ca. 81) belongs to Mycena, a large and hyperdiverse genus of small mushroom-forming fungi in the Agaricales order (Basidiomycota). The genus counts ca. 1300 described species (names in https://www.indexfungorum.org/ 2023) and the non-bioluminescent members do not seem to form a monophyletic group. This implies a complex history of bioluminescence loss across Mycena species, potentially involving convergent and lineage-specific mechanisms. However, Mycena lacks a solid phylogenetic framework to test this hypothesis and due to its elusive nature, it remains one of the most under sampled fungal genera.
In Fungi genes involved in the bioluminescent form a cluster (Bioluminescence gene cluster – BGC) composed by 4 known core genes plus a variable number of accessory and uncharacterized genes. Extensive rearrangements of the genes within the BGC and relocation of the cluster itself have been observed along the diversification of Mycena and its sister marasmioid clade, that includes Armillaria and Omphalotus species. Knowing the genomic context of the BCG across several species, might reveal the evolutionary circumstances of its diversification and loss and provide new clues on the interplay between lifestyle and genome architecture in fungi.
The absence of a robust and densely sampled phylogenetic framework for the Mycena genus, together with the scarcity of chromosomal-level assemblies, impedes the reconstruction of the evolutionary history that led to the birth and death of the fungal bioluminescence gene cluster.
The goal of this project is to generate the first, genome-based, and species-rich classification of Mycena that integrates present-day and historical (type-)specimens from museum collections.
In Fungi genes involved in the bioluminescent form a cluster (Bioluminescence gene cluster – BGC) composed by 4 known core genes plus a variable number of accessory and uncharacterized genes. Extensive rearrangements of the genes within the BGC and relocation of the cluster itself have been observed along the diversification of Mycena and its sister marasmioid clade, that includes Armillaria and Omphalotus species. Knowing the genomic context of the BCG across several species, might reveal the evolutionary circumstances of its diversification and loss and provide new clues on the interplay between lifestyle and genome architecture in fungi.
The absence of a robust and densely sampled phylogenetic framework for the Mycena genus, together with the scarcity of chromosomal-level assemblies, impedes the reconstruction of the evolutionary history that led to the birth and death of the fungal bioluminescence gene cluster.
The goal of this project is to generate the first, genome-based, and species-rich classification of Mycena that integrates present-day and historical (type-)specimens from museum collections.
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.
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.
When all the results are combined in a publication in 2025, we expect to reveal the first genome-based phylogenetic hypothesis based on more than 200 species for this hypervariable genus. We will also use this new phylogenomic framework to study the diversification of the bioluminescence cluster based on the presence/absence of its key components across a large set of species.
After our publication(s) the methodology to develop and apply target capture in fungi will be available for the scientific community including the possibility to purchase the actual bait.
In general, despite the highly fundamental nature of this project, we provided methods that unlocked a vast natural diversity that can be used in the near future, to decipher evolution, characterize new biosynthetic pathways and redefine species assignments in a genus that needs urgent prioritization in conservation.
After our publication(s) the methodology to develop and apply target capture in fungi will be available for the scientific community including the possibility to purchase the actual bait.
In general, despite the highly fundamental nature of this project, we provided methods that unlocked a vast natural diversity that can be used in the near future, to decipher evolution, characterize new biosynthetic pathways and redefine species assignments in a genus that needs urgent prioritization in conservation.