Unraveling intragenomic polymorphisms in the high-quality genome of Hypoxylaceae: a comprehensive study of the rDNA cistron

Abstract

Sequencing ribosomal DNA loci, such as ITS and LSU, are among the first steps to characterize and identify newly isolated fungal strains. Substantial doubt has recently been raised concerning the unequivocal suitability of using this sequence information for inferring inter- and intraspecific relationships, as especially the ITS locus was repeatedly shown to harbor high intragenomic plasticity. The evolutionary mechanisms leading to this phenomenon are poorly understood, but this is about to change, owing to the availability of third generation sequencing techniques. The current study attempts a comprehensive analysis of rDNA cistrons in the genomes of over 40 selected species of Hypoxylaceae (Xylariales), using a combination of 2nd generation (Illumina) and 3rd generation sequencing technologies (Oxford Nanopore), along with extensive usage of bioinformatic tools to obtain high-quality genomes. Our findings revealed high intragenomic polymorphisms in distinct Hypoxylaceae species, indicating the presence of deep rDNA paralogs. Phylogenetic inference of the ITS rDNA showed low support for the primary clades within Hypoxylaceae, while inference using the protein encoding gene RPB2 showed better support. We also found that proteinogenic genes are not prone to intraspecific and intragenomic polymorphisms as much as the rDNA loci. Our study highlights the limitations of solely relying on the rDNA cistron as a barcode for fungi. We propose TUB2 as a new primary barcoding marker for Hypoxylaceae and other Xylariales. Moreover, we demonstrate the feasibility to retrieve some DNA loci that had hitherto not been used for classical phylogenetic studies (such as TEF1) in Xylariales directly from genomes without the necessity to work with primers.

Charting Taxa inAmanitaSectionAmidella(Basidiomycota:Amanitaceae)

“…It is worth mentioning that, because of nrDNA cistron heterogeneity, a single sequence might not be representative of the collection itself (as so well exemplified in Figure 3 and Online Resource 4a with the Amanita hiltonii ITS sequences in the outgroup (Davison and Giustiniano 2020)). Genomic analyses have demonstrated that the same can happen in the LSU (Paloi et al 2022;Cedeño-Sanchez et al 2024). Nevertheless, a recent survey has obtained a >99% pairwise identity between ITS cistrons in Amanita (Bradshaw et al 2023), therefore we feel that nrDNA cistron heterogeneity would affect, if anything, the separations at interspecies level rather than higher-level relationships.…”