Can whole genome sequencing resolve taxonomic ambiguities in fungi? The case study of Colletotrichum associated with ferns

Introduction: The genus Colletotrichum comprises numerous fungal species with diverse ecological roles, including plant pathogenic, endophytic, and saprophytic lifestyles. Accurate species identification is crucial for understanding host-pathogen interactions, disease epidemiology, and fungal ecology. However, taxonomic classification within Colletotrichum remains challenging due to genetic complexity and phenotypic plasticity. Conventional approaches such as single-gene analyses and multilocus sequence typing (MLST) frequently fail to resolve closely related taxa, leading to misidentifications that hinder species delimitation and comparative evolutionary studies. Whole-genome sequencing (WGS) offers a promising alternative by providing genome-wide resolution for phylogenetic analysis. This study investigates Colletotrichum isolates associated with the fern Rumohra adiantiformis and evaluates the effectiveness of WGS in addressing taxonomic uncertainties. Methods: A total of 18 Colletotrichum isolates, including fern-associated strains, were analyzed. Genomic DNA was extracted and sequenced using the Illumina NovaSeq platform. High-quality genome assemblies were generated, and gene prediction was conducted using AUGUSTUS. Orthogroup assignment and phylogenomic analysis were performed based on single-copy orthologs, and phylogenetic trees were constructed using MLST and WGS-based approaches. Comparative analyses were carried out to assess the taxonomic resolution provided by WGS in relation to traditional methods. Results: Genome-wide phylogenomic analysis revealed distinct evolutionary lineages among Colletotrichum isolates that MLST failed to resolve, highlighting host and geographic differentiation. High-quality genome assemblies were obtained, with 98.3% of genes assigned to orthogroups, indicating strong genomic conservation. Phylogenetic analyses confirmed a close relationship between Rumohra adiantiformis-associated isolates and Colletotrichum filicis, reinforcing the effectiveness of WGS in species identification. Discussion: These findings demonstrate the superiority of WGS over MLST in resolving species boundaries and reconstructing evolutionary relationships. The enhanced resolution provided by genome-wide data enables more accurate taxonomic classification, reducing misidentifications and improving our understanding of fungal biodiversity. By refining Colletotrichum taxonomy, WGS facilitates ecological and pathogenic studies, offering a robust framework for future research in fungal systematics and plant pathology. As sequencing technologies continue to advance, WGS is expected to become a standard tool for fungal species delineation and evolutionary studies.