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

Andrea Menicucci ¹ Salvatore Iacono ¹ Madalena Ramos ^1,2 Chiara Fiorenzani ¹ Natalia Peres ³ Lavern Wayne Timmer ³ Antonio Prodi ¹ Riccardo Baroncelli ^1*

• ¹ University of Bologna, Bologna, Italy
• ² Higher Institute of Agronomy, University of Lisbon, Lisbon, Portugal
• ³ Gulf Coast Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Wimauma, Florida, United States

The genus Colletotrichum encompasses numerous fungal species with diverse ecological roles, including significant plant pathogens. These species pose serious taxonomic challenges due to their genetic complexity and phenotypic plasticity. Conventional methods such as single-gene analyses and multilocus sequence typing (MLST) often fail to resolve closely related taxa, leading to misidentifications and hindering efforts to understand hostpathogen interactions.This study focuses on isolates associated with the fern Rumohra adiantiformis and evaluates the potential of whole-genome sequencing (WGS) to address taxonomic uncertainties within the genus Colletotrichum. Through genome-wide phylogenomic analyses of 18 isolates, we identified distinct evolutionary lineages reflecting both host and geographic differentiation that MLST could not resolve. High-quality genome assemblies were generated, with 98.3% of genes assigned to orthogroups, underscoring significant genomic conservation across the isolates. Using single-copy orthologs, robust phylogenies were constructed, confirming the close relationship between fern-associated isolates and Colletotrichum filicis.Our findings demonstrate the advantages of WGS over traditional techniques, offering enhanced resolution for species identification and deeper insights into evolutionary relationships. This comprehensive genomic approach not only refines fungal taxonomy but also provides a robust foundation for ecological and pathogenic studies in Colletotrichum and other complex fungal genera. Colletotrichum tamarilloi CBS 129955 Solanum betaceum Colombia Baroncelli et al., 2024 Colletotrichum sp. H-24*** Rumohra adiantiformis Costa Rica This work Colletotrichum sp. H-19*** Rumohra adiantiformis Costa Rica This work Colletotrichum sp. H-8*** Rumohra adiantiformis Costa Rica This work Colletotrichum sp. 05-161*** Rumohra adiantiformis USA This work Colletotrichum sp. 05-200*** Rumohra adiantiformis USA This work Colletotrichum sp. H-3*** Rumohra adiantiformis Costa Rica This work Colletotrichum sp. 05-155*** Rumohra adiantiformis USA This work *Ex-type or holo-type. **Colletotrichum nymphaeae was selected as outgroup. ***Isolates originally described by MacKenzie et al., 2009 DNA extraction