| Summary: | The genus Phyllosticta comprises diverse plant-associated fungi, many of which are significant pathogens or endophytes with complex taxonomic histories. Traditional classification, reliant on morphology and host associations, has long been challenged by overlapping traits, necessitating integrative approaches combining molecular and phenotypic data. In this study, four new Phyllosticta species—P. decaspermi sp. nov. (from Decaspermum montanum and dead leaves), P. morellae sp. nov. (from Morella rubra), P. clematidea sp. nov. (from Clematis vitalba), and P. pittosporicola sp. nov. (from Pittosporum illicioides and dead leaves)—are described based on multi-locus phylogenetic analysis (ITS, LSU, tef1, act, and gpdh) and morphological characterization. These taxa are assigned to the P. capitalensis and P. concentrica species complexes, expanding the known diversity of these clades. Phylogenomic analysis using 5,399 orthologous protein clusters confirmed robust species boundaries within Phyllosticta, mirroring patterns observed in other fungal groups where phylogenomics resolves ambiguous relationships. Gene family analysis revealed a high proportion of conserved single-copy orthologs, indicating stable core functions, alongside unique gene families likely underlying ecological specialization. KEGG metabolic pathway analysis highlighted species-specific adaptations: for example, enhanced “Protein processing in endoplasmic reticulum” in saprophytic species and active “Amino sugar and nucleotide sugar metabolism” in host-associated taxa, reflecting functional divergence linked to lifestyle. These findings align with broader fungal evolutionary trends, where nuclear genomic divergence and potential mitochondrial contributions (e.g., intron dynamics) shape metabolic strategies enabling adaptation to diverse hosts. This study enhances our understanding of Phyllosticta taxonomy and evolution, emphasizing the utility of integrative approaches in resolving fungal diversity.
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