Phylogenomic and Comparative Genomic Analyses Reveal Deep Evolutionary Structure and Cryptic Diversity in the Genus Trichoderma

The genus Trichoderma comprises ecologically and biotechnologically important fungi that have been widely investigated and used in agriculture, industrial biotechnology, and biological control. However, publicly available genomes reveal substantial taxonomic inconsistencies across the genus, which can complicate strain identification, reproducibility, and the comparison and selection of strains for applied research and biotechnology. Here, we present a comprehensive phylogenomic and comparative genomic analysis integrating one of the largest collections of Trichoderma genomes analyzed to date. Phylogenomic reconstruction based on 920 conserved single-copy orthologs recovered four major evolutionary clades with strong statistical support and revealed widespread taxonomic inconsistencies affecting multiple species complexes, including T. harzianum, T. asperellum, T. viride, and T. longibrachiatum. Comparative analyses demonstrated marked clade-associated differences in genome size, GC content, repetitive DNA content, gene content, and whole-genome conservation patterns. Genome size was positively associated with repetitive-element accumulation and gene number, whereas GC content showed a negative association with genome size. We additionally characterized a novel Colombian isolate that clustered within a highly supported and divergent lineage together with three inconsistently annotated public genomes. This lineage, provisionally designated Trichoderma sp. “CB2”, formed a sister group to the Longibrachiatum complex and exhibited strong internal genomic cohesion and clear divergence from neighboring lineages. Orthology-based analyses identified lineage-associated proteins with predicted functions related to transcriptional regulation, plant biomass degradation, secondary metabolism, and detoxification. Overall, this study provides a genome-scale framework for resolving Trichoderma diversity and highlights the extent of taxonomic inconsistencies in public genomic resources. Improved phylogenomic characterization of strains can facilitate more reliable strain identification, reproducibility, comparative genomic studies, and the selection and evaluation of Trichoderma strains for agricultural and biotechnological applications.