Qian Zhang
Pestalotiopsis-like taxa encompass a diverse group of fungi that are often associated with plant diseases, unique ecological interactions such as endophytism, and the production of novel chemical metabolites. Traditionally, classifying pestalotiopsis-like taxa has been challenging due to their significant phenotypic plasticity and the overlapping morphological characteristics among species. Identifying certain isolates at the species level remains problematic, or even impossible, with current DNA sequencing methods, which highlights issues with defining species boundaries. To refine species boundaries, we integrated evidence from single-gene phylogenies (ITS, tef1, and tub2), multi-gene phylogenetic analyses combined with species delimitation methods (GCPSR—genealogical concordance phylogenetic species recognition; PTP—Poisson tree processes; mPTP—multi-rate Poisson tree processes; ABGD—automatic barcode gap discovery; and ASAP—assemble species by automatic partitioning), and genomic metrics including average nucleotide identity (ANI) and the proportion of shared core gene clusters within the pangenome. Two novel species, Neopestalotiopsis camelliae and Pseudopestalotiopsis dasymaschalonis, are introduced based on integrative analyses. Evidence of over-splitting was detected across all three genera. Consequently, five species complexes—Pe. adusta species complex, Pe. brassicae species complex, Pe. clavata species complex, Pe. rosea species complex, and Ps. cocos species complex—are established to accommodate lineages that form monophyletic clades in both multi-gene and genome-scale phylogenies but exhibit short internal branches, low statistical support, and indistinct species boundaries. Furthermore, 28 species are synonymized, including seven in Neopestalotiopsis, 18 in Pestalotiopsis, and three in Pseudopestalotiopsis. Overall, our findings highlight the necessity of integrating genomic evidence with traditional phylogenetic approaches to achieve reliable species delimitation and prevent taxonomic inflation in pestalotiopsis-like fungi.