Understanding the genetic characteristics of these species has become a vital necessity for their precise identification and classification. In this comprehensive study, we conducted an in-depth analysis of the genetic composition of various Cytospora species, focusing on four distinct gene regions: ITS, ACT, TUB, and RPB2. Our primary objective was to elucidate the evolutionary relationships among these species and their capability to differentiate from other taxonomic groups. Within the ITS region, our analysis revealed the presence of 68 clades. However, this region was able to effectively separate only 43% of the studied species. Notably, a basal clade featuring C. valsoide was observed, with relatively modest bootstrap values for species containing multiple sequences. Turning our attention to the ACT region, we identified 45 clades, achieving a 53% success rate in species separation. Importantly, this region exhibited higher bootstrap values for certain species, including C. populinopsis, C. japonica, and C. leucostoma sequences. The TUB region emerged as particularly successful, presenting 34 clades and achieving an impressive 76% success rate in species separation. High bootstrap values were observed for species such as C. chrysosperma, C. davidiana, and C. brevispora. In the RPB2 region, we identified 63 clades, effectively separating 80% of the studied species. Species with multiple sequences formed cohesive clusters with high bootstrap values. The combination of gene regions yielded robust results. In the combined phylogenetic analysis of gene regions, ITS+ACT, ITS+RPB2, ITS+TUB, ITS+ACT+TUB and ITS+TUB+RPB2+ACT were able to separate 97, 97, 100, 116 and 125 species among 137, respectively.
