Genome Comparison Reveals a High-Resolution DNA Marker for Phylogenetic Discrimination Within the <i>Colletotrichum gloeosporioides</i> Species Complex

The <i>Colletotrichum gloeosporioides</i> species complex (CGSC), comprising more than 50 closely related species, constitutes a globally significant phytopathogenic group. Current species delimitation within this complex predominantly relies on multilocus phylogeny, an approach that is time-consuming and technically demanding. To address these limitations, we screened for novel high-resolution markers by performing comparative analyses of public CGSC genomes. Candidate loci being universally present across the CGSC and showing a clock-like evolutionary rate and rapid sequence divergence were further evaluated based on characteristics of the DNA barcoding gap, gene tree-species tree concordance, and phylogenetic topological support. This multi-criteria screening pipeline identified a 3,111-bp locus (M28), which encodes a PMS1 homolog in the DNA mismatch repair pathway with good potential. In phylogenetic reconstruction using 103 representative isolates spanning 41 validated CGSC species, single-locus M28 phylogeny showed high topological congruence with genome-based phylogeny and demonstrated good species-level discrimination capacity, except for very shallow phylogenetic nodes. Consequently, a degenerate primer pair targeting the M28 locus was designed, which demonstrated high PCR amplification efficiency. Together, this study establishes M28 as an efficient species discrimination barcode for cost-effective, high-throughput species diversity assessments in the CGSC.