Divergence Time Determines the Performance of the Fungal Internal Transcribed Spacer (ITS) DNA Barcode in the Lichen-Forming Ascomycete Genus Parmelina

Molecular studies have repeatedly demonstrated that morphology-based species circumscriptions often underestimate the diversity of lichenized fungi by masking distinct evolutionary lineages under a single species name. In Parmelina, a lichen-forming ascomycete genus whose extant diversity originated during the Miocene and Pliocene, several cryptic species have recently been recognized. Here, we evaluated the performance of the fungal DNA barcode marker, the internal transcribed spacer (ITS) region, in discriminating species that diverged at different evolutionary times. ITS reliably discriminated all the species that originated during the Miocene, for which intra- and inter-lineage distances form a clean, non-overlapping barcode gap (0.0137–0.0162 substitutions/site), but it failed to resolve the two species complexes that radiated during the Pliocene. Barcode-gap analyses based on genetic-distance thresholds previously established for parmelioid lichens produced congruent results, confirming species discrimination among Miocene lineages while highlighting inconsistencies in the current circumscription of two Pliocene species complexes. We also provide a curated ITS DNA barcode reference library comprising 749 accurately identified specimens sampled across the geographic range of Parmelina. Expanded taxon sampling further revealed new country records for P. clandestina in Greece, Hungary, India, Norway, Slovakia, and Switzerland, and for P. mediterranea in France, Greece, Morocco, Turkey, and Switzerland. Our results demonstrate that the discriminatory power of the fungal ITS barcode depends on the evolutionary age of species divergence, successfully identifying older lineages while revealing limitations for more recently diverged taxa. Automated, distance-based delimitation with ABGD and ASAP independently recovered every Miocene lineage, split P. atricha into two entities and assigned the whole P. tiliacea aggregate to a single partition, corroborating this conclusion.