Abstract
Phylogenetic studies in lichenized fungi need reliable primers, as off-target amplification of environmental fungi or the lichen photobiont presents a challenge. In this study, new mycobiont-specific primers were developed and evaluated for four genetic markers (nrITS, RPB2, MCM7, and mtSSU) within the diverse genus Cladonia. A dataset of 110 specimens was used to assess amplification success, sequence quality, and taxonomic resolution. All markers showed high PCR success rates, with nrITS, MCM7, and mtSSU primer pairs exceeding 90%. nrITS displayed the highest sequence variability and nucleotide diversity, whereas mtSSU was the most conserved. Pairwise genetic distance analyses revealed that nrITS provided the strongest species-level resolution, RPB2 offered intermediate divergence, MCM7 was informative at the clade level but limited for closely related subclades, and mtSSU was best suited for deeper phylogenetic splits. This study shows the value of targeted primer design, and the new primers proved to be robust tools for future molecular identification and evolutionary research in Cladonia.