New Phylogenetic Markers with Enhanced Phylogenetic Resolution for Cladoniaceae and Parmeliaceae

Taxonomic revisions of fungi have traditionally relied on molecular phylogenies using universal markers such as nuclear and mitochondrial rRNA, cytochrome oxidase, RNA polymerase II, minichromosome maintenance complex component 7, and elongation factor 1. However, these markers often fail to resolve the phylogenetic relationships of closely related fungal species, and further development of fungal universal markers for classification is limited. To address this, the study attempted to develop semi-universal markers targeting specific fungal families rather than the entire kingdom, assessing their potential for taxonomic purposes. Cladoniaceae and Parmeliaceae were selected due to their high species diversity and significant taxonomic challenges. High-resolution phylogenetic markers for single-copy genes with high substitution rates were developed for these families. In this study, primer sequences for nine newly developed phylogenetic markers are presented: cell division control protein 12 (CDC12), histone acetyltransferase esa1 (ESA1), GPI-anchor transamidase (GPITA), the intergenic spacer between hypothetical protein and elongation factor 3 (HYP2-EF3), isocitrate dehydrogenase (ICDH), mitochondrial-processing peptidase-like protein subunit beta (MPP2), SNF7 family protein (SNF7), zf-C2HC5-domain-containing protein (ZFCP), and V-type ATP synthase subunit B (VATPS). These markers exhibited substitution rates at least three times and up to seven times higher than the ITS domain. The phylogeny of Cladonia gracilis (Cladoniaceae), reconstructed using concatenated sequences of ten markers (ITS, CDC12, ESA1, GPITA, HYP2-EF3, ICDH, MPP2, SNF7, VATPS, and ZFCP), displayed well-resolved branches with high bootstrap support. These markers are expected to provide sufficient resolution for the phylogenetic analysis of closely related species in Cladoniaceae and Parmeliaceae. Experimental conditions and bioinformatic tools for multiplex PCR with barcode primers and sequencing on the MiSeq platform were established, ensuring labor- and cost-effective sequencing performance. This approach demonstrates that semi-universal phylogenetic markers targeting the family level can be used to draw taxonomic conclusions for problematic taxa and potentially be applied to other taxonomic groups.