The enigmatic fungal genus Ceraceosorus provides a theoretical framework for studying intragenomic variation in ribosomal DNA sequences

Multicopy nuclear ribosomal (rDNA) genes have been used as markers for fungal identification for three decades. The rDNA sequences in a genome are thought to be homogeneous due to concerted evolution. However, intragenomic variation of rDNA sequences has recently been observed in many fungi, which cause problems in fungal identification and species abundance estimation. Various sequence-based methods have been used to demonstrate rDNA sequence heterogeneity, but there is no technical assessment of the comparability of results from these methods. In this article, we sampled smut fungi representing all major lineages of subphylum Ustilaginomycotina as a system to examine sequence heterogeneity in the rDNA repeats. Three methods were used: PCR-cloning-Sanger sequencing, targeted amplicon high-throughput sequencing, and WGS high-throughput sequencing. Based on our analyses, Ceraceosorus is the only sampled fungal genus in Ustilaginomycotina showing intragenomic variation, with up to 27 nucleotide variant sites in the ITS1–5.8S–ITS2 region and 2.6% divergence among analyzed ITS haplotypes. We found many conflicting patterns across the three detection methods, with up to 28 conflicting variant sites in one sample. Surprisingly, at least 40% of these conflicts are due to PCR-cloning-sequencing errors, as the corresponding variant sites were not observed in the other methods. Based on our data and the literature, we evaluated the characteristics and advantages/disadvantages of each detection method. A model for how intragenomic variation may arise in the rDNA region is presented. Finally, we describe the fourth known species of Ceraceosorus, C. americanus, isolated from an asymptomatic rosemary leaf collected in Louisiana, USA. We anticipate that our study will provide a framework for future research in rDNA regions as well as other similar multicopy genes. ### Competing Interest Statement The authors have declared no competing interest.