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. Author SummaryRibosomal DNA (rDNA) genes are one of the most ancient multicopy genes in cellular organisms. They function as a part of the protein synthesis machinery in a cell. The rDNA sequences have also been used in species identification and microbial community profiling. Despite these utilities, little is known how the rDNA genes have evolved. Biologists initially thought the sequences among rDNA copies are homogeneous, but many recent cases illustrated rDNA sequence heterogeneity. In this article, we utilized the fungal genus Ceraceosorus together with allied smut fungi as a system to study sequence heterogeneity in the rDNA genes using various detection methods. Our system found rDNA sequence homogeneity as a common form, while sequence heterogeneity is taxon-specific. Based on our data and literature review, we explained possible sources for sequence heterogeneity in the rDNA genes. Our study also noticed result discrepancies across variant detection methods. These include artefactual variants from the PCR-cloning-sequencing method, inconsistent detected variants from the independent runs of high-throughput sequencing, and technical errors in bioinformatic analyses. We therefore emphasize the importance of methodological choices which have different pros and cons for studying intragenomic variation of rDNA genes, as well as other multicopy gene families.