Elucidation of intragenomic variation of ribosomal DNA sequences in the enigmatic fungal genus Ceraceosorus, including a newly described species Ceraceosorus americanus.

Multicopy nuclear ribosomal DNA (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 may make fungal identification and species abundance estimation based on these loci problematic. Ceraceosorus is an enigmatic genus in the smut lineage Ustilaginomycotina for which very limited distribution data exist. Our previous research demonstrated intragenomic variation in the internal transcribed spacer (ITS1-5.8S-ITS2) region of two Ceraceosorus species. In this study, we described the fourth known species of Ceraceosorus, C. americanus, isolated from an asymptomatic rosemary leaf collected in Louisiana, USA. This is the first report of this genus in the Americas. We then selected all four known Ceraceosorus species, plus exemplar smut fungi representing all major lineages of subphylum Ustilaginomycotina, to examine sequence heterogeneity in three regions of the rDNA repeat (partial 18S, ITS, and partial 28S regions). Three methods were used: PCR-cloning-Sanger sequencing, targeted amplicon high-throughput sequencing, and whole-genome shotgun high-throughput sequencing. Our results show that Ceraceosorus is the only sampled fungal genus in Ustilaginomycotina with significant intragenomic variation at the ITS, with up to 25 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 27 conflicting variant sites recorded from a single individual. At least 40% of the conflicting patterns are possibly due to PCR-cloning-sequencing errors, as the corresponding variant sites were not observed in the other detection methods. Based on our data and the literature, we evaluated the characteristics and advantages/disadvantages of each detection method. Finally, a model for how intragenomic variation in the rDNA copies within a genome may arise is presented.