Sequence heterogeneity of large subunit and internal transcribed spacer domains in the DNA operon encoding for the ribosomal RNA of Ogataea uvarum Sp. Nov.

A yeast strain isolated during a large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy) was subjected to sequence analysis of the large subunit (LSU) and internal transcribed spacer (ITS) domains of its DNA operon encoding for the ribosomal RNA (rDNA). The two molecular marker sequences indicated that this strain could not be attributed to any known species and it was described as the type strain of Ogatea uvarum sp.nov. Moreover, the molecular assays showed several secondary peaks in the ITS2 sequence, but not in the LSU D1/D2. In the aim to test whether these peaks were due to the internal heterogeneity of the DNA operon encoding for the rDNA, the two domains themselves and the clones from them derived after PCR amplification were sequenced. The analyses on the internal variants of ITS and LSU showed a significant variability, although within that predictable among different strains of the same yeast species. In this Ogatea uvarum Sp. Nov., ITS was more variable than LSU especially in the ITS2 region. The heterogeneity revealed by this strain was then judged in the frame of its potential consequence in NGS-based environmental metagenomic studies, in which the variability among operons can lead to biodiversity overestimation and to incorrect identification at the species level. The above findings are discussed in the light of the diverse analytical approaches for fungi identification based on sequence similarity. The results of this study show that the internal variability of the rDNA operon requires careful consideration before being used in future NGS metagenomic investigations and emphasizes the need of specific models to interpret the concept of fungal species, when the reproductive barriers represented by exclusively sexual reproduction are not present.