Challenges in ITS-based identification of Cunninghamella bertholletiae and Rhizopus microsporus due to intragenomic ITS sequence heterogeneity

Cunninghamella bertholletiae and Rhizopus microsporus are clinically important Mucorales that frequently exhibit mixed peaks in chromatograms from conventional Sanger sequencing of the internal transcribed spacer (ITS) region. Intragenomic ITS heterogeneity has been reported in R. microsporus but has not yet been characterized in Cunninghamella species. This study investigated ITS patterns in nine C. bertholletiae , three Cunninghamella echinulata , and two R. microsporus isolates collected in Taiwan. The ITS-D1/D2 regions were examined by PCR, cloning, and Sanger sequencing; one C. bertholletiae isolate was further studied using PacBio HiFi sequencing. In C. bertholletiae , cloning and Sanger sequencing revealed length variation at six homopolymeric tracts ≥ 8 bp within the ITS region, consistent with PacBio findings. C. echinulata exhibited none or minimal intragenomic ITS variation. Both R. microsporus isolates contained divergent ITS and D1/D2 sequence patterns; one strain showed substantial indels, resulting in intragenomic divergence of 27.4% in ITS and 3.10% in D1/D2. These hybrid ITS sequence patterns were highly similar to those observed in strains from Hong Kong, suggesting that ancient hybridization has been stably inherited. Overall, intragenomic divergence ranged 0.14%–0.55% in C. bertholletiae , 0%–0.23% in C. echinulata , and 1.19%–27.37% in R. microsporus for ITS, and 0%–0.41%, 0%–0.41 %, and 1.43%–3.10% for D1/D2, respectively. In conclusion, our study revealed the patterns of ITS heterogeneity, including length variation in long homopolymeric tracts in C. bertholletiae and hybrid ITS patterns in R. microsporus , which contribute to Sanger chromatogram ambiguity and pose challenges for ITS-based identification. Although D1/D2 sequencing offers an alternative, its utility may also be affected by intragenomic divergence in R. microsporus , highlighting the need for single-copy genes to improve species identification. IMPORTANCE C unninghamella bertholletiae and Rhizopus microsporus are clinically important Mucorales that often yield ambiguous internal transcribed spacer (ITS) chromatograms in Sanger sequencing. We showed that in C. bertholletiae , chromatogram ambiguity arises from length variation in six long homopolymeric tracts (≥8 bp), while in R. microsporus, it results from hybrid ITS types, as observed in isolates from Hong Kong. Such intragenomic ITS sequence heterogeneity poses challenges to ITS-based identification. Although D1/D2 sequencing offers an alternative, its utility may also be affected by intragenomic divergence in R. microsporus , highlighting the need for single-copy genes to improve species identification.