Evaluation of ITS2 molecular morphometrics effectiveness in species delimitation of Ascomycota - A pilot study

Exploring the secondary structure information of nuclear ribosomal internal transcribed spacer 2 (ITS2) has been a promising approach in species delimitation. However, Compensatory base changes (CBC) concept employed in this approach turns futile when CBC is absent. This prompted us to investigate the utility of insertion/deletion (INDELs) and substitutions in fungal delineation at species level. Upon this rationale, 116 strains representing 97 species, belonging to 6 genera (Colletotrichum, Boeremia, Leptosphaeria, Peyronellaea, Plenodomus and Stagonosporopsis) of Ascomycota were retrieved from Q-bank for molecular morphometric analysis. CBC, INDELs and substitutions between the species of their respective genus were recorded. Most species combinations lacked CBC. Among the substitution events, transitions were predominant. INDELs were less frequent than the substitutions. These evolutionary events were mapped upon the helices to discern species specific variation sites. In 68 species unique variation sites were recognised. The remaining 29 species shared absolute similarity with distinctly named species. The variation sites catalogued in them overlapped with other distinct species and resulted in the blurring of species boundaries. Species specific variation sites recognized in this study are the preliminary results and they could be discerned with absolute confidence when larger datasets encompassing all described species of genera were investigated. They could be of potential use in barcoding fungi at species level. This study also concludes that the ITS2 molecular morphometric analysis is an efficient third dimensional study of the fungal species delimitation. This may help to avoid the false positives in species delimitations and to alleviate the challenges in molecular characterization.