DNA barcoding of fungi: a case study using ITS sequences for identifying aquatic hyphomycete species

Abstract

Aquatic hyphomycetes are a polyphyletic group of fungi that play a crucial role in organic matter turnover in streams. They have been traditionally identified based on the morphology of conidia collected in stream water or obtained from leaves colonized in nature upon aeration in the laboratory. Therefore, species identification is limited by our ability to induce conidium production and to establish pure cultures. Conidial shapes are believed to be the result of convergent evolution, so similar conidia may be produced by different conidiogenesis processes, which may prevent unambiguous identification. Currently, a great effort in fungal taxonomy is being made at introducing a set of criteria based on comparisons of selected nucleotide sequences instead of or in addition to phenotypic characters. We examined the suitability of ITS1-5.8S-ITS2 rRNA gene region or its subregions (ITS1 and ITS2) to identify aquatic hyphomycetes, by sequencing and comparing these regions in 94 fungal isolates belonging to 19 species collected in Portuguese streams with different environmental conditions during 8 years. Sequences of ITS1, ITS2 and ITS1-5.8S-ITS2 rRNA genes of the Portuguese isolates of aquatic hyphomycetes and those from the GenBank exhibited taxonomic cohesiveness, the isolates grouped with their respective species but all Tricladium species did not group within the Tricladium genus. Cohesiveness was not observed between isolates with respect to location, condition of stream or date of collection. Evolutionary divergences (ITS1-5.8S-ITS2 sequences; Kimura 2-parameter distance) between con-specific isolates were shallow and a deep divergence between species was generally observed. The NJ trees based on ITS1 or ITS2 rRNA gene sequences had lower statistical support for some internal nodes, we therefore propose ITS1-5.8S-ITS2 rRNA gene as barcode for identifying species of aquatic hyphomycetes.