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Aquatic hyphomycete identification by ITS2 sequencing

folhas_em_decomposio.jpgAquatic hyphomycetes play a pivotal role in organic matter turnover in headwater streams. These fungi produce extracellular enzymes that break down complex plant polymers and transform plant material into a more suitable and nutritious food source for invertebrate shredders.

The group of Fernandes and collaborators compared the efficiency of molecular methods namely DGGE (Denaturing Gradient Gel Electrophoresis) and 454 pyrosequencing of the transcribed spacer 2 region (ITS2) of the ribosomal RNA gene to the classical spore identification through microscopy to assess aquatic hyphomycete diversity on Quercus robur leaves decomposing in five Portuguese streams.

The researchers found that pyrosequencing was more efficient than DGGE to identify OTUs (Operational Taxonomic Units). However, only 53% of the hyphomycetes species were recovered from both spore morphology and pyrosequencing, while 26% and 21% were recovered only by spore morphology or only pyrosequencing, respectively.

Two problems were identified for hyphomycetes identification. One of the problems of pyrosequencing failure was the absence of ITS barcodes in the database for some of the species identified by spore morphology. Another problem was the 3% phylogenetic distance commonly used to generate OTUs.  Some aquatic hyphomycete species that are easily distinguishable by spore morphology have ITS sequences differing by less than 3%. For instance, Lemonniera alabamensis and L. aquatica have ITS sequences differing by only 1.3% and even species belonging to different genera, such as Heliscus submersus and Flagellospora penicillioides, differ by only 1.1%.

Overall, 454 pyrosequencing is a powerful tool for revealing aquatic hyphomycete diversity, but there is a need to continually barcode fungal species ITS to cover all the extent of fungal diversity in the databases. 

Fernandes I, Pereira A, Trabulo J, Pascoal C, Cássio F, Duarte S. 2015. Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity? Fungal Ecology, 18:130-134.

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