Sequencing DNA extracted from single conidia of aquatic hyphomycetes

Taxonomy and identification of aquatic hyphomycetes have traditionally been based on the morphology and development of conidia. Conidium morphology has been shaped by natural selection to facilitate attachment to substrata in running water, and convergent evolution has favoured two major types, scolecoid and multiradiate spores. As a result, phylogenetically distinct species often produce morphologically very similar spores. This raises the danger of misidentification when relying on spores, and pure cultures are often needed. An alternative is the use of unique DNA sequences (barcodes) that allow identification regardless of the presence of spores or their shape. We successfully extracted DNA from single, stained spores (cotton blue in lactophenol) and amplified a partial ITS region with a sequence similarity of up to 99.6 % to deposited GenBank sequences. With single spores released from pure culture, the success rate (DNA extraction, PCR, sequence similarity ≥99 %) was close to 50 % after optimizing the initial steps of spore recovery. Magnetic beads and Whatman FTA cards were equally successful, but FTA cards potentially allow successive amplifications of different sequences from a single extraction. A simple modification of the foam sampling technique may allow us to take a much deeper and broader look at the taxonomy and biogeography of aquatic hyphomycetes.