An assessment of the fungal conservation value of Hardcastle Crags (Hebden Bridge, West Yorkshire) using NextGen DNA sequencing of soil samples.
Soil samples were collected from 14 grassland areas (each ca. 900 m2) in the Hardcastle Crags area, near Hebden Bridge, West Yorkshire (Hollin Hall, Crimsworth Dene and along the Widdop road sub-areas). After freeze-drying, grinding, DNA extraction and PCR (polymerase chain reaction) amplification of the ribosomal RNA fungal DNA barcode region, high throughput DNA sequencing was conducted using an Ion Torrent PGM sequencer. This yielded approximately 331,000 fungal DNA sequences (mean 23,686 sequences per sample [range 10,887-- 40,487]), allowing the fungi present in these soils to be both identified and quantified. Basidiomycete fungi dominated all samples (63-92% of all sequences) with the Hygrophoraceae (waxcap family) being most abundant (mean 32% of all sequences), followed by the coral fungi (Clavariaceae; mean 23%). More detailed analysis of the Hygrophoraceae revealed the presence of 25 species, of which six species had not been observed to fruit at the site, during recent autumn surveys. However, three Hygrophoraceae species which had been previously recorded fruiting at the site were not detected in soil DNA. Several species present at the site were found in different areas from those where they had been detected in fruitbody surveys. The fluid taxonomic status of many members of the other CHEGD taxa (e.g. Clavariaceae, Entolomataceae and to some extent Geoglossaceae and Dermoloma spp.) make it difficult to provide exact species counts for these. Based on comparison of the eDNA and data from past fruitbody surveys, these areas have high conservation value for grassland fungi and merit consideration for designation as a Site of Special Scientific Interest (SSSI).