It is our intention to integrate our molecular methods, research and results into Hebeloma.org in the future. At the current stage of development however we present just a overview and invite the interested reader to consult the references for more details.
If permitted, DNA is extracted from each database collection and, if possible, the barcode (ITS, internal transcribed spacer region of the nuclear ribosomal gens) is generated. There are plenty of standard methods for DNA extraction and all of these are likely to work well for Hebeloma, as do the standard ITS primers used for Agaricales. For older material, or if the initial polymerase chain reaction (PCR) fails, the ITS is amplified in two parts. In some species of Hebeloma, the intragenomic variation of the ITS (and other nuclear loci!) is high and often includes indels, while the interspecific variation is often low, so we standardly sequence PCR products from both directions to achieve high quality data.
As in many other groups of fungi, not all species of Hebeloma can be easily identified by their barcode sequence, although we have observed that even in species that are very similar in their barcode sequence to other species, collections with identical ITS tend to be from the same species. However, this cannot be taken for granted. The most important source of molecular identification is comparison with our own set of sequences, either for BLAST searches or in Neighbour Joining trees including all complete (ITS1 and ITS2) sequences. The use of Neighbour Joining is dictated by computational resources and the size of the dataset with well over 10,000 sequences, also including data published by other parties. Any conflicts between molecular and morphological identification are rigorously followed up, if necessary, repeating the molecular analysis, starting from a new DNA extract.
In some taxa, the amplification of additional molecular markers is useful to aid identification. Typically, we use two variable domains of the mitochondrial small subunit of the ribosomal RNA genes, V6 and V9, as well as partial sequences of some nuclear single copy genes, the second largest subunit of RNA polymerase II (RPB2), a partial sequence of the translation elongation factor 1α (Tef1a), and occasionally MCM7 (a partial sequence of minichromosome maintenance complex component 7). Again, for every collection, potential conflicts between the results for different loci are investigated.
To date, our focus was on the limits of species and infrageneric taxa rather than on reconstructing the phylogeny of the genus, realizing that even including the data from all (nuclear) loci does not resolve the infrageneric phylogeny. The most inclusive species tree (Maximum Likelihood) is presented in Beker et al. (2016), based on concatenated data of five loci. There remain conflicts and challenges that need to be resolved. Phylogenetic reconstructions including non-European species can be found in various subsequent papers by our group.