Phylogenetic Analyses of the Litter Decomposing Fungi Clitocybe Found in the Cle Elum Ranger District

Fungi are some of the most diverse and understudied organisms on the planet. In this experiment we studied a genus of litter decomposing fungi, Clitocybe, which are found in the Cle Elum Ranger District of the Wenatchee National Forest of Kittitas County using phylogenetic techniques. Clitocybe are part of a diverse and ecologically important guild of fungi that influence nutrient cycling and soil fertility in the forest. A phylogenetic analysis was done using DNA barcoding, a technique which uses short universal sequences (~640-680 base pairs) to establish species identifications which is possible through polymerase chain reaction (PCR). Due to the biochemical nature of these fungi, PCR is greatly inhibited and, as a result, the PCR solution was adjusted to optimize the effectiveness of the PCR. Each sample was then quantified using a 16 well DNA plate reader, and each sample was adjusted to 10 ng/L; additionally, a copy of each primer for each sample set at 3mM and sent to High Throughput Sequencing at the University of Washington. The sequence data was then screened for quality and, using a blast search, species identification was assigned based upon sequence similarities with known species. A phylogenetic tree was then constructed using PAUP and MrBayes software, to show the evolutionary relationships of the specimens