Background/Question/Methods Understanding nutritional ecology and evolution of fungi gives greater insight into global nutrient cycling and forest ecology, as fungi are critical components of carbon and nutrient cycles.  We used isotopic and molecular methods to examine fungal evolution with respect to carbon acquisition strategies in the Hygrophoraceae clade.  Ectomycorrhizal fungi are typically enriched in ¹⁵N and depleted in ¹³C relative to saprotrophic fungi, allowing these groups to be distinguished by analyzing their sporocarps.  We collected Hygrocybe and Humidicutis spp. at Harvard Forest in central Massachusetts in 2009.  Molecular analysis of the specimens was done by targeting the ITS and 28S sequences.  Results were compared with the existing sequences in GenBank for phylogenetic identification.  Mapping isotopic signatures onto the phylogenetic tree may reveal whether fungi switch carbon acquisition strategies spontaneously or gradually in evolutionary time, through a series of mutations.  Insight into the ancestral history yields better understanding of the phylogenetic traits associated with isotopic signatures. 
Results/Conclusions The results indicate that the typically saprotrophic Hygrocybe spp. are unusually depleted in ¹³C and enriched ¹⁵N.  The trend, which is usually observed in ectomycorrhizal species, suggests unusual nutrient acquisition in the Hygrophoraceae clade.  This may be due to a number of biotic intrigues such as exhibiting both lichenizing and saprotrophic behavior.  Hygrocybe spp., once locally normalized, exhibits isotopic signatures comparable to that of ectomycorrhizal fungi.  These data reveal the complex evolutionary development of carbon and nutrient acquisition that can be discovered by coupling genetic and isotopic techniques.