Pooled samples bias fungal community descriptions

We tested the accuracy of molecular analyses for recovering the species richness and structure of pooled fungal communities of known composition. We constructed replicate pools of 2-20 species and analysed these pools by two separate pooling-DNA extraction procedures and three different molecular analyses (Automated Ribosomal Intergenic Spacer Analysis (ARISA), terminal restriction fragment length polymorphism (T-RFLP) and clone library-sequencing). None of the methods correctly described the known communities. Only clone library-sequencing with high sequencing per pool (similar to 100 clones) recovered reasonable estimates of richness. Frequency data were skewed with all procedures and analyses. These results indicate that the error introduced by pooling samples is significant and problematic for ecological studies of fungal communities.