Analyzing salt-marsh fungal diversity: comparing ARISA fingerprinting with clone sequencing and pyrosequencing

In a previous study from our laboratory we used automated ribosomal intergenic spacer analysis (ARISA) to assess salt-marsh fungal diversity (Torzilli et al. 2006). The results demonstrated that different salt-marsh plants harbor distinct fungal communities, thereby supporting the hypothesis that substratum type is an important factor in determining fungal community composition. However, ARISA of several pure cultures of salt-marsh fungi indicated that an operational taxonomic unit (OUT) in an ARISA community profile may represent more than one taxon. To assess the extent to which such ambiguity might have affected the interpretation of our ARISA fingerprinting, we have now fingerprinted and sequenced clones derived from the same fungal DNA used for our ARISA community profiles. Results from this confirmed that an ARISA OTU may represent multiple taxa and that a given taxon may be represented by more than one OTU. Nonetheless, sequencing still confirmed the importance of substratum in determining community composition, and indicated that despite ambiguities associated with OTU's, ARISA may be used to provide a quick snapshot of diversity which can be further refined using sequencing methods. In addition, we compared the fungal diversity from short-form Spartina alterniflora as revealed by clone sequencing with that obtained from pyrosequencing, which avoids the cloning biases of traditional sequencing, and provide greatly expanded depth of coverage. Pyrosequencing significantly enhanced the characterization of fungal diversity compared to traditional clone sequencing.