INTERPRETING ECOLOGICAL DIVERSITY INDICES APPLIED TO T-RFLP DATA: INSIGHTS FROM SIMULATED MICROBIAL COMMUNITIES

Authors: BLACKWOOD, CHRISTOPHER - KENT STATE UNIVERSITY; HUDLESTON, DEBORAH - UNIVERSITY OF MICHIGAN; ZAK, DONALD - UNIVERSITY OF MICHIGAN; Buyer, Jeffrey

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2007
Publication Date: 6/29/2007
Citation: Blackwood, C.B., Hudleston, D., Zak, D.R., Buyer, J.S. 2007. Interpreting ecological diversity indices applied to t-rflp data: insights from simulated microbial communities. Applied and Environmental Microbiology. 73:5276-5283.

Interpretive Summary: Ecologists summarize data about groups of organisms (communities) by tabulating the total number of organisms, the number of species, and the number of organisms per species. These numbers are combined into different numerical indices that provide information about the diversity of the community. These indices were originally developed for plants and animals, and are now being applied to microorganisms despite the fact that little work has been done to validate their use with microbial communities. Soil bacteria are exceedingly abundant and diverse, with approximately one billion bacteria per gram of soil divided into approximately one million species. A variety of methods are used to identify the species and the number of organisms per species, but none of these methods are entirely successful. Terminal restriction fragment length polymorphism (T-RFLP) profiling is one method often used, and a number of papers have been published applying diversity indices to T-RFLP data. In this paper we used mathematically simulations to see how successful various diversity indices were, when applied to T-RFLP data, at accurately representing the actual community being studied. We found that most diversity indices did not work well with T-RFLP data, and that conclusions derived from those diversity indices about the underlying community were inaccurate. Previously published work using these methods need to be re-evaluted in light of these findings, and scientists using T-RFLP to characterize soil microbial communities will need to find other ways to determine the diversity of those communities.

Technical Abstract: Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to detect differences in diversity of the microbial communities sampled. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying community even though multiple taxa can generate the same TRF and rare TRFs are excluded by a relative abundance (fluorescence) threshold. True community diversity was defined using the lognormal abundance distribution. Simulated T-RFLP profiles were then generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. Using a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-Evar) and true Shannon diversity (H’), with correlations between 0.71 and 0.81. TRF-H’ did correlate well with the true H’ in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H’ cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were also shown to be sensitive to the relative abundance threshold. In general, the use of diversity indices on T-RFLP data cannot be recommended (with the possible exception of TRF-Evar). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition, rather than a true difference in community diversity.