Abstract
Summary Exploring the variation in microbial community diversity between locations ( β diversity) is a central topic in microbial ecology. Currently, there is no consensus on how to set the significance threshold for β diversity. Here, we describe and quantify the technical components of β diversity, including those associated with the process of subsampling. These components exist for any proposed β diversity measurement procedure. Further, we introduce a strategy to set significance thresholds for β diversity of any group of microbial samples using rarefaction, invoking the notion of a meta‐community. The proposed technique was applied to several in silico generated operational taxonomic unit (OTU) libraries and experimental 16 S r RNA pyrosequencing libraries. The latter represented microbial communities from different biological rapid sand filters at a full‐scale waterworks. We observe that β diversity, after subsampling, is inflated by intra‐sample differences; this inflation is avoided in the proposed method. In addition, microbial community evenness ( G ini > 0.08) strongly affects all β diversity estimations due to bias associated with rarefaction. Where published methods to test β significance often fail, the proposed meta‐community‐based estimator is more successful at rejecting insignificant β diversity values. Applying our approach, we reveal the heterogeneous microbial structure of biological rapid sand filters both within and across filters.