Abstract
Estimating the species richness of microorganisms is of great importance in predicting, maintaining and managing microbial communities. Although the roles of environmental heterogeneity and geographical distance in structuring soil microbial communities have been studied intensively, the effects of environmental and spatial variation on the species richness estimation have not been examined. To this end, we have explored their effects on estimating the belowground soil bacterial species richness within a 50 ha forest dynamic plot (FDP) using a published massive sequencing dataset with intensive sampling scheme. Our resampling analyses showed that, for a given sequencing depth, increasing the sample size could significantly enhance the detection of rare species by capturing more of the environmental and spatial variation, thus obtaining higher observed and estimated species richness. Additionally, the estimates of bacterial species richness were significantly and positively correlated with environmental variation among samples, indicating that environmental filtering was the main mechanism driving the processes of community assembly for belowground soil bacterial communities in the plot. Moreover, this effect of environmental variation could be markedly alleviated when the sample size was higher than 450, and thus we predicted that there were at least 42,866 soil bacterial species based on 8,296,878 sequences from 550 samples in the whole 50 ha FDP. Furthermore, we built a power law environmental heterogeneity equation (EHE) as a decision-tool to determine an approximate sample size for comprehensively capturing the environmental gradient within a given habitat. Collectively, this work further links the inherent environmental and spatial variation to the estimation of soil bacterial species richness within a given region, and provides a useful tool of sampling design for a better understanding of microbial biogeographic patterns and estimation of microbial biodiversity.