Pyrosequencing enumerates and contrasts soil microbial diversity

Abstract

Estimates of the number of species of bacteria per gram of soil vary between 2000 and 8.3 million (Gans et al., 2005; Schloss and Handelsman, 2006). The highest estimate suggests that the number may be so large as to be impractical to test by amplification and sequencing of the highly conserved 16S rRNA gene from soil DNA (Gans et al., 2005). Here we present the use of high throughput DNA pyrosequencing and statistical inference to assess bacterial diversity in four soils across a large transect of the western hemisphere. The number of bacterial 16S rRNA sequences obtained from each site varied from 26,140 to 53,533. The most abundant bacterial groups in all four soils were the Bacteroidetes, Betaproteobacteria and Alphaproteobacteria. Using three estimators of diversity, the maximum number of unique sequences (operational taxonomic units roughly corresponding to the species level) never exceeded 52,000 in these soils at the lowest level of dissimilarity. Furthermore, the bacterial diversity of the forest soil was phylum rich compared to the agricultural soils, which are species rich but phylum poor. The forest site also showed far less diversity of the Archaea with only 0.009% of all sequences from that site being from this group as opposed to 4%-12% of the sequences from the three agricultural sites. This work is the most comprehensive examination to date of bacterial diversity in soil and suggests that agricultural management of soil may significantly influence the diversity of bacteria and archaea.

Figure 1

The effect of the sequencing effort on the estimation of the number of OTUs.

Figure 2

Rarefaction curves depicting the effect of % dissimilarity on the number of OTUs identified. Note the comparatively high species richness of the agricultural samples while the Canadian forest soil has very high phylum richness.

Figure 3

Estimated number of OTUs for each sample using parametric (rarefaction) and nonparametric estimators (Chao1 and ACE) compared to the observed OTUs resolved from the sequences.

Figure 4

Relative abundance of phyla and proteobacterial classes for each soil library, in which 16S sequences were classified according to the nearest neighbor in the Greengenes database (http://greengenes.lbl.gov).