Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil

Abstract

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.

FIG. 1.

Rarefaction curves for the bacterial, fungal, and archaeal clone libraries constructed from each of the soil samples. Rarefaction curves were generated using EstimateS (version 7.5; R. K. Colwell, http://purl.oclc.org/estimates). In all nine libraries, there is no apparent asymptote in the rarefaction curves, suggesting that the libraries do not encompass the full extent of OTU richness in each of the communities with an OTU defined at the ≥97% sequence similarity level.

FIG. 2.

Estimation of OTU richness (M in equation 1) (panel a) and the abundance of the most common OTU (a in equation 1) (panel b) in each of the three soils. Symbols correspond to soil type (▴, prairie; ▪, rainforest; •, desert). Parameters were estimated by fitting a power law function to OTU abundance distributions. Maximum-likelihood values are denoted with symbols, and bars indicate 68% confidence regions for the parameter estimates of the actual community (see Materials and Methods). Due to the high range of isolikelihood estimates for OTU richness in the desert archaeal, prairie fungal, and rainforest viral communities, we can conclude only that the number of OTUs in each of these communities is likely to exceed 10⁶. The asterisks indicate that the maximum-likelihood estimates of OTU richness for the desert archaeal and rainforest viral communities exceeded 10¹⁰ OTUs.

FIG. 3.

Predicted values of Simpson's diversity index for each of the 12 communities. Since Simpson's index (D) is defined as the probability that two individuals taken at random from the community belong to the same species (or, in this case, OTU) (41), higher values of D⁻¹ indicate higher overall diversity. Symbols correspond to soil type (▴, prairie; ▪, rainforest; •, desert). The mean value for D⁻¹ (with one standard error in parentheses) for each taxonomic group is denoted above each set of symbols.

FIG. 4.

Hierarchical clustering showing the phylogenetic distance between viral communities from soil (this study), marine sediment (8), human fecal samples (9), and seawater environments (11). Distances were estimated with the weighted Unifrac algorithm (38, 39) using only those sequences from the metagenomic libraries with significant hits to the Phage Proteomic Tree (http://phage.sdsu.edu/oceanviruses) to generate the input phylogenetic trees. A sequence jackknifing technique was applied to each cluster to determine the sensitivity of the relationships to sample size. Asterisks indicate that the nodes are well supported, having been observed in >95% of the jackknifing runs. The soil viral communities were significantly different from the viral communities in the other environments (P < 0.02 in all cases with the UniFrac significance test) (39).