The rarefaction results (Fig. ) indicate that only a portion of the richness in the bacterial, fungal, and archaeal communities (at the ≥97% sequence similarity level) was surveyed with the clone libraries, as none of the curves reached an asymptote. However, coarse estimates of microbial diversity can be obtained without sampling every individual OTU in a given community (15
), and we can compare relative levels of community richness and evenness in the targeted microbial taxa. Nonparametric estimators (i.e., Chao I and ACE) (41
) are frequently used to estimate the total number of OTUs in a given community (6
). However, in all cases, the nonparametric estimates of total OTU richness failed to stabilize or reach an asymptote (data not shown), so they cannot be used to estimate the total number of OTUs within each community (34
). Instead, we used a parametric technique, based on the observed OTU abundance distribution, to predict the community-level diversity of these three groups, assuming that the form of the OTU abundance distribution is the same for both the libraries and the communities as a whole. For the viral communities, which were surveyed by constructing metagenomic libraries, the OTU abundance distribution was predicted by mathematically modeling the contig spectra.
We tested four different models that are commonly used to describe microbial community structure (23
) and used the most appropriate model (a power law function [Table ]) to estimate the OTU richness and evenness of each community. While more complex parametric models have been used to estimate OTU richness (23
), these models were not tested because there is no a priori reason to choose one type of model over another and because less parsimonious models (those with a larger number of parameters) are likely to underestimate model error. The power law model yielded the lowest model error in 9 of the 12 cases (Table ). Table shows the close correspondence between the observed number of OTUs and the power law model prediction of OTU numbers for each library. The second-best-performing model, the log-normal model, yielded estimates of OTU richness across soils and taxonomic groups that were generally similar to the estimates obtained using the power law model (Table ). Since the levels of diversity are estimated from the OTU abundance curve, the estimates of OTU richness should be relatively robust to changes in library size (Table ). However, for some of the OTU richness estimates, there was a wide range in the 70% confidence regions around the maximum-likelihood values (Fig. ). This high degree of uncertainty in richness estimates reflects the difficulties associated with reliably fitting the tail of a given distribution. This is readily apparent in Table and in the extremely high richness estimates for the desert archaeal and prairie fungal communities (Fig. ). Although our clone libraries are larger than most clone libraries published to date, they are still miniscule considering the overwhelming complexity of the soil microbial communities, making it difficult to estimate the exact number of OTUs in each taxonomic group. Due to this high degree of uncertainty, the richness estimates should be considered carefully, as they are likely to be more useful for comparing richness levels between taxonomic groups than for defining the exact number of OTUs in each of the collected soil samples. However, it is worth noting that there is far less uncertainty associated with the estimates of evenness for the individual communities (Fig. ), as the evenness estimates are less susceptible to errors associated with predicting the specific shape of the tail end of the OTU distribution.
The model results suggest that the total OTU-level richness of bacteria, archaea, fungi, and viruses was extremely high at all sites (Fig. ), with the estimated richness of the last three groups equaling or exceeding the richness of soil bacteria in all habitats. The desert archaeal, prairie fungal, and rainforest viral communities were particularly OTU rich, with a minimum estimate of >106 unique OTUs each (Fig. ), more than an order of magnitude higher than bacterial richness at the same sites. Of course, given the caveats detailed above, it is important to recognize the high degree of uncertainty inherent in these richness estimates.
The estimated differences in evenness between taxa are likely to be more robust than our estimates of total OTU richness (Fig. ). Of the four taxonomic groups, the archaeal communities were the least even, with a single OTU accounting for >8% of the population in a given community (Fig. ). The fungal and archaeal communities had lower evenness levels than bacterial communities, an observation consistent with results reported elsewhere (43
). There was no apparent correlation between the estimated evenness and richness of the communities (r2
= 0.05; P
> 0.5). Interestingly, the estimated probabilities of selecting two individuals of the same OTU from a community (Simpson's diversity index) (41
) were relatively consistent within each taxonomic group regardless of soil type (Fig. ). This consistency suggests that the overall structure of each of these communities is controlled by the type of microbe in question rather than the specific features of the soil environment.
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 (more ...)
Although the slopes of the rarefaction curves were lower for archaea and fungi than for bacteria (Fig. ), the differences in slopes reflect a lower community-level evenness in these groups (Fig. ), not necessarily a lower overall OTU richness. This point is worth reiterating; the slopes of rarefaction curves reflect both the richness and evenness of communities, and therefore, in most cases, rarefaction analyses alone cannot be used to compare richness levels of different microbial communities (30
Not only are soil bacteria, archaea, fungi, and viruses locally diverse, but our results indicate that these groups are also globally diverse, as we observed little phylogenetic overlap between soils. None of the identified archaeal, fungal, or bacterial OTUs was found at more than one site, and we observed only one instance of an overlapping viral sequence (≥98% identity over 20 bp) between sites when all viral sequences (4,577 in total) were assembled together. While we have no way of estimating the global richness of these groups, the lack of overlap in observed OTUs between sites tells us that the global diversity of each of these groups must be very high. The century-old speculation that the global diversity of the smallest organisms should be relatively low (22
) appears to be incorrect.
The estimated number of bacterial OTUs in the three plots (≈104
unique OTUs [Fig. ]) closely matches the estimates obtained in other studies (59
). Our estimates of fungal richness are substantially higher than estimates obtained using classical taxonomic approaches (a maximum of 3,000 fungal species identified from a single 400-ha site) (25
), confirming the results of other studies showing that molecular surveys can uncover a large pool of fungal diversity that has been overlooked (2
). Soil archaea also appear to have an equivalent, if not greater, OTU richness than soil bacterial communities, consistent with the high levels of phylogenetic diversity observed in other studies of soil archaea (46
). To our knowledge, there are no comparable studies of phylogenetic richness in soil viral communities. However, it is important to note that because we examined only viruses with double-stranded DNA, the true richness of viral communities at each site is likely to be even higher than our estimates.
Of the three soils examined, no individual soil harbored the most diverse community of microorganisms. The estimated number of OTUs was highest in the desert soil for archaea, the prairie soil for fungi, and the rainforest soil for viruses, while the richness of bacterial OTUs was very similar across the three soils (Fig. ). Due to a paucity of studies comparing microbial diversity across soils from different ecosystems and the large number of possible mechanisms that may influence levels of taxonomic richness, it is unclear how to interpret these results. Fierer and Jackson (21
) found the lowest levels of bacterial diversity in rainforest soils, but their study (which estimated diversity by terminal restriction fragment length polymorphism fingerprinting) was not necessarily examining diversity at the same level of taxonomic resolution as in this study. The high estimated richness of archaeal OTUs in the desert soil is surprising considering the challenging nature of this environment, but other studies have also observed high levels of archaeal diversity in soils and other environments that are likely to be suboptimal for microbial growth (50
). The fungal results (Fig. ) are consistent with a study by Jumpponen and Johnson (33
) in which high fungal diversity was also observed in soils collected from Konza Prairie, KS.
To our knowledge, this is the first study to use sequencing to characterize soil viral communities. TBLASTX comparison of the soil sequences against the GenBank nonredundant database revealed that the majority of the viral sequences showed no significant similarity to previously described sequences (E value of <0.001). Among the identifiable hits, there were numerous similarities to phages (viruses that infect bacteria) (Table ) and to herpesviruses (data not shown). While there was very little overlap in viral sequences (≥98% identity over 20 bp) between sites (see above), comparison of the sequences against a database containing the genomes of 510 completely sequenced phages demonstrated that similar types of phages were found in all three soil types (Table ; Fig. ). The most abundant phage types observed in the soil samples were similar to phages that infect the soil bacteria Actinoplanes
, and Streptomyces
, as well as the halophilic archaeon Haloarcula
(Table ). The phage types observed in the soil samples were significantly different from the dominant types found in marine or fecal samples (8
) (Table ; Fig. ), suggesting that distinct habitat types harbor distinct viral communities.
Comparison of viral communities from soil and other environments
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 (more ...)
A number of mechanisms may contribute to the surprising local richness of soil microbial communities (Fig. ). Such factors may include a high degree of microscale variability in soil properties, rapid rates of speciation, high immigration rates, and low rates of extinction (14
). In addition, it is important to recognize that small body size alone may partially account for the high diversity of soil microorganisms at individual sites. Since richness is often correlated with the abundance of a taxon in a given area (16
), which is largely a function of body size (42
), surveying microbial diversity in individual soils may be similar in magnitude to surveying the diversity of “macro-organisms” at continental scales. For example, estimating microbial richness in our 100-m2
plots is likely to be analogous in terms of scale to estimating bird species richness (assume a body size of 10−3
) in a 108
area. While body size alone is not likely to account for the high diversity of soil microorganisms, once we reconcile differences in spatial scale, the local richness of soil microorganisms may be more comparable to the observed levels of plant and animal richness.
Together our results confirm that we have only begun to explore the diversity of soil microorganisms. In an individual sample, our data suggest that the actual number of archaeal, fungal, bacterial, and viral “species” (or OTUs) exceeds the total number of microbial species that have been named to date (≈7,500 named archaea and bacteria combined, ≈80,000 fungi, and ≈2,000 viruses) (12
). Clearly, the majority of the microbial diversity on Earth remains undiscovered.