The diversity and biogeography of soil bacterial communities Noah Fierer*��� and Robert B. Jackson*��� *Department of Biology and ���Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708 Edited by Christopher B. Field, Carnegie Institution of Washington, Stanford, CA, and approved December 5, 2005 (received for review August 29, 2005) For centuries, biologists have studied patterns of plant and animal diversity at continental scales. Until recently, similar studies were impossible for microorganisms, arguably the most diverse and abundant group of organisms on Earth. Here, we present a conti- nental-scale description of soil bacterial communities and the environmental factors influencing their biodiversity. We collected 98 soil samples from across North and South America and used a ribosomal DNA-fingerprinting method to compare bacterial com- munity composition and diversity quantitatively across sites. Bac- terial diversity was unrelated to site temperature, latitude, and other variables that typically predict plant and animal diversity, and community composition was largely independent of geo- graphic distance. The diversity and richness of soil bacterial com- munities differed by ecosystem type, and these differences could largely be explained by soil pH (r2 0.70 and r2 0.58, respectively P 0.0001 in both cases). Bacterial diversity was highest in neutral soils and lower in acidic soils, with soils from the Peruvian Amazon the most acidic and least diverse in our study. Our results suggest that microbial biogeography is controlled primarily by edaphic variables and differs fundamentally from the biogeography of ������macro������ organisms. biodiversity microbial ecology soil bacteria terminal-restriction fragment length polymorphism Aand lthough microorganisms are perhaps the most diverse (1, 2) abundant (3) type of organism on Earth, the distribu- tion of microbial diversity at continental scales is poorly under- stood. Ecologists describing microbial biogeography typically invoke Beijerinck (4) from a century ago: ������Everything is every- where, the environment selects.������ However, few studies have attempted to verify this statement or specify which environmen- tal factors exert the strongest influences on microbial commu- nities in nature (5, 6). With the advent of ribosomal DNA- analysis methods that permit the characterization of bacterial communities without culturing (7, 8), it is now possible to examine the full extent of microbial diversity and describe the biogeographical patterns exhibited by microorganisms at large spatial scales. Scientific understanding of microbial biogeography is partic- ularly weak for soil bacteria, even though the diversity and composition of soil bacterial communities is thought to have a direct influence on a wide range of ecosystem processes (9, 10). Much of the recent work in soil microbial ecology has focused on cataloging the diversity of soil bacteria and documenting how soil bacterial communities are affected by specific environmental changes or disturbances. As a result, we know that soil bacterial diversity is immense (11, 12) and that the composition and diversity of soil bacterial communities can be influenced by a wide range of biotic and abiotic factors (13). However, almost all of this work has been site-specific, limiting our understanding of the factors that structure soil bacterial communities across biomes and regions. We hypothesize that soil bacterial communities do exhibit biogeographical patterns at the continental scale of inquiry and that these patterns are predictable. Whereas previous studies have examined the biogeographical distributions of soil fungal communities (14) and individual strains of soil bacteria (15, 16), to our knowledge, no previous study has examined how entire soil bacterial communities are structured across large spatial scales. We hypothesize that the biogeographical patterns exhib- ited by soil bacteria will be fundamentally similar to the patterns observed with plant and animal taxa and that those variables which are frequently cited as being good predictors of animal and plant diversity, particularly those variables related to energy, water, or the water���energy balance (17���19), will also be good predictors of bacterial diversity. To test these hypotheses, we used a ribosomal DNA-fingerprinting method to compare the composition and diversity of bacterial communities in 98 soils collected from across North and South America. Results and Discussion Soil bacterial diversity, as estimated by phylotype richness and diversity (Shannon index) (20), varied across ecosystem types (Fig. 1). Of all soil and site variables examined, soil pH was, by far, the best predictor of both soil bacterial diversity (r2 0.70, P 0.0001 Table 1 and Fig. 1A) and richness (r2 0.58, P 0.0001 Fig. 1B) with the lowest levels of diversity and richness observed in acid soils (Fig. 1). Because soils with pH levels 8.5 are rare, it is not clear whether the relationship between bacterial diversity is truly unimodal, as indicated in Fig. 1, or whether diversity simply plateaus in soils with near-neutral pHs. Like- wise, because our fingerprinting method underestimates total bacterial diversity (see Methods), we cannot predict how the absolute diversity of bacteria changes across the pH gradient. When we compare paired sampling locations with similar veg- etation and climate but very different soil pHs, we find evidence for the strong correlation between bacterial diversity and soil pH at the local scale. For example, two deciduous forest soils collected in the Duke Forest, North Carolina (see Table 3, which is published as supporting information on the PNAS web site), showed that the soil with the higher pH (DF2, pH 6.8) had an estimated bacterial richness 60% higher than the more acidic soil (DF3, pH 5.1). Similarly for two tropical forest soils collected 1 km apart in the Peruvian Amazon, the soil with the higher pH (PE8, pH 5.5) had an estimated bacterial richness 26% higher than the more acidic soil (PE7, pH 4.1). Qualitatively, there was no clear relationship between soil bacterial diversity and plant diversity at the continental scale. Although plant diversity was not determined at each sampling site, ecosystems with the highest levels of bacterial diversity (semiarid ecosystems in the continental U.S.) have relatively low levels of plant diversity (21). Likewise, soils from terra firme sites in the Peruvian Amazon in our analysis had relatively low levels of bacterial diversity (H 2.5���2.7), but Conflict of interest statement: No conflicts declared. This paper was submitted directly (Track II) to the PNAS office. Abbreviations: MAT, mean average temperature PET, potential evapotranspiration. ���To whom correspondence should be addressed at: Department of Ecology and Evolution- ary Biology, Campus Box 334, University of Colorado, Boulder, CO 80309-0334. E-mail: noahfierer@gmail.com. �� 2006 by The National Academy of Sciences of the USA 626���631 PNAS January 17, 2006 vol. 103 no. 3 www.pnas.org cgi doi 10.1073 pnas.0507535103

these sites have some of the highest recorded levels of plant diversity on Earth (22). In fact, we added the tropical sites at Manu National Park, Peru (PE, Table 3) and Missiones, Argentina (AR, Table 3) to test our initial relationship and to contrast microbial diversity at two tropical sites with high plant diversity but contrasting soil pH. There was also no apparent latitudinal gradient in diversity (Table 1 and Fig. 2), unlike diversity observations for plants and animals (18). Consequently, the environmental factors fre- quently cited as good predictors of plant and animal diversity at continental scales, particularly mean annual temperature (MAT) and potential evapotranspiration (PET) (17���19), had little effect on measured soil bacterial diversity (Fig. 2). Sam- pling resolution can have an important influence on the assess- ment of diversity patterns (23, 24), and, in this study, soils were collected from plots of 100 m2 that are smaller in size than those commonly used to quantify large-scale patterns of plant and animal diversity (17). However, because individual soil bacteria are many orders-of-magnitude smaller than individual plants or animals (25), the number of individuals per plot may be directly comparable. It is also possible that the small size of our plots causes us to overestimate the importance of local parameters, such as soil pH, on bacterial community composi- tion and underestimate the importance of parameters, such as PET and MAT, which are more regional in scale. Nonetheless, our results do suggest that the biogeographical patterns observed in soil bacterial communities are fundamentally different from those observed in well studied plant and animal communities Fig. 1. The relationship between soil pH and bacterial phylotype diversity (A) and phylotype richness (B), defined as the number of unique phylotypes. Diversity was estimated by using the Shannon index, a summary variable that incorporates the richness and evenness of phylotypes (20). Symbols correspond to general ecosystem categories, and labels denote individual soils (see Table 3). Detailed information on the individual soils is provided in Table 3. Both quadratic regressions (H 0.08pH2 1.12 pH 0.5, r2 0.70 for A and Richness 1.65pH2 23.2pH 42.3, r2 0.58 for B) were statistically significant (P 0.0001). There was no significant correlation between the residuals of the two regressions shown here and any of the other soil and site variables listed in Table 1 (P 0.25 in all cases). Fierer and Jackson PNAS January 17, 2006 vol. 103 no. 3 627 ECOLOGY