Although amplicon pyrosequencing approaches have revolutionized studies on microbial community composition and diversity, it should be noted that studies employing this approach are subject to an ever-growing list of experimental and interpretive caveats. For example, pyrosequencing error rates (47
) and clustering methods (44
) have been shown to artificially inflate representatives of the rare biosphere, and PCR primer choice can influence diversity estimates (22
). Moreover, a recent study by Zhou et al. (81
) showed that experimental reproducibility using amplicon-based methods is low, with very little taxonomic overlap between technical replicates, and difficulties in reliably comparing communities using common β diversity measures. Our study aimed to alleviate some of these concerns and reduce the variability inherent in stochastic sampling by employing a collection scheme that pooled biological replicates in silico
from the three soil or rhizosphere samples from each cactus. The use of biological replicates has been shown to reduce sampling artifact and improve quantitative analyses of α diversity and β diversity distances between samples (81
In contrast to studies that rely on standard cloning methods and that consequently reveal modest levels of taxonomic diversity of microorganisms in desert soils (for example, see references 19
, and 66
but see reference 26
), our results showed that microbial communities in this extreme environment are both abundant and diverse. This is perhaps unsurprising, as other typically extreme environments likewise harbor diverse microbial communities (26
). Both α and β diversity measures are much higher than previous estimates for desert soil (e.g., compare Shannon diversity measures between the current results and the 2005 study of Nagy and colleagues [57
]). Further, rarefaction analysis indicated that even by applying culture-independent high-throughput methods, we detected less than half of the species diversity likely present (see Fig. S1 and S2 in the supplemental material; coverage in ). These diverse bacterial communities have potentially important implications for the abundance of bacterial pathogens in desert soils (76
) and are likely to play an important role in soil formation, nutrient cycling, plant diversity, and primary productivity of desert environments (24
Our experimental design aimed to determine the relative influence of location, associated soil properties, and plant associations on microbial communities in the Sonoran Desert of southern Arizona at two different scales, tens and thousands of meters. Our replicate sampling approach enabled us to determine that the primary driver of microbial diversity and community associations is the general location from which samples are chosen. All of our samples were more similar to those collected within the surrounding 100-m2
collection area, regardless of whether they were collected from the rhizosphere or from the bulk soil. An ANOVA test for OTU abundance by location shows that only a few abundant OTUs are significantly associated with the principal location of samples, including the Thermoprotei
< 0.001) and the class Alphaproteobacteria
< 0.05). This observation supports the idea that local soil characteristics shape the bulk soil and rhizosphere communities comparably by influencing several common taxa and that cacti have relatively less influence on the community of microbes found in the rhizosphere. This scale has implications for microorganisms affecting human health transported by wind in deserts (35
) and for estimating and conserving the desert microbiome (33
). Indeed, the microbial communities from the Tumamoc Hill and Biosphere2 sites, despite sampling the rhizosphere from two different cactus species, showed more similarities to each other than either did to those samples from the Finger Rock site, which sampled the same cactus species as at Tumamoc Hill.
Because of our limited sampling depth and high diversity of OTUs in our samples, we were unable to determine whether these spatial differences in microbial communities are due merely to differences in the relative abundance of OTUs between sites or actual differences in the contingent of OTUs. A recent deep sequencing study of marine microbial communities shows that differences in community composition in that environment is driven by changes in the relative abundance of a widely shared and dynamic set of OTUs, and not by temporal differences in the presence or absence of OTUs (14
). Deeper sequencing will be necessary to determine whether similar dynamics determine community differences in Sonoran Desert soils.
Several physical and chemical soil properties were correlated with the differences between communities as measured by UniFrac, including pH, percent carbon, electric conductivity, cation exchange capacity, and even soil particle size distribution (e.g., percent silt and sand). Some of these characteristics have previously been implicated in correlations with diversity, particularly pH (17
). Although the percent carbon in soil significantly correlated with β diversity measurements, neither the percent nitrogen (%N) nor the ratio of carbon to nitrogen (C:N) available in the soil showed significant correlation between rhizosphere or soil communities (in the rhizosphere, P
= 0.85 for %N and P
= 0.89 for C:N; in soil, P
= 0.28 for %N and P
= 0.62 for C:N). These observations suggest that available carbon, not nitrogen, is a limiting factor in driving local microbial diversity in these environments. Interestingly, given the scarcity of water in this desert ecosystem, available water content was not correlated with microbial diversity.
Only within the two natural sites surveyed did the presence of plant roots drive community structure enough to group rhizosphere and interplant samples into distinct communities based on UPGMA clustering and principal coordinate analysis. This suggests that, at the scale of a few meters, bacterial communities found in soil and associated with roots are not random samples from a common pool of species but that these two habitats differ in their microbial composition. As stated above, however, this conclusion may be premature and predicated on our limited sequencing depth. The failure of the artificial environment of Biosphere2 to replicate the pattern of more-natural environs hints that the latter scenario may be the case. It should also be noted that the soil characteristics of the two cacti sampled at Biosphere2 were very different from each other (see Table S2 in the supplemental material), as this artificial environment was created using nonhomogenous soil sources and is daily exposed to high levels of traffic, experimentation, and other human influences.
We did not find support for the resource island hypothesis related to species diversity or abundance. Although we observed relatively more OTUs in rhizosphere samples than in soil samples, the difference was not significant. This contradicts the view that a high nutrient concentration in the vicinity of roots results in higher abundance and diversity of microorganisms compared to interplant soil. Our results, however, show that rhizosphere and soil communities sustain distinct microbial taxa, as the vast majority of OTUs are found exclusively in one or the other communities, but not both. These observations suggest that seedlings of cacti growing under trees might benefit from many types of bacteria unique to the rhizosphere but found in low abundance, rather than by bacterial abundance or diversity per se, or by other physical factors (e.g., shade, temperature, nutrient availability) not related to the microorganisms.
Although archaea are now recognized as important and diverse contingents of many types of soil (4
), to our knowledge this is the first report to describe such a large fraction of Thermoprotei
in desert soil samples. The direct contradiction to prior studies on soils similar to those from our study area (57
), or in cold deserts (65
), which failed to observe a significant proportion of Archaea
, highlights the ability of pyrosequencing to more completely capture the diversity of nonculturable microbes. Our data suggest that Thermoprotei
, particularly the family Desulfurococcaceae
, may play an important role in the soils of the Sonoran Desert. Further experiments are necessary to confirm the veracity of our observations (i.e., test the possibility of primer bias for increased Thermoprotei
amplification) and to fully elucidate the impact of Thermoprotei
on the microbial communities of this region.
One observation consistent to all samples was the dominance of a core set of abundant taxa. This was true whether we were considering the overlap of soil and rhizosphere samples or geographic groupings. The core microbiome of this study has a different composition than those reads found only in individual samples, with the aforementioned preponderance of Thermoprotei
. This is perhaps not unexpected, as Crenarchaeota
contains radio- and thermotolerant species detected previously in other extreme environments, including desert soils (16
), hot springs (75
), permafrost (80
), and marine environments near the Sonoran Desert (8
). The rare OTUs appear to be relatively similar in terms of the proportion of various phyla between sample sites. However, there are a large number of rare phyla that appear to be particular for a specific sample site. For example, the Firmicutes
taxa were far more abundant in soil samples at the Finger Rock site than other sites, suggesting that these rare variants exhibit a more stochastic distribution than the common and abundant core microbiome. This observation is in agreement with other recent pyrosequencing studies that have found soils to be dominated by a relatively small number of microbial taxa but that harbor a wide array of rare, yet highly diverse microbes (74
Despite constituting an artificial environment primarily enclosed and isolated from the surrounding desert where the cardón cacti were introduced (3
), the contingent of soil microbes found in the coastal fog desert biome of Biosphere2 closely resemble natural surrounding communities dominated by saguaro cacti. Indeed, Biosphere2 shared a larger number of OTUs with the more distantly sampled and natural setting of Tumamoc Hill (22,239 reads from 2,719 OTUs) than the latter did with the other natural sampling site in our study, Finger Rock (6,275 reads from 803 OTUs). Interestingly, there was no more apparent significant differences between the soil and rhizosphere samples from Biosphere2 and the natural collection sites based on weighted UniFrac β diversity significance test, and overall OTU diversity measures were higher than at the natural sites. These two observations are consistent with the altered nature of this human-made environment. For instance, the diversity of the B2 samples could have been enhanced by microorganisms from a remote site where the cardóns were located before they were transplanted in addition to microorganisms from a diverse mix of local soils used to create the desert fog biome of B2. Members of the Thermoprotei
were no less prevalent in Biosphere2 than in natural settings, highlighting the importance of this class in distinct locations of the Sonoran Desert ecosystem and on the two dominant columnar cacti species. These observations bode well for previous and future ecological studies in this large synthetic community (78
), as soil microbial populations are known to exert a wide range of effects on plant communities and their ecological success (5
Our results clearly indicate that culture-independent approaches are able to reveal large portions of previously undetected microbial communities and highlight the potential importance of these previously cryptic taxa to extreme environments. By far, the largest percentage of reads from both soil and rhizosphere samples were from unclassified Thermoprotei, suggesting that this abundant class of archaea plays a crucial, if previously unrecognized role in soil and rhizosphere ecology of the Sonoran Desert. The influence of one dominant plant type of the Sonoran Desert, the columnar saguaro and cardón cacti, on microbial communities was slight in comparison to local soil influences on abundant taxa, suggesting that the extreme physical factors in this environment play critical roles in shaping microbial communities. This study reveals a clear gap in the current understanding of desert microbial ecology and stresses that future studies should focus their efforts on isolating and classifying the abundant Thermoprotei, particularly the Desulfurococcales, so dominant in this community.