We investigated community diversity in four soil environments by determining phylotype richness, distribution, and similarity for approximately 50 isolates and nearly 200 16S rDNA clones from each environment. Both plating and 16S rDNA cloning suffer from biases that can distort community composition, richness, and structure. We assumed that the biases operated uniformly for our four environmental samples and that therefore the soil samples could be compared. Although our data were not replicated, they provided a starting point for assessing the merits of 16S rDNA cloning compared to cultivation. The data demonstrated that the 16S rDNA clone libraries provided more comprehensive sampling of the phylogenetic diversity in the four soil environments than the culture collections provided. Use of the two methods to assess community diversity demonstrated that the methods provided contradictory measures of relative phylotype richness in the Cosnino interspace environment, but the methods identified roughly similar relationships among the four communities when phylotype distributions were compared (that is, evenness was high in three of the environments but was comparatively lower in the Sunset Crater interspace environment). Both methods also identified the Cosnino interspace and rhizosphere environments as the environments that were most similar in terms of phylotype composition and the Sunset Crater interspace as the most dissimilar environment.
The ability of 16S rDNA cloning to sample the phylogenetic diversity in natural communities more comprehensively than cultivation is characteristic of the method. Our results are entirely consistent with this characteristic. The phylogenetic breadth of any one of our clone libraries is comparable to the phylogenetic breadth observed in previous studies in which 16S rDNA cloning was used to characterize soil microbial communities (3
). An extensive phylogenetic analysis of 56 clones from our libraries and a comparison of the data with data obtained in other studies of cloned 16S rDNA sequences from soil have been described previously (24
). In addition to multiple representatives of well-known bacterial divisions, members of a new division (now called the Acidobacterium
division) were described. The Acidobacterium
division was the most abundant and diverse group in our 16S rDNA clone libraries. This division was also the most abundant division in a clone library established from Wisconsin soil (3
) despite the use of different PCR primers (primer 530 forward instead of primer 8-27 forward) and DNA extraction procedures (see reference 24
for placement of the Wisconsin sequences in the Acidobacterium
division). Phylogenetic information for an additional 112 clones obtained in this study has not substantially altered the division level diversity noted previously (24
). Sequences affiliated with the genus Nitrospira
have been identified, thus expanding the number of divisions detected in the four soil samples from six to seven. Additional divisions may be represented by the 36 sequences (placed in uncertain category [Fig. ]) that had extremely low Sab
values with RDP sequences. For example, an RDP sequence representing candidate division OP9 (20
) had an Sab
value of 0.474 with one of the S0 16S rDNA clones. We are currently collecting full-length sequences from some of the 36 rDNA clones in order to accurately define their phylogenetic affiliations. The phylogenetic data obtained in the current study augment the impressive diversity described previously (24
). When compared with data in other reports, our data demonstrate results typical of 16S rDNA cloning.
The phylogenetic diversity observed among our four culture collections is also typical of the cultivation method. Gram-positive bacteria and proteobacteria are the predominant bacteria cultivated from soil on general-purpose, aerobic-growth media (1
). The skewed abundance of gram-positive isolates may have been due in part to the fact that isolates were selected after plates were incubated for only 3 days. In a study of two soils in Japan, 71% of the isolates that formed visible colonies within 18 h of inoculation were members of gram-positive species (22
). As the length of the incubation period increased, the relative abundance of gram-positive organisms in the total collection of isolates decreased. With two soils from New Mexico the proportion of cultivated gram-positive organisms decreased from 96 to 67% in a collection obtained after 24 h of incubation and then augmented after 3 weeks of incubation (9
). Gram-positive organisms have also been found to predominate in the outer layer of soil aggregates (17
) and are preferentially recovered during extraction procedures that do not adequately disrupt aggregates (through sonication, for example). Nonetheless, since the culture collections used in the current study were established in the same way from the four communities, they should allow comparisons between communities despite their narrow phylogenetic scope.
Measures of diversity among the clone libraries and culture collections revealed similar relationships among three of the four environmental samples. The results obtained with both methods used indicated that the levels of diversity in the Sunset Crater and Cosnino rhizosphere environments were comparable, while phylotype richness was lower and more skewed in the Sunset Crater interspace. Relatively similar values for phylotype richness (S, D, and H) and evenness (H/Hmax and D/Dmax) were obtained for the rhizosphere environments compared to the Sunset Crater interspace (Table ). Culture collections from samples obtained in September 1994 exhibited the same pattern of diversity as the culture collections obtained in April 1994 (data not shown), suggesting that the diversity in the Sunset Crater interspace was in fact less than the diversity in the rhizosphere environments. This observation is consistent with the idea that pinyon pine roots (matched for age) provide a homogeneous environment for microbial growth and community development and modulate the effects of different soil types. The lower diversity observed in the Sunset Crater interspace was likewise consistent with our knowledge of the physical characteristics of this environment. The Sunset Crater interspaces are devoid of macroscopic plant life. The black cinder gravel that constitutes the soil at Sunset Crater creates a hot, exceptionally arid environment due to low water retention. The apparently stressful conditions in the interspace are expected to reduce microbial community diversity, and the low moisture content and rapid water drainage in the cinder gravel should limit horizontal migration of bacteria from the rhizosphere environment to the interspace.
The 16S rDNA cloning and plate cultivation methods provided inconsistent assessments of relative phylotype richness in the fourth environment, the Cosnino interspace. Unlike the barren Sunset Crater interspaces, the interspaces at Cosnino are covered by grass and forb species, and the sandy loam soil at Cosnino retains more water than the Sunset Crater cinder gravel. Grass roots are abundant in the Cosnino interspace soil. Phylotype richness appeared to be as high in this environment as it was in the pinyon pine rhizospheres when it was measured by 16S rDNA cloning. However, based on plate cultivation, the Cosnino interspace appeared to have approximately one-half as many phylotypes (like the Sunset Crater interspace) as the rhizosphere environments (Fig. ). This result was surprising in light of the physical characteristics of the Cosnino interspace. Interestingly, 16S rDNA cloning and plate cultivation both demonstrated that the frequency distribution of phylotypes (evenness) in the Cosnino interspace was relatively similar to that observed in the pinyon pine rhizosphere environments. In the absence of replicated data and larger sample sizes, it is difficult to accurately interpret the discrepancy between the 16S rDNA cloning and plate cultivation data. The most probable explanation, however, is that the discrepancy arose from sampling different fractions of the microbial community. Whereas the C0 clone library contained a wide variety of eubacteria, the iC0 culture collection contained primarily fast-growing, cultivable, aerobic heterotrophs belonging to the gram-positive and proteobacteria divisions. Thus, it seems reasonable to assume that one subcomponent of the interspace community may have lower species richness but that the total community diversity in the Cosnino interspace is probably similar to the community diversity in the rhizosphere environment.
Interpretation of phylotype composition similarity between communities was more problematic than analysis of phylotype richness or distribution. The similarity values were uniformly low (11 to 31%). However, the low values obtained from standard similarity comparisons may have been largely the result of each library containing a large proportion of rare phylotypes that had a low probability of being detected. Indeed, the average similarity of the culture collections (which contained smaller proportions of rare phylotypes) was higher than the average similarity of the clone libraries. The detection frequency of a phylotype in two or more libraries was positively correlated with phylotype abundance. We observed this relationship among the clone libraries as well as among the culture collections, although in the latter case the small sample size (34 phylotypes) provided a marginal number of data points. This relationship was expected for replicate samples but not for samples which had substantially different compositions. For replicate or very similar samples, abundant phylotypes should have greater probabilities of being resampled than rare phylotypes have. If community similarity values were calculated by using only the subset of phylotypes that appeared in at least two libraries, the average levels of similarity of the clone libraries and culture collections rose from 15 to 53% and from 26 to 52%, respectively. These data suggest that the phylotype compositions of the four environments may be substantially similar but that 16S rDNA clone libraries and culture collections document the similarities inadequately due to the large abundance of rare phylotypes typical for such collections.
In summary, 16S rDNA cloning appears to be as valid as plate cultivation for investigating diversity in environments despite the numerous biases that can occur in the 16S rDNA cloning method. Although the two approaches in some cases provide different assessments of relative community diversity, the discrepancies probably arise from sampling different segments of microbial communities. Identifying consistent relationships between environments based on comparisons of culture collections and culture-independent techniques may be highly dependent on the habitats sampled due to the limited ability of a single cultivation method to survey the bacterial domain and the influence of bacterial physiology in situ on the success of cultivation in the laboratory. Previous studies comparing the compositions of microbial communities by analysis of 16S rDNA clone libraries have been confined almost exclusively to comparisons of marine environments (2
). Overall community similarities have not been described. However, identical or nearly identical (<1% mismatch) 16S rDNA sequences have been retrieved from marine samples obtained thousands of miles apart (8
). Thus far, such sequences have not been identified in clone libraries derived from different soil samples. Analyses of community diversity by using clone libraries have not been replicated to date. Key questions about sampling probabilities and the range of composition similarity need to be addressed by using replicate samples in order to facilitate the interpretation of clone diversity in different environments. Other techniques based on direct 16S rDNA amplification that are more amenable to replication should provide powerful tools for rapidly investigating diversity in communities despite the inability of such techniques to accurately assess community structure.