Shifts in Rhizoplane Communities of Aquatic Plants after Cadmium Exposure

In this study we present the comparative molecular analysis of bacterial communities of the aquatic plant Lemna minor from a contaminated site (RCP) and from a laboratory culture (EPA), as well as each of these with the addition of cadmium. Plants were identified as L. minor by analysis of the rpl16 chloroplast region. Comparative bacterial community studies were based on the analyses of 16S rRNA clone libraries, each containing about 100 clones from the root surfaces of plants. Bacterial communities were compared at three phylogenetic levels of resolution. At the level of bacterial divisions, differences in diversity index scores between treatments, with and without cadmium within the same plant type (EPA or RCP), were small, indicating that cadmium had little effect. When we compared genera within the most dominant group, the β-proteobacteria, differences between unamended and cadmium-amended libraries were much larger. Bacterial diversity increased upon cadmium addition for both EPA and RCP libraries. Analyses of diversity at the phylotype level showed parallel shifts to more even communities upon cadmium addition; that is, percentage changes in diversity indices due to cadmium addition were the same for either plant type, indicating that contamination history might be independent of disturbance-induced diversity shifts. At finer phylogenetic levels of resolution, the effects of cadmium addition on bacterial communities were very noticeable. This study is a first step in understanding the role of aquatic plant-associated microbial communities in phytoremediation of heavy metals.     

Recovery of novel bacterial diversity from a forested wetland impacted by reject coal

Sulphide mineral mining together with improperly contained sulphur-rich coal represents a significant environmental problem caused by leaching of toxic material. The Savannah River Site's D-area harbours a 22-year-old exposed reject coal pile (RCP) from which acidic, metal rich, saline runoff has impacted an adjacent forested wetland. In order to assess the bacterial community composition of this region, composite sediment samples were collected at three points along a contamination gradient (high, middle and no contamination) and processed for generation of bacterial and archaeal 16S rDNA clone libraries. Little sequence overlap occurred between the contaminated (RCP samples) and unimpacted sites, indicating that the majority of 16S rDNAs retrieved from the former represent organisms selected by the acidic runoff. Archaeal diversity within the RCP samples consisted mainly of sequences related to the genus Thermoplasma and to sequences of a novel type. Bacterial RCP libraries contained 16S rRNA genes related to isolates (Acidiphilium sp., Acidobacterium capsulatum, Ferromicrobium acidophilium and Leptospirillum ferrooxidans) and environmental clones previously retrieved from acidic habitats, including ones phylogenetically associated with organisms capable of sulphur and iron metabolism. These libraries also exhibited particularly novel 16S rDNA types not retrieved from other acid mine drainage habitats, indicating that significant diversity remains to be detected in acid mine drainage-type systems.     

It's all relative: ranking the diversity of aquatic bacterial communities

The study of microbial diversity patterns is hampered by the enormous diversity of microbial communities and the lack of resources to sample them exhaustively. For many questions about richness and evenness, however, one only needs to know the relative order of diversity among samples rather than total diversity. We used 16S libraries from the Global Ocean Survey to investigate the ability of 10 diversity statistics (including rarefaction, non-parametric, parametric, curve extrapolation and diversity indices) to assess the relative diversity of six aquatic bacterial communities. Overall, we found that the statistics yielded remarkably similar rankings of the samples for a given sequence similarity cut-off. This correspondence, despite the different underlying assumptions of the statistics, suggests that diversity statistics are a useful tool for ranking samples of microbial diversity. In addition, sequence similarity cut-off influenced the diversity ranking of the samples, demonstrating that diversity statistics can also be used to detect differences in phylogenetic structure among microbial communities. Finally, a subsampling analysis suggests that further sequencing from these particular clone libraries would not have substantially changed the richness rankings of the samples.     

Phylogenetic analysis of bacteria associated with Laminaria saccharina

Bacterial communities associated with the brown alga Laminaria saccharina from the Baltic Sea and from the North Sea were investigated using denaturing gradient gel electrophoresis and 16S rRNA gene clone libraries. The rhizoid, cauloid, meristem and phyloid revealed different 16S rRNA gene denaturing gradient gel electrophoresis banding patterns indicating a specific association of bacterial communities with different parts of the alga. Associations with cauloid and meristem were more specific, while less specific associations were obtained from the old phyloid. In addition, seasonal and geographical differences in the associated communities were observed. Results from 16S rRNA gene libraries supported these findings. Bacterial phylotypes associated with the alga were affiliated with the Alphaproteobacteria (nine phylotypes), Gammaproteobacteria (nine phylotypes) and the Bacteroidetes group (four phylotypes). A number of bacteria associated with other algae and other marine macroorganisms were among the closest relatives of phylotypes associated with L. saccharina.     

Environmental and biotic drivers of soil microbial β‐diversity across spatial and phylogenetic scales

Soil microbial communities play a key role in ecosystem functioning but still little is known about the processes that determine their turnover (β‐diversity) along ecological gradients. Here, we characterize soil microbial β‐diversity at two spatial scales and at multiple phylogenetic grains to ask how archaeal, bacterial and fungal communities are shaped by abiotic processes and biotic interactions with plants. We characterized microbial and plant communities using DNA metabarcoding of soil samples distributed across and within eighteen plots along an elevation gradient in the French Alps. The recovered taxa were placed onto phylogenies to estimate microbial and plant β‐diversity at different phylogenetic grains (i.e. resolution). We then modeled microbial β‐diversities with respect to plant β‐diversities and environmental dissimilarities across plots (landscape scale) and with respect to plant β‐diversities and spatial distances within plots (plot scale). At the landscape scale, fungal and archaeal β‐diversities were mostly related to plant β‐diversity, while bacterial β‐diversities were mostly related to environmental dissimilarities. At the plot scale, we detected a modest covariation of bacterial and fungal β‐diversities with plant β‐diversity; as well as a distance–decay relationship that suggested the influence of ecological drift on microbial communities. In addition, the covariation between fungal and plant β‐diversity at the plot scale was highest at fine or intermediate phylogenetic grains hinting that biotic interactions between those clades depends on early‐evolved traits. Altogether, we show how multiple ecological processes determine soil microbial community assembly at different spatial scales and how the strength of these processes change among microbial clades. In addition, we emphasized the imprint of microbial and plant evolutionary history on today's microbial community structure.     

Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity

This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.     

Comparative microbial diversity analyses of modern marine thrombolitic mats by barcoded pyrosequencing

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities.     

Drastic changes in aquatic bacterial populations from the Cuatro Cienegas Basin (Mexico) in response to long-term environmental stress

Understanding the changes of aquatic microbial community composition in response to changes in temperature and ultraviolet irradiation is relevant for predicting biogeochemical modifications in the functioning of natural microbial communities under global climate change scenarios. Herein we investigate shifts in the bacterioplankton composition in response to long-term changes in temperature and UV radiation. For this purpose, 15 mesocosms were seeded with composite aquatic microbial communities from natural pools within the Cuatro Cienegas Basin (Mexican Chihuahuan desert) and were subject to different temperatures and UV conditions. 16S rRNA gene clone libraries were obtained from water samples at the mid-point (4 months) and the end of the experiment (8 months). An increase in bacterial diversity over time was found in the treatment of constant temperature and UV protection, which suggests that stable environments promote the establishment of complex and diverse bacterial community. Drastic changes in the phylogenetic bacterioplankton composition and structure were observed in response to fluctuating temperature and increasing UV radiation and temperature. Fluctuating temperature induced the largest decrease of bacterial richness during the experiment, indicating that frequent temperature changes drive the reduction in abundance of several species, most notably autotrophs. The long-term impact of these environmental stresses reduced diversity and selected for generalist aquatic bacterial populations, such as Porphyrobacter. These changes at the community level occur at an ecological time scale, suggesting that under global warming scenarios cascade effects on the food web are possible if the microbial diversity is modified.     

Are Archaea inherently less diverse than Bacteria in the same environments?

Like Bacteria, Archaea occur in a wide variety of environments, only some of which can be considered 'extreme'. We compare archaeal diversity, as represented by 173 16S rRNA gene libraries described in published reports, to bacterial diversity in 79 libraries from the same source environments. An objective assessment indicated that 114 archaeal libraries and 45 bacterial libraries were large enough to yield stable estimates of total phylotype richness. Archaeal libraries were seldom as large or diverse as bacterial libraries from the same environments. However, a relatively larger proportion of libraries were large enough to effectively capture rare as well as dominant phylotypes in archaeal communities. In contrast to bacterial libraries, the number of phylotypes did not correlate with library size; thus, 'larger' may not necessarily be 'better' for determining diversity in archaeal libraries. Differences in diversity suggest possible differences in ecological roles of Archaea and Bacteria; however, information is lacking on relative abundances and metabolic activities within the sampled communities, as well as the possible existence of microhabitats. The significance of phylogenetic diversity as opposed to functional diversity remains unclear, and should be a high priority for continuing research.     

Bacterial Communities Involved in Soil Formation and Plant Establishment Triggered by Pyrite Bioweathering on Arctic Moraines

In arctic glacier moraines, bioweathering primed by microbial iron oxidizers creates fertility gradients that accelerate soil development and plant establishment. With the aim of investigating the change of bacterial diversity in a pyrite-weathered gradient, we analyzed the composition of the bacterial communities involved in the process by sequencing 16S rRNA gene libraries from different biological soil crusts (BSC). Bacterial communities in three BSC of different morphology, located within 1 m distance downstream a pyritic conglomerate rock, were significantly diverse. The glacier moraine surrounding the weathered site showed wide phylogenetic diversity and high evenness with 15 represented bacterial classes, dominated by Alphaproteobacteria and pioneer Cyanobacteria colonizers. The bioweathered area showed the lowest diversity indexes and only nine bacterial families, largely dominated by Acidobacteriaceae and Acetobacteraceae typical of acidic environments, in accordance with the low pH of the BSC. In the weathered BSC, iron-oxidizing bacteria were cultivated, with counts decreasing along with the increase of distance from the rock, and nutrient release from the rock was revealed by environmental scanning electron microscopy-energy dispersive X-ray analyses. The vegetated area showed the presence of Actinomycetales, Verrucomicrobiales, Gemmatimonadales, Burkholderiales, and Rhizobiales, denoting a bacterial community typical of developed soils and indicating that the lithoid substrate of the bare moraine was here subjected to an accelerated colonization, driven by iron-oxidizing activity.     

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of <Emphasis Type="Italic">Methylobacterium</Emphasis> in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism–plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR–denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR–DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.     

Bacterial community dynamics across a floristic gradient in a temperate upland grassland ecosystem

Alterations in soil bacterial communities across a transect between a semi natural upland grassland and an agriculturally improved enclosure were assessed using culture-based methods and a nucleic-acid-based method, terminal restriction fragment length polymorphism (TRFLP). While plant diversity decreased across the transect towards the improved area, numbers of 16S rDNA terminal restriction fragments increased, indicating an increase in numbers of bacterial ribotypes. Bacterial numbers, microbial activity, and potential functional diversity also followed a similar trend, increasing with decreasing plant diversity. Alterations in bacterial community structure were coincident with changes in soil physicochemical properties which also changed across the transect. Increases in soil pH, nitrate, phosphorus, potassium, and calcium occurred toward the improved grassland, while organic matter and ammonium declined. The inverse relationship between floristic diversity and bacterial ribotype numbers suggests that soil physicochemical factors may be as influential in determining bacterial diversity in soils of upland grassland communities as floristic diversity.     

Richness and diversity of bacteria in the Nansha carbonate platform (Core MD05-2896), South China Sea

We explored the bacterial diversity and vertical distribution along a sediment core (MD05-2896) from the coral reefs of the Nansha carbonate platform in the South China Sea. Bacterial diversity is determined by 16S rRNA molecular survey from twelve subsamples A, obtained via cloning, sequencing and phylogenetic analyses. We estimated the species richness by parametric and nonparametric models, which identified 326 ± 40 (SE) bacteria species. The dominant bacterial groups included Planctomycetes, Deltaproteobacteria, and candidate division OP3, which constituting 23.7, 10.4, and 9.5 % of bacterial 16S rRNAclone libraries, respectively. The observed stratification of bacterial communities was correlated with C/N ratio. This study improves our understanding of the species–environment relationship in the sub-sea floor sediment.     

Resilience to fire of phylogenetic diversity across biological domains

Fire alters the structure and composition of above‐ and belowground communities with concurrent shifts in phylogenetic diversity. The inspection of postfire trends in the diversity of ecological communities incorporating phylogenetic information allows to better understand the mechanisms driving fire resilience. While fire reduces plant phylogenetic diversity based on the recruitment of evolutionarily related species with postfire seed persistence, it increases that of soil microbes by limiting soil resources and changing the dominance of competing microbes. Thus, during postfire community reassembly, plant and soil microbes might experience opposing temporal trends in their phylogenetic diversity that are linked through changes in the soil conditions. We tested this hypothesis by investigating the postfire evolution of plant and soil microbial (fungi, bacteria and archaea) communities across three 20‐year chronosequences. Plant phylogenetic diversity increased with time since fire as pioneer seeders facilitate the establishment of distantly related late‐successional shrubs. The postfire increase in plant phylogenetic diversity fostered plant productivity, eventually recovering soil organic matter. These shifts over time in the soil conditions explained the postfire restoration of fungal and bacterial phylogenetic diversity, which decreased to prefire levels, suggesting that evolutionarily related taxa with high relative fitness recover their competitive superiority during community reassembly. The resilience to fire of phylogenetic diversity across biological domains helps preserve the evolutionary history stored in our ecosystems.     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

Bacterial Communities in the Rhizosphere of Biofuel Crops Grown on Marginal Lands as Evaluated by 16S rRNA Gene Pyrosequences

Microbes are key components of the soil environment and are important contributors to the sustainability of agricultural systems, which is especially significant for biofuel crops growing on marginal lands. We studied bacterial communities in the rhizosphere of five biofuel crops cultivated in four locations in Michigan to determine which factors were correlated to changes in the structure of those communities. Three of these sites were marginal lands in that two were not suitable for conventional agriculture and one was regulated as a brownfield due to prior industrial pollution. Bacterial community composition and structure were assessed by 454 sequencing of the 16S rRNA gene. A total of 387,111 sequences were used for multivariate statistical analysis and to test for correlation between community structure and environmental variables such as plant species, soil attributes, and location. The most abundant bacterial phyla found in the rhizosphere of all crops were Acidobacteria, Proteobacteria, Actinobacteria, and Verrucomicrobia. Bacterial communities grouped by location rather than by crop and their structures were correlated to soil attributes, principally pH, organic matter, and nutrients. The effect of plant species was low but significant, and interactions between locations, plant species, and soil attributes account for most of the explained variation in the structure of bacterial communities, showing a complex relationship between bacterial populations and their environment. Bacterial diversity was higher in the agricultural sites compared to adjacent forest sites, indicating that the cultivation of those biofuel crops increased the rRNA diversity.     

Attached bacterial populations shared by four species of aquatic angiosperms

Symbiotic relationships between microbes and plants are common and well studied in terrestrial ecosystems, but little is known about such relationships in aquatic environments. We compared the phylogenetic diversities of leaf- and root-attached bacteria from four species of aquatic angiosperms using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of PCR-amplified 16S rRNA genes. Plants were collected from three beds in Chesapeake Bay at sites characterized as freshwater (Vallisneria americana), brackish (Potomogeton perfoliatus and Stuckenia pectinata), and marine (Zostera marina). DGGE analyses showed that bacterial communities were very similar for replicate samples of leaves from canopy-forming plants S. pectinata and P. perfoliatus and less similar for replicate samples of leaves from meadow-forming plants Z. marina and V. americana and of roots of all species. In contrast, bacterial communities differed greatly among plant species and between leaves and roots. DNA sequencing identified 154 bacterial phylotypes, most of which were restricted to single plant species. However, 12 phylotypes were found on more than one plant species, and several of these phylotypes were abundant in clone libraries and represented the darkest bands in DGGE banding patterns. Root-attached phylotypes included relatives of sulfur-oxidizing Gammaproteobacteria and sulfate-reducing Deltaproteobacteria. Leaf-attached phylotypes included relatives of polymer-degrading Bacteroidetes and phototrophic Alphaproteobacteria. Also, leaves and roots of three plant species hosted relatives of methylotrophic Betaproteobacteria belonging to the family Methylophilaceae. These results suggest that aquatic angiosperms host specialized communities of bacteria on their surfaces, including several broadly distributed and potentially mutualistic bacterial populations.     

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico

Background

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.

Methodology/Principal Findings

We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil).

Conclusions/Significance

The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient environment, allowed us to differentiate resident symbionts from potentially transient or prey bacteria. Pairing replicated clone library construction with rapid community profiling via T-RFLP analyses will greatly facilitate future studies of sponge-microbe symbioses.     

Phylogenetic Comparisons of Bacterial Communities from Serpentine and Nonserpentine Soils

I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale (~100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.