Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities

Ecological diversity indices are frequently applied to molecular profiling methods, such as terminal restriction fragment length polymorphism (T-RFLP), in order to compare diversity among microbial communities. We performed simulations to determine whether diversity indices calculated from T-RFLP profiles could reflect the true diversity of the underlying communities despite potential analytical artifacts. These include multiple taxa generating the same terminal restriction fragment (TRF) and rare TRFs being excluded by a relative abundance (fluorescence) threshold. True community diversity was simulated using the lognormal species abundance distribution. Simulated T-RFLP profiles were generated by assigning each species a TRF size based on an empirical or modeled TRF size distribution. With a typical threshold (1%), the only consistently useful relationship was between Smith and Wilson evenness applied to T-RFLP data (TRF-E(var)) and true Shannon diversity (H'), with correlations between 0.71 and 0.81. TRF-H' and true H' were well correlated in the simulations using the lowest number of species, but this correlation declined substantially in simulations using greater numbers of species, to the point where TRF-H' cannot be considered a useful statistic. The relationships between TRF diversity indices and true indices were sensitive to the relative abundance threshold, with greatly improved correlations observed using a 0.1% threshold, which was investigated for comparative purposes but is not possible to consistently achieve with current technology. In general, the use of diversity indices on T-RFLP data provides inaccurate estimates of true diversity in microbial communities (with the possible exception of TRF-E(var)). We suggest that, where significant differences in T-RFLP diversity indices were found in previous work, these should be reinterpreted as a reflection of differences in community composition rather than a true difference in community diversity.     

Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from γ-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from α-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4′,6′-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for α-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. α-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.     

Richness and Diversity of Bacterioplankton Species along an Estuarine Gradient in Moreton Bay, Australia

Bacterioplankton community diversity was investigated in the subtropical Brisbane River-Moreton Bay estuary, Australia (27°25′S, 153°5′E). Bacterial communities were studied using automated rRNA intergenic spacer analysis (ARISA), which amplifies 16S-23S ribosomal DNA internally transcribed spacer regions from mixed-community DNA and detects the separated products on a fragment analyzer. Samples were collected from eight sites throughout the estuary and east to the East Australian Current (Coral Sea). Bacterioplankton communities had the highest operational taxonomic unit (OTU) richness, as measured by ARISA at eastern bay stations (S [total richness] = 84 to 85 OTU) and the lowest richness in the Coral Sea (S = 39 to 59 OTU). Richness correlated positively with bacterial abundance; however, there were no strong correlations between diversity and salinity, NO₃⁻ and PO₄³⁻ concentrations, or chlorophyll a concentration. Bacterioplankton communities at the riverine stations were different from communities in the bay or Coral Sea. The main differences in OTU richness between stations were in taxa that each represented 0.1% (the detection limit) to 0.5% of the total amplified DNA, i.e., the “tail” of the distribution. We found that some bacterioplankton taxa are specific to distinct environments while others have a ubiquitous distribution from river to sea. Bacterioplankton richness and diversity patterns in the estuary are potentially a consequence of greater niche availability, mixing of local and adjacent environment communities, or intermediate disturbance. Furthermore, these results contrast with previous reports of spatially homogeneous bacterioplankton communities in other coastal waters.     

Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing

Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species—the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E−9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E−14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356–896 OTUs) was >2-fold higher than in the MI (112–567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that can effectively breakdown a wide variety of complex polysaccharides.     

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [¹⁴C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H₂-utilizing and ¹⁴CO₂-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H₂-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H₂ utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Comparison of Diversities and Compositions of Bacterial Populations Inhabiting Natural Forest Soils

The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes. The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates. The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils. Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition. Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the α-Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%). Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the α-Proteobacteria (27%), respectively. The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny. In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition.     

Aspects of Diversity Measurement for Microbial Communities

A useful measure of diversity was calculated for microbial communities collected from lake water and sediment samples using the Shannon index (H′) and rarefaction [E(S)]. Isolates were clustered by a numerical taxonomy approach in which limited (<20) tests were used so that the groups obtained represented a level of resolution other than species. The numerical value of diversity for each sample was affected by the number of tests used; however, the relative diversity compared among several sampling locations was the same whether 11 or 19 characters were examined. The number of isolates (i.e., sample size) strongly influenced the value of H′ so that unequal sized samples could not be compared. Rarefaction accounts for differences in sample size inherently so that such comparisons are made simple. Due to the type of sampling carried out by microbiologists, H′ is estimated and not determined and therefore requires a statement of error associated with it. Failure to report error provided potentially misleading results. Calculation of the variance of H′ is not a simple matter and may be impossible when handling a large number of samples. With rarefaction, the variance of E(S) is readily determined, facilitating the comparison of many samples.     

The biogeography of fungal communities in wetland sediments along the Changjiang River and other sites in China

Whether fungal community structure depends more on historical factors or on contemporary factors is controversial. This study used culture-dependent and -independent (polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)) methods to assess the influence of historical and contemporary factors on the distributions of fungi in the wetland sediments at 10 locations along the Changjiang River and at 10 other locations in China. The culture-dependent approach detected greater species diversity (177 operational taxonomic units (OTUs)) than PCR-DGGE analysis (145 OTUs), and the species in the genera of Penicillium (relative frequency=16.8%), Fusarium (15.4%), Aspergillus (7.6%), Trichoderma (5.8%) and Talaromyces (4.2%) were dominant. On the basis of DGGE data, fungal diversity along the Changjiang River increased from upstream to downstream; altitude explained 44.8% of this variation in diversity. And based on the data from all 20 locations, the fungal communities were geographically clustered into three groups: Southern China, Northern China and the Qinghai-Tibetan Plateau. Multivariate regression tree analysis for data from the 20 locations indicated that the fungal community was influenced primarily by location (which explained 61.8% of the variation at a large scale), followed by total potassium (9.4%) and total nitrogen (3.5%) at a local scale. These results are consistent with the concept that geographic distance is the dominant factor driving variation in fungal diversity at a regional scale (1000–4000 km), whereas environmental factors (total potassium and total nitrogen) explain variation in fungal diversity at a local scale (<1000 km).     

生物肥与甲壳素和恶霉灵配施对香蕉枯萎病的防治效果

通过盆栽试验研究了生物肥与甲壳素和恶霉灵配施防治香蕉枯萎病效果,试验结果表明,生物肥与恶霉灵配施(H+F)处理香蕉枯萎病病情指数最高,生物肥与甲壳素配施(C+F)处理病情指数最低。单独生物肥处理防病效果为32.8%,生物肥与甲壳素配施处理为42.5%,而生物肥与恶霉灵配施加重了香蕉枯萎病病情。Biolog Eco微平板研究发现,AWCD(平均每孔颜色变化率)和Shannon等4个多样性指数变化趋势与防病效果相反:防病效果好的处理,土壤细菌功能多样性指数反而低,经检测发现病原真菌(Fusarium oxysporum f. sp. cubense)可利用Biolog Eco微平板上碳源底物并发生颜色变化,干扰测定结果。T-RFLP分析土壤细菌DNA多样性,对照(灭菌生物肥)土壤中TRFs末端限制性片段最少,生物肥与甲壳素配施处理最多。与网上数据库比较,生物肥与甲壳素配施增加了土壤中芽胞杆菌种类,与恶霉灵配施降低了芽胞杆菌种类。分析发现,T-RFLP和Biolog的主成份分析载荷图具有较高一致性。因此,生物肥与生物农药甲壳素配施,从生态角度控制土传病害,优势互补,提高了土壤细菌多样性,改善了土壤细菌群落结构,有利于提高防病效果。     

Effect of warming and drought on grassland microbial communities

The soil microbiome is responsible for mediating key ecological processes; however, little is known about its sensitivity to climate change. Observed increases in global temperatures and alteration to rainfall patterns, due to anthropogenic release of greenhouse gases, will likely have a strong influence on soil microbial communities and ultimately the ecosystem services they provide. Therefore, it is vital to understand how soil microbial communities will respond to future climate change scenarios. To this end, we surveyed the abundance, diversity and structure of microbial communities over a 2-year period from a long-term in situ warming experiment that experienced a moderate natural drought. We found the warming treatment and soil water budgets strongly influence bacterial population size and diversity. In normal precipitation years, the warming treatment significantly increased microbial population size 40–150% but decreased diversity and significantly changed the composition of the community when compared with the unwarmed controls. However during drought conditions, the warming treatment significantly reduced soil moisture thereby creating unfavorable growth conditions that led to a 50–80% reduction in the microbial population size when compared with the control. Warmed plots also saw an increase in species richness, diversity and evenness; however, community composition was unaffected suggesting that few phylotypes may be active under these stressful conditions. Our results indicate that under warmed conditions, ecosystem water budget regulates the abundance and diversity of microbial populations and that rainfall timing is critical at the onset of drought for sustaining microbial populations.     

Quantifying Community Dynamics of Nitrifiers in Functionally Stable Reactors

A sequential batch reactor (SBR) and a membrane bioreactor (MBR) were inoculated with the same sludge from a municipal wastewater treatment plant, supplemented with ammonium, and operated in parallel for 84 days. It was investigated whether the functional stability of the nitrification process corresponded with a static ammonia-oxidizing bacterial (AOB) community. The SBR provided complete nitrification during nearly the whole experimental run, whereas the MBR showed a buildup of 0 to 2 mg nitrite-N liter⁻¹ from day 45 until day 84. Based on the denaturing gradient gel electrophoresis profiles, two novel approaches were introduced to characterize and quantify the community dynamics and interspecies abundance ratios: (i) the rate of change [Δ_t(week)] parameter and (ii) the Pareto-Lorenz curve distribution pattern. During the whole sampling period, it was observed that neither of the reactor types maintained a static microbial community and that the SBR evolved more gradually than the MBR, particularly with respect to AOB (i.e., average weekly community changes of 12.6% ± 5.2% for the SBR and 24.6% ± 14.3% for the MBR). Based on the Pareto-Lorenz curves, it was observed that only a small group of AOB species played a numerically dominant role in the nitritation of both reactors, and this was true especially for the MBR. The remaining less dominant species were speculated to constitute a reserve of AOB which can proliferate to replace the dominant species. The value of these parameters in terms of tools to assist the operation of activated-sludge systems is discussed.     

An index of functional diversity

Abstract. Functional diversity has been seen as the key to predicting the stability, invasibility, resource capture, nutrient cycling and productivity of communities. However, it has been unclear how to estimate it. Ten criteria for an index of functional diversity are developed. These include that it should reflect the range of characters present and the abundance of the species with those characters in the community, and be unaffected by the measurement units used or by the number of species. An index that meets all ten criteria, FD_var, is investigated. It is based on the variance in characters, weighted by the abundance of the species with those characters. Tested with artificial and randomly generated data, it showed reasonable use of the 0 ‐ 1 range (mean 0.60, range 0.0009 ‐ 0.975) and intuitive behaviour. Tested with field data from eight sites in New Zealand, it gave a good spread of values (mean 0.65, range across sites 0.34 ‐ 0.84), showed good ability to distinguish between the communities and its performance was ecologically intuitive. Illustrative correlations are made with mean annual temperature and soil fertility, determined by a bio‐assay. FD_var is recommended for general use.     

Effect of hydrogen on soil bacterial community structure in two soils as determined by terminal restriction fragment length polymorphism

The metabolism of hydrogen evolved from HUP^? legume nodules can alter bacterial community structures in the rhizosphere. Our earlier experiments demonstrated increased hydrogen uptake and appearance of white spots within bacterial colonies in H₂-treated soil. We were also able to isolate hydrogen-oxidizing bacteria from soil samples exposed to hydrogen, but not from samples exposed to air. To further understand the effect of hydrogen metabolism on soil microbial communities, in this study 16S rRNA terminal restriction fragment (TRF) profiles of different soil samples exposed to hydrogen gas under laboratory, greenhouse, and field conditions were analyzed. Relationships between soil bacterial community structures from hydrogen-treated soil samples and controls, illustrated by UPGMA (unpaired group mathematical averages) dendrograms, indicated a significant contribution of hydrogen metabolism to the variation in bacterial community. The intensity variation of TRF peaks includes both hydrogen-utilizing bacteria, whose growth were stimulated by hydrogen exposure, and other bacterial species whose growth was inhibited. Comparison of TRF profiles between laboratory and greenhouse samples showed that T-RFLP is a useful technique in the detection of root-related effects on soil bacterial community structure.     

Recovery of ciliate communities from an oligotrophic mountain stream after a catastrophic wind damage

We studied the recovery of ciliate taxoceonoses in a mountain stream after a catastrophic windstorm that strongly affected mountainous regions of Slovakia in 2004. To this end, we analyzed changes in the community structure of ciliate assemblages from the Zubrovica stream in the Low Tatra Mts during the time frame between 2003 and 2008 by various statistical methods, including diversity and similarity indices, hierarchical clustering, multidimensional scaling, and PCA. The climax ciliate communities were characteristic for oligotrophic running waters, i.e., they were equitable (E_H = 0.75–1.00) and harbored a comparatively low number of species (2–9) with typically low abundances (10–125 ind./ml). However, the community structure changed dramatically after the devastating windstorm. There was a significant increase of species number (10–30) and abundance (260–1480 ind./ml), concomitant with a decrease of the equitability (E_H = 0.43–0.83). These changed quantitative and qualitative community parameters wore off comparatively quickly, i.e., about six months after the catastrophic windstorm, the ciliate taxocoenoses had reached a community structure similar to that before the wind damage. The present observations and those from terrestrial habitats indicate that ciliate communities have a good capability to comparatively quickly reach a climax even after a strong disturbance.     

Non-Additive Effects of Genotypic Diversity Increase Floral Abundance and Abundance of Floral Visitors

Background

In the emerging field of community and ecosystem genetics, genetic variation and diversity in dominant plant species have been shown to play fundamental roles in maintaining biodiversity and ecosystem function. However, the importance of intraspecific genetic variation and diversity to floral abundance and pollinator visitation has received little attention.

Methodology/Principal Findings

Using an experimental common garden that manipulated genotypic diversity (the number of distinct genotypes per plot) of Solidago altissima, we document that genotypic diversity of a dominant plant can indirectly influence flower visitor abundance. Across two years, we found that 1) plant genotype explained 45% and 92% of the variation in flower visitor abundance in 2007 and 2008, respectively; and 2) plant genotypic diversity had a positive and non-additive effect on floral abundance and the abundance of flower visitors, as plots established with multiple genotypes produced 25% more flowers and received 45% more flower visits than would be expected under an additive model.

Conclusions/Significance

These results provide evidence that declines in genotypic diversity may be an important but little considered factor for understanding plant-pollinator dynamics, with implications for the global decline in pollinators due to reduced plant diversity in both agricultural and natural ecosystems.     

Effects of maternal genotypic identity and genetic diversity of the red mangrove Rhizophora mangle on associated soil bacterial communities: A field‐based experiment

Loss of plant biodiversity can result in reduced abundance and diversity of associated species with implications for ecosystem functioning. In ecosystems low in plant species diversity, such as Neotropical mangrove forests, it is thought that genetic diversity within the dominant plant species could play an important role in shaping associated communities. Here, we used a manipulative field experiment to study the effects of maternal genotypic identity and genetic diversity of the red mangrove Rhizophora mangle on the composition and richness of associated soil bacterial communities. Using terminal restriction fragment length polymorphism (T‐RFLP) community fingerprinting, we found that bacterial community composition differed among R. mangle maternal genotypes but not with genetic diversity. Bacterial taxa richness, total soil nitrogen, and total soil carbon were not significantly affected by maternal genotypic identity or genetic diversity of R. mangle. Our findings show that genotype selection in reforestation projects could influence soil bacterial community composition. Further research is needed to determine what impact these bacterial community differences might have on ecosystem processes, such as carbon and nitrogen cycling.     

Sediment microbial communities in Great Boiling Spring are controlled by temperature and distinct from water communities

Great Boiling Spring is a large, circumneutral, geothermal spring in the US Great Basin. Twelve samples were collected from water and four different sediment sites on four different dates. Microbial community composition and diversity were assessed by PCR amplification of a portion of the small subunit rRNA gene using a universal primer set followed by pyrosequencing of the V8 region. Analysis of 164 178 quality-filtered pyrotags clearly distinguished sediment and water microbial communities. Water communities were extremely uneven and dominated by the bacterium Thermocrinis. Sediment microbial communities grouped according to temperature and sampling location, with a strong, negative, linear relationship between temperature and richness at all taxonomic levels. Two sediment locations, Site A (87–80 °C) and Site B (79 °C), were predominantly composed of single phylotypes of the bacterial lineage GAL35 ($\widehat{p}$=36.1%), Aeropyrum ($\widehat{p}$=16.6%), the archaeal lineage pSL4 ($\widehat{p}$=15.9%), the archaeal lineage NAG1 ($\widehat{p}$=10.6%) and Thermocrinis ($\widehat{p}$=7.6%). The ammonia-oxidizing archaeon ‘Candidatus Nitrosocaldus'' was relatively abundant in all sediment samples <82 °C ($\widehat{p}$=9.51%), delineating the upper temperature limit for chemolithotrophic ammonia oxidation in this spring. This study underscores the distinctness of water and sediment communities in GBS and the importance of temperature in driving microbial diversity, composition and, ultimately, the functioning of biogeochemical cycles.     

Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes

Steep vertical gradients of oxidants (O₂ and NO₃⁻) in Puget Sound and Washington continental margin sediments indicate that aerobic respiration and denitrification occur within the top few millimeters to centimeters. To systematically explore the underlying communities of denitrifiers, Bacteria, and Archaea along redox gradients at distant geographic locations, nitrite reductase (nirS) genes and bacterial and archaeal 16S rRNA genes (rDNAs) were PCR amplified and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis. The suitablility of T-RFLP analysis for investigating communities of nirS-containing denitrifiers was established by the correspondence of dominant terminal restriction fragments (T-RFs) of nirS to computer-simulated T-RFs of nirS clones. These clones belonged to clusters II, III, and IV from the same cores and were analyzed in a previous study (G. Braker, J. Zhou, L. Wu, A. H. Devol, and J. M. Tiedje, Appl. Environ. Microbiol. 66:2096–2104, 2000). T-RFLP analysis of nirS and bacterial rDNA revealed a high level of functional and phylogenetic diversity, whereas the level of diversity of Archaea was lower. A comparison of T-RFLPs based on the presence or absence of T-RFs and correspondence analysis based on the frequencies and heights of T-RFs allowed us to group sediment samples according to the sampling location and thus clearly distinguish Puget Sound and the Washington margin populations. However, changes in community structure within sediment core sections during the transition from aerobic to anaerobic conditions were minor. Thus, within the top layers of marine sediments, redox gradients seem to result from the differential metabolic activities of populations of similar communities, probably through mixing by marine invertebrates rather than from the development of distinct communities.     

Influence of Soil Characteristics on the Diversity of Bacteria in the Southern Brazilian Atlantic Forest

The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Although the diversity of its fauna and flora has been studied fairly well, little is known of its microbial communities. In this work, we analyzed the Atlantic Forest ecosystem to determine its bacterial biodiversity, using 16S rRNA gene sequencing, and correlated changes in deduced taxonomic profiles with the physicochemical characteristics of the soil. DNAs were purified from soil samples, and the 16S rRNA gene was amplified to construct libraries. Comparison of 754 independent 16S rRNA gene sequences from 10 soil samples collected along a transect in an altitude gradient showed the prevalence of Acidobacteria (63%), followed by Proteobacteria (25.2%), Gemmatimonadetes (1.6%), Actinobacteria (1.2%), Bacteroidetes (1%), Chloroflexi (0.66%), Nitrospira (0.4%), Planctomycetes (0.4%), Firmicutes (0.26%), and OP10 (0.13%). Forty-eight sequences (6.5%) represented unidentified bacteria. The Shannon diversity indices of the samples varied from 4.12 to 3.57, indicating that the soils have a high level of diversity. Statistical analysis showed that the bacterial diversity is influenced by factors such as altitude, Ca²⁺/Mg²⁺ ratio, and Al³⁺ and phosphorus content, which also affected the diversity within the same lineage. In the samples analyzed, pH had no significant impact on diversity.The Brazilian Atlantic Forest is one of the 25 biodiversity hot spots in the world. Altogether, these hot spots contain more than 60% of the total terrestrial species of the planet (17). The Atlantic Forest is a dense ombrophilous forest with several variations, including coastal (3 to 50 m), submontane (50 to 500 m), montane (500 to 1,200 m), and high montane (1,200 to 1,400 m) forests, creating a vegetation gradient ranging from shrubs to well-developed montane forest (4). The Serra do Mar is a mountainous system that shelters the main remainder of the Atlantic Forest following the Brazilian east coast, from north to south along the coastal line, and it is divided into diverse sections of high and low blocks, which have regional denominations.The most important law-protected conservation area of the Brazilian Atlantic Forest is located in the Serra do Mar of the southern state of Paraná. This conservation area (∼5,000 km²) shelters 72% of the fauna and flora species that occur in Paraná and was declared a Biosphere Reserve by UNESCO in 1992. Much is known about the diversity of its fauna and flora, but little is known of its microbial diversity, particularly the soil microbial diversity and the soil characteristics that influence it.The soil microbial diversity is vast, and it is estimated that >99% of species remain unidentified (1, 28). Acidobacteria and Proteobacteria are the most abundant groups in soil (15). However, the Proteobacteria lineage is more diverse and stable than the Acidobacteria lineage, suggesting that the latter group is more susceptible to variation in soil properties and to disturbing factors (33). Seasonal, physical, and physicochemical factors can be relevant to the structure and diversity of microbial communities. For example, seasonal changes in vegetation and temperature led to replacement of dominant groups in a wheat field (25) and in grassland soils (16). The particle size also has an influence on the bacterial diversity of soils. The clay fraction has a more diverse bacterial community than do silt or sand fractions (23). Finally, analyses of communities from North and South American soils showed that pH plays a major role in bacterial diversity, with less diverse communities associated with a lower pH (9).Human activity can also change the microbial diversity of soils, both qualitatively and quantitatively. Analyses of microbial communities on coral atolls in the central Pacific Ocean under different degrees of human impact showed that the least-impacted atoll had autotrophs and heterotrophs equally distributed in the community, whereas the most-impacted atoll had a dominance of heterotrophs and about 10 times more microbial cells and virus-like particles in the water column, including a large percentage of potential pathogens (7). A comparison between bacterial communities in forest and pasture soil showed that there is a less diverse and more restricted community in pasture soils. The vegetation shift from forest to pasture resulted in changes to G+C% contents of soil bacterial DNA and amplified rRNA gene restriction analysis (ARDRA) profiles (18). Similar changes occurred with communities of soils submitted to agroindustrial treatments and pollutants (3, 30).In this work, we used a culture-independent approach based on 16S rRNA gene sequences to survey the bacterial community of the Atlantic Forest soils and determined the physicochemical factors affecting its bacterial biodiversity.