Robust estimation of microbial diversity in theory and in practice

Quantifying diversity is of central importance for the study of structure, function and evolution of microbial communities. The estimation of microbial diversity has received renewed attention with the advent of large-scale metagenomic studies. Here, we consider what the diversity observed in a sample tells us about the diversity of the community being sampled. First, we argue that one cannot reliably estimate the absolute and relative number of microbial species present in a community without making unsupported assumptions about species abundance distributions. The reason for this is that sample data do not contain information about the number of rare species in the tail of species abundance distributions. We illustrate the difficulty in comparing species richness estimates by applying Chao''s estimator of species richness to a set of in silico communities: they are ranked incorrectly in the presence of large numbers of rare species. Next, we extend our analysis to a general family of diversity metrics (‘Hill diversities''), and construct lower and upper estimates of diversity values consistent with the sample data. The theory generalizes Chao''s estimator, which we retrieve as the lower estimate of species richness. We show that Shannon and Simpson diversity can be robustly estimated for the in silico communities. We analyze nine metagenomic data sets from a wide range of environments, and show that our findings are relevant for empirically-sampled communities. Hence, we recommend the use of Shannon and Simpson diversity rather than species richness in efforts to quantify and compare microbial diversity.     

A New Framework to Accurately Quantify Soil Bacterial Community Diversity from DGGE

Denaturing gradient gel electrophoresis (DGGE) has been and remains extensively used to assess and monitor the effects of various treatments on soil bacterial communities. Considering only abundant phylotypes, the diversity estimates produced by this technique have been proven to be uncorrelated to true community diversity. The aim of this paper was to develop a framework to estimate a community’s true diversity from DGGE. Developed using in silico DGGE profiles generated from published pyrosequencing datasets, this framework elongates the rank-abundance distributions (RADs) drawn by band quantification using the peak-to-signal ratio (PSR) parameter, which was proven to be related to bacterial richness. The ability to compare DGGE-based diversity estimates to the true diversity of communities led to a unique opportunity to identify potential pitfalls when analyzing DGGE gels with commercial analysis software programs and gain insight into the process of DNA band clustering in the profiles. Bacterial diversity was compared through richness, Shannon, and Simpson’s 1/D indices. Intermediate results demonstrated that, even though commercial gel analysis software programs were unable to produce consistent results throughout all samples, a newly developed Matlab-based framework unraveled the dominance profiles of communities from band quantification. Elongating these partial RADs using the PSRs extracted from the DGGE profiles chiefly made it possible to accurately estimate the true diversity of communities. For all the samples analyzed, the estimated Shannon and Simpson’s 1/D were accurate at ±10 %. Richness estimations were less accurate, ranging from ?11 to 31 % of the expected values. The framework showed great potential to study the structure and diversity of soil bacterial communities.     

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.     

Diversity of geobacteraceae species inhabiting metal-polluted freshwater lake sediments ascertained by 16S rDNA analyses

The abundance, distribution, and phylogenetic diversity of members of the Fe(III)-reducing family Geobacteraceae were studied along a gradient of metal contaminants in Lake Coeur d’Alene, Idaho. Partial 16S rRNA gene fragments were amplified by PCR using primers directed toward conserved regions of the gene within the family Geobacteraceae. Analysis of amplicons separated by denaturing gradient gel electrophoresis (DGGE) suggested within-site variation was as great as between-site variation. Amplicons were cloned and grouped by RFLP type and DGGE migration distance and representatives were sequenced. Grouping clones with 3% or less sequence dissimilarity, 15 distinct phylotypes were identified compared to 16 distinct DGGE bands. Only 1 phylotype was recovered from all sites. This clone, B14, is most closely related to Geobacter metallireducens and constituted a greater portion of the pristine community than of the contaminated communities. A second phylotype, Q2, predominated in the contaminated communities and was notably absent from the pristine libraries. Clone Q2 presents a high degree of sequence similarity to two Geobacter spp. previously isolated from this region of Lake Coeur d’Alene. Six phylotypes were unique to the contaminated sediments, whereas two were found only in the pristine sediments. Indices of diversity (Shannon and Simpson) were consistently higher when calculated with DGGE data than when clone library data were used. Most-probable-number PCR and real-time PCR suggested that the Geobacteraceae phylotypes were spread relatively evenly across all three sites along the gradient. Our data indicate that the Geobacteraceae are diverse and abundant in Lake Coeur d’Alene sediments, regardless of metals content. These results provide insight into the ability of dissimilatory Fe(III)-reducing bacteria to colonize habitats with elevated metal concentrations, and they have important implications for the management and remediation of metal-contaminated sites.     

Community assessment techniques and the implications for rarefaction and extrapolation with Hill numbers

Diversity estimates play a key role in ecological assessments. Species richness and abundance are commonly used to generate complex diversity indices that are dependent on the quality of these estimates. As such, there is a long‐standing interest in the development of monitoring techniques, their ability to adequately assess species diversity, and the implications for generated indices. To determine the ability of substratum community assessment methods to capture species diversity, we evaluated four methods: photo quadrat, point intercept, random subsampling, and full quadrat assessments. Species density, abundance, richness, Shannon diversity, and Simpson diversity were then calculated for each method. We then conducted a method validation at a subset of locations to serve as an indication for how well each method captured the totality of the diversity present. Density, richness, Shannon diversity, and Simpson diversity estimates varied between methods, despite assessments occurring at the same locations, with photo quadrats detecting the lowest estimates and full quadrat assessments the highest. Abundance estimates were consistent among methods. Sample‐based rarefaction and extrapolation curves indicated that differences between Hill numbers (richness, Shannon diversity, and Simpson diversity) were significant in the majority of cases, and coverage‐based rarefaction and extrapolation curves confirmed that these dissimilarities were due to differences between the methods, not the sample completeness. Method validation highlighted the inability of the tested methods to capture the totality of the diversity present, while further supporting the notion of extrapolating abundances. Our results highlight the need for consistency across research methods, the advantages of utilizing multiple diversity indices, and potential concerns and considerations when comparing data from multiple sources.     

Assessment of microbial diversity bias associated with soil heterogeneity and sequencing resolution in pyrosequencing analyses

It is important to estimate the true microbial diversities accurately for a comparative microbial diversity analysis among various ecological settings in ecological models. Despite drastically increasing amounts of 16S rRNA gene targeting pyrosequencing data, sampling and data interpretation for comparative analysis have not yet been standardized. For more accurate bacterial diversity analyses, the influences of soil heterogeneity and sequence resolution on bacterial diversity estimates were investigated using pyrosequencing data of oak and pine forest soils with focus on the bacterial 16SrRNA gene. Soil bacterial community sets were phylogenetically clustered into two separate groups by forest type. Rarefaction curves showed that bacterial communities sequenced from the DNA mixtures and the DNAs of the soil mixtures hadmidsize richness compared with other samples. Richness and diversity estimates were highly variable depending on the sequence read numbers. Bacterial richness estimates (ACE, Chao 1 and Jack) of the forest soils had positive linear relationships with the sequence read number. Bacterial diversity estimates (NPShannon, Shannon and the inverse Simpson) of the forest soils were also positively correlated with the sequence read number. One-way ANOVA shows that sequence resolution significantly affected the a-diversity indices (P<0.05), but the soil heterogeneity did not (P>0.05). For an unbiased evaluation, richness and diversity estimates should be calculated and compared from subsets of the same size.     

A conceptual guide to measuring species diversity

Three metrics of species diversity – species richness, the Shannon index and the Simpson index – are still widely used in ecology, despite decades of valid critiques leveled against them. Developing a robust diversity metric has been challenging because, unlike many variables ecologists measure, the diversity of a community often cannot be estimated in an unbiased way based on a random sample from that community. Over the past decade, ecologists have begun to incorporate two important tools for estimating diversity: coverage and Hill diversity. Coverage is a method for equalizing samples that is, on theoretical grounds, preferable to other commonly used methods such as equal-effort sampling, or rarefying datasets to equal sample size. Hill diversity comprises a spectrum of diversity metrics and is based on three key insights. First, species richness and variants of the Shannon and Simpson indices are all special cases of one general equation. Second, richness, Shannon and Simpson can be expressed on the same scale and in units of species. Third, there is no way to eliminate the effect of relative abundance from estimates of any of these diversity metrics, including species richness. Rather, a researcher must choose the relative sensitivity of the metric towards rare and common species, a concept which we describe as ‘leverage.' In this paper we explain coverage and Hill diversity, provide guidelines for how to use them together to measure species diversity, and demonstrate their use with examples from our own data. We show why researchers will obtain more robust results when they estimate the Hill diversity of equal-coverage samples, rather than using other methods such as equal-effort sampling or traditional sample rarefaction.     

Detection of novel oral phylotypes associated with periodontitis

A phylogenetic approach based on 16S rRNA (rDNA) has been recently applied to investigate the diversity of cultivable and uncultivable species in the human oral cavity without cultivation. In a previous study [Sakamoto et al. (2000) Microbiol. Immunol. 44, 643-652], we identified a number of novel oral phylotypes, representing as yet uncultured organisms. The purpose of this study was to design specific PCR primers for five phylotypes AP12, AP21, AP24, AP50, and RP58, which are deeply branched particularly in the phylogenetic tree, and determine the prevalence of these phylotypes in 45 patients with periodontitis and 18 healthy subjects. The specificity of each primer was validated by the sequence analysis of PCR products obtained from saliva and subgingival plaque samples. Among phylotypes tested, phylotype AP24, which is closely related to oral clone DA014 reported previously [Paster et al. (2001) J. Bacteriol. 183, 3770-3783], was significantly associated with saliva and subgingival plaque samples from patients with periodontitis (P<0.01), but the difference was not statistically significant in the presence of other phylotypes. These data suggest that phylotype AP24 may play an important role in periodontal disease.     

Differential effects of abiotic factors and host plant traits on diversity and community composition of root-colonizing arbuscular mycorrhizal fungi in a salt-stressed ecosystem

Arbuscular mycorrhizal fungi (AMF) were investigated in roots of 18 host plant species in a salinized south coastal plain of Laizhou Bay, China. From 18 clone libraries of 18S rRNA genes, all of the 22 AMF phylotypes were identified into Glomus, of which 18 and 4 were classified in group A and B in the phylogenetic tree, respectively. The phylotypes related to morphologically defined Glomus species occurred generally in soil with higher salinity. AMF phylotype richness, Shannon index, and evenness were not significantly different between root samples from halophytes vs. non-halophytes, invades vs. natives, or annuals vs. perennials. However, AMF diversity estimates frequently differed along the saline gradient or among locations, but not among pH gradients. Moreover, UniFrac tests showed that both plant traits (salt tolerance, life style or origin) and abiotic factors (salinity, pH, or location) significantly affected the community composition of AMF colonizers. Redundancy and variation partitioning analyses revealed that soil salinity and pH, which respectively explained 6.9 and 4.2 % of the variation, were the most influential abiotic variables in shaping the AMF community structure. The presented data indicate that salt tolerance, life style, and origin traits of host species may not significantly affect the AMF diversity in roots, but do influence the community composition in this salinized ecosystem. The findings also highlight the importance of soil salinity and pH in driving the distribution of AMF in plant and soil systems.     

太白红杉群落物种多样性与环境因子的关系

定量研究了太白红杉群落物种多样性与环境因子的关系,结果表明:(1)海拔与物种丰富度和种间相遇机率呈极显著的负相关;与Shannon指数、Simpson指数和Pielou指数表现为"中间高度膨胀"的规律,即中等海拔高度上多样性高而低海拔和高海拔多样性较低。(2)岩石裸露度与物种丰富度呈显著的负相关。(3)土壤含水量与物种丰富度呈极显著的正相关;与种间相遇机率呈先降后升的趋势。(4)环境因子与物种多样性逐步回归结果是:海拔和岩石裸露度与物种丰富度关系密切,其回归方程为:S=90.62-0.02E-14.14B(r=0.769,P<0.05)。     

不同种植年限香榧根际土壤微生物多样性

为探明不同种植年限对香榧根际土壤微生物群落特征的影响,采用高通量测序技术,分析种植5 a、10 a和15 a香榧根际土壤细菌、真菌的群落结构和多样性特征.结果表明: 在种植15 a的香榧土壤中细菌Chao1指数、ACE指数和Shannon指数显著降低,Simpson指数无显著变化.NMDS分析显示,种植年限对细菌群落结构变化有显著影响,而种植5 a和10 a香榧林地土壤具有相似的细菌群落.细菌相对丰度、多样性以及群落结构(基本上由变形菌、放线菌、酸杆菌和绿弯菌组成)的变化与有机质、C/N、全氮呈极显著相关.香榧根际土壤真菌Chao1指数和ACE指数随种植年限的增加显著降低,Shannon指数和Simpson指数在种植10 a香榧林地中较高.真菌NMDS分析显示,相同种植年限土壤真菌群落聚在一起,不同种植年限之间能明显分开.真菌优势菌群主要包括子囊菌门、担子菌门、接合菌门.有机质是影响真菌丰富度、多样性和群落结构变化的主要因子.综上,香榧根际土壤微生物群落随种植年限不同而发生明显变化,种植年限、C/N、土壤全氮和有机质含量是影响香榧根际土壤微生物群落结构的主要因子.     

Analysis of the Rumen Bacterial Diversity under two Different Diet Conditions using Denaturing Gradient Gel Electrophoresis,Random Sequencing,and Statistical Ecology Approaches

Bacterial community structure and diversity in the rumen of steers in conditions of hay and corn diets was assessed by in vitro retrieval and analysis of the variable region (V3) of 16S rDNA. Two types of libraries were generated in this study: DGGE libraries, which further were analysed by excising, reamplification, and sequencing, and random shotgun sequence libraries. Phylogenetic and sequence similarity analyses of the resultant 68 clone sequences in DGGE libraries revealed the presence of 42 operational taxonomic units (OTUs) or phylotypes defined as having more than 97% of sequence similarity. One hundred and thirty four clone sequences in shotgun libraries were clustered into 72 phylotypes. The phylotype similarity, diversity, richness, and evenness in these libraries were estimated using a variety of diversity indices. In relation to diet, the corn-fed animals displayed more diverse and rich bacterial populations, which were mostly contributed by CFB-related phylotypes. Proteobacteria were also numerically prevalent on this diet (27%) but were represented by a few phylotypes thus diminishing the overall diversity and species richness values. On hay diet, the principal contributors to general diversity and species richness appeared to be low-G + C gram-positives. Although the ruminal Treponemaes were encountered only in hay-fed animals, their impact on species diversity on hay diet was low because of the limited number of phylotypes.     

Diversity measures in macroinvertebrate and zooplankton communities related to the trophic status of subtropical reservoirs: Contradictory or complementary responses?

Macroinvertebrate communities have been widely used as a tool for assessing the environmental quality of freshwater ecosystems, whereas zooplankton communities have been to some extent neglected. However, the importance of using different indicators to achieve a more comprehensive framework of assessment regarding water quality has been recognized. This study compared estimates of species richness (number of species) and the Shannon–Wiener index for data on macroinvertebrate and zooplankton communities in tropical reservoirs and related them to their trophic state. The trop+hic classification was obtained by applying the Carlson index (1977) modified by Toledo et al. (1983), and the index of the Brazilian Society of the Environmental Technology Agency. The comparative response of the different indicators was analyzed using a series of bivariate correlations (Draftsman’s plot). The results illustrate that diversity measures, namely species richness, responded differently when related to the trophic classification of reservoirs, depending on the community considered. The species richness of zooplankton was positively related to hypereutrophic conditions, due to the higher number of rotifer species, including tolerant generalist species and at the same time, as a result of the exclusion of species from other groups, whereas for macroinvertebrates, species richness was negatively related to hypereutrophic conditions. Melanoides tuberculatus, which exhibits a high tolerance and competitive ability under such conditions, was the dominant species in macroinvertebrate communities, which excluded endemic species and reduced local richness and diversity. The same indicators applied to the zooplankton and macroinvertebrate communities might therefore provide contradictory responses regarding ecological quality assessment in tropical reservoirs, which suggest that zooplankton should be taken into account among the biological quality elements considered in the ecological quality assessment, management, and restoration of water bodies.     

Biodiversity, Community Structural Shifts, and Biogeography of Prokaryotes within Antarctic Continental Shelf Sediment

16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region. Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions). The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs). At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes. At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria. Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core. Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.     

佛坪国家级自然保护区植物群落物种多样性特征

用物种多样性指数（Simpson指数和Shannon指数）、均匀度指数（Pielou均匀度）和物种丰富度,对佛坪国家级自然保护区主要高等植物群落物种多样性进行了测度。结果表明：森林群落的多样性程度高于灌丛和草甸,落叶阔叶林高于针叶林,栎林高于杨桦林。群落物种多样性有随海拔高度的升高而降低的趋势。物种多样性在森林群落垂直结构上的分布特点,表现为一般乔木层与灌木层接近但低于草本层。     

Chironomus plumosus larvae increase fluxes of denitrification products and diversity of nitrate-reducing bacteria in freshwater sediment

Benthic invertebrates affect microbial processes and communities in freshwater sediment by enhancing sediment-water solute fluxes and by grazing on bacteria. Using microcosms, the effects of larvae of the widespread midge Chironomus plumosus on the efflux of denitrification products (N₂O and N₂ + N₂O) and the diversity and abundance of nitrate- and nitrous-oxide-reducing bacteria were investigated. Additionally, the diversity of actively nitrate- and nitrous-oxide-reducing bacteria was analyzed in the larval gut. The presence of larvae increased the total effluxes of N₂O and N₂ + N₂O up to 8.6- and 4.2-fold, respectively, which was mostly due to stimulation of sedimentary denitrification; incomplete denitrification in the guts accounted for up to 20% of the N₂O efflux. Phylotype richness of the nitrate reductase gene narG was significantly higher in sediment with than without larvae. In the gut, 47 narG phylotypes were found expressed, which may contribute to higher phylotype richness in colonized sediment. In contrast, phylotype richness of the nitrous oxide reductase gene nosZ was unaffected by the presence of larvae and very few nosZ phylotypes were expressed in the gut. Gene abundance of neither narG, nor nosZ was different in sediments with and without larvae. Hence, C. plumosus increases activity and diversity, but not overall abundance of nitrate-reducing bacteria, probably by providing additional ecological niches in its burrow and gut.     

Mechanical soil disturbance as a determinant of arbuscular mycorrhizal fungal communities in semi-natural grassland

While the effect of disturbance on overall abundance and community composition of arbuscular mycorrhizal (AM) fungi has been researched in agricultural fields, less is known about the impact in semi-natural grasslands. We sampled two AM plant species, Festuca brevipila and Plantago lanceolata, from an ongoing grassland restoration experiment that contained replicated plowed and control plots. The AM fungal community in roots was determined using nested PCR and LSU rDNA primers. We identified 38 phylotypes within the Glomeromycota, of which 29 belonged to Glomus A, six to Glomus B, and three to Diversisporaceae. Only three phylotypes were closely related to known morphospecies. Soil disturbance significantly reduced phylotype richness and changed the AM fungal community composition. Most phylotypes, even closely related ones, showed little or no overlap in their distribution and occurred in either the control or disturbed plots. We found no evidence of host preference in this system, except for one phylotype that preferentially seemed to colonize Festuca. Our results show that disturbance imposed a stronger structuring force for AM fungal communities than did host plants in this semi-natural grassland.     

When species accumulation curves intersect: implications for ranking diversity using small samples

Diversity in biological communities frequently is compared using species accumulation curves, plotting observed species richness versus sample size. When species accumulation curves intersect, the ranking of communities by observed species richness depends on sample size, creating inconsistency in comparisons of diversity. We show that species accumulation curves for two communities are expected to intersect when the community with lower actual species richness has higher Simpson diversity (probability that two random individuals belong to different species). This may often occur when comparing communities that differ in habitat heterogeneity or disturbance, as we illustrate using data from neotropical butterflies. In contrast to observed species richness, estimated Simpson diversity always produces a consistent expected ranking among communities across sample sizes, with the statistical accuracy to confidently rank communities using small samples. Simpson diversity should therefore be particularly useful in rapid assessments to prioritize areas for conservation.     

Community Dynamics of Arbuscular Mycorrhizal Fungi in High-Input and Intensively Irrigated Rice Cultivation Systems

Application of a mycorrhizal inoculum could be one way to increase the yield of rice plants and reduce the application of fertilizer. We therefore studied arbuscular mycorrhizal fungi (AMF) in the roots of wetland rice (Oryza sativa L.) collected at the seedling, tillering, heading, and ripening stages in four paddy wetlands that had been under a high-input and intensively irrigated rice cultivation system for more than 20 years. It was found that AMF colonization was mainly established in the heading and ripening stages. The AMF community structure was characterized in rhizosphere soils and roots from two of the studied paddy wetlands. A fragment covering the partial small subunit (SSU), the whole internal transcribed spacer (ITS), and the partial large subunit (LSU) rRNA operon regions of AMF was amplified, cloned, and sequenced from roots and soils. A total of 639 AMF sequences were obtained, and these were finally assigned to 16 phylotypes based on a phylogenetic analysis, including 12 phylotypes from Glomeraceae, one phylotype from Claroideoglomeraceae, two phylotypes from Paraglomeraceae, and one unidentified phylotype. The AMF phylotype compositions in the soils were similar between the two surveyed sites, but there was a clear discrepancy between the communities obtained from root and soil. The relatively high number of AMF phylotypes at the surveyed sites suggests that the conditions are suitable for some species of AMF and that they may have an important function in conventional rice cultivation systems. The species richness of root-colonizing AMF increased with the growth of rice, and future studies should consider the developmental stages of this crop in the exploration of AMF function in paddy wetlands.