Estimating Bacterial Diversity for Ecological Studies: Methods,Metrics, and Assumptions

Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques.     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

Comparing the hyper-variable V4 and V9 regions of the small subunit rDNA for assessment of ciliate environmental diversity

The hyper-variable V4 and V9 regions of the small subunit (SSU) rDNA have been targeted for assessing environmental diversity of microbial eukaryotes using next generation sequencing technologies. Here, we explore how the genetic distances among these short fragments compare with the distances obtained from near full-length SSU-rDNA sequences by comparing all pairwise estimates, as well as within and among species of ciliates. Results show that pairwise distances from V4 more closely match the near full-length SSU-rDNA and are more comparable with previous studies based on much longer SSU-rDNA fragments, then pairwise distances from V9. Thus, studies that use the V4 will estimate similar values of phylotype richness and community structure as would have been estimated using the full-length SSU-rDNA.     

中国狼不同地理种群遗传多样性及系统进化分析

为了解中国狼不同地理种群遗传多样性及系统发育情况,从中国境内狼的主要分布区青海、新疆、内蒙古和吉林4个地区采集样品,用分子生物学技术手段成功地获得44个个体线粒体DNA控制区第一高变区(HVRⅠ)序列和40个线粒体Cyt b部分序列。线粒体控制区HVRⅠ共检测到51个变异位点,位点变异率为8.76%;线粒体Cyt b部分序列发现31个变异位点,位点变异率为5.33%,未见插入及缺失现象,变异类型全部为碱基置换。共定义了16个线粒体HVRⅠ单倍型,其中吉林与内蒙种群存在共享单倍型,估计这两地间种群亲缘关系较近。4个地理种群中新疆种群拥有较高的遗传多样性(0.94)。中国狼种群总体平均核苷酸多态性为2.27%,与世界其他国家地区相比,中国狼种群拥有相对较高的遗传多样性。通过线粒体HVRⅠ单倍型构建的系统进化树可以看出,中国狼在进化上分为2大支,其中位于青藏高原的青海种群独立为一支,推测其可能长期作为独立种群进化。基于青海种群与新疆,内蒙种群的线粒体Cyt b遗传距离,推测中国狼2个世系可能在更新世冰川时期青藏高原受地质作用急速隆起后出现分歧,分歧时间大约在1.1 MY前。     

Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.Culture-independent 16S rRNA gene surveys are now routinely utilized to examine the microbial diversity in various environmental habitats. However, in surveys of highly diverse ecosystems, the size of clone libraries typically constructed (100 to 500 clones) allows for the identification only of members of the community that are present in high abundance (2, 13, 14, 17, 24, 51). In addition to the failure to detect the rare members of the ecosystem, these relatively small datasets provide inaccurate estimates when used for computing species richness within an ecosystem. Regardless of the approach utilized to estimate species richness, the estimates obtained are highly dependent on sample size, and smaller datasets typically result in the underestimation of species richness (14, 44, 47, 55).The use of a pyrosequencing-based approach (40) in 16S gene-based diversity surveys promises to overcome both of the above-mentioned problems associated with inadequate sampling. The large number of 16S rRNA gene sequences produced (hundreds of thousands) allows access to rare members of the community (25; J. M. Tiedje, presented at the 108th General Meeting of the American Society for Microbiology, Boston, MA, 2008), as well as a relatively more accurate estimation of species richness. However, with the introduction of this new technology, it is necessary to correlate the results obtained from newer pyrosequencing-based surveys to the extensive collection of longer, capillary sequence-generated 16S rRNA gene sequences that has been deposited in public databases during the last 2 decades. Several recent studies have examined the utility of pyrosequencing fragments in providing an accurate survey of overall community structure (36) and investigated the ability of various fragments spanning the 16S rRNA gene to accurately predict the phylogenetic affiliation of pyrosequencing-generated fragments at various taxonomic cutoffs (35, 54). As such, these admirable efforts gave useful insights into the advantages and limitations of the pyrosequencing approach in 16S-based community surveys, pinpointed specific regions that provide better phylogenetic resolution than other pyrosequencing-generated regions, and provided a quantitative assessment of binning accuracy at various empirical cutoffs.However, while issues regarding correlating phylogenies of shorter and longer fragments are actively being addressed, efforts to calibrate species richness data obtained from various pyrosequencing fragments at various taxonomic cutoffs to estimates obtained using longer 16S rRNA gene fragments are still lacking. It is unclear how pairwise distances and, hence, operational taxonomic unit (OTU) assignments and species richness estimates computed using various shorter fragments spanning various regions of the 16S rRNA gene will correlate to pairwise distances computed using the nearly complete 16S rRNA gene. Elucidating such differences between shorter and nearly complete fragments, as well as between shorter fragments representing different regions in the 16S rRNA gene, is absolutely necessary for accurate meta-analysis of species richness in previously published and future datasets constructed using various sequencing approaches.Here, we constructed, sequenced, and analyzed a 16S rRNA library of 1,132 clones generated from an undisturbed tallgrass prairie soil in central Oklahoma and compared the numbers of OTUs and species richness values obtained using the full-length data sets (with and without the application of the Lane mask filter that excludes hypervariable regions from the phylogenetic analysis) (32) and fragments simulating pyrosequencing output generated by clipping where known conserved bacterial primers are encountered in the 16S rRNA gene. The lengths of the chosen simulated-pyrosequencing fragments represent amplicons that have been generated using the original GS20 pyrosequencing platform (≈100 bp) (25, 44, 48), similar to those currently being generated using the GS FLX pyrosequencing platform (≈250 bp) (1, 20, 35) or amplicons produced using the anticipated increase in the new GS XLR pyrosequencing platform (>250 bp). We show that the choice of the pyrosequenced fragment could indeed impact the number of OTUs calculated at different taxonomic cutoffs, with some fragments underestimating and others overestimating such parameters compared to the results with longer, nearly complete 16S rRNA gene fragments. We also show that even more marked differences could be encountered when comparing two pyrosequencing fragments within the same molecule. Further, we established a regression analysis that explains the nature of the observed discrepancies using the proportions of the hypervariable, variable, and conserved bases within fragments.     

Sequence of the Small Subunit Ribosomal RNA Gene Expressed in the Bloodstream Stages of Plasmodium berghei: Evolutionary Implications1

ABSTRACT. We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene expressed by bloodstream stages of the apicomplexan Plasmodium berghei. It is 2059 nucleotides long. Elements contributing to its relatively large size are all concentrated in regions known to be variable in length among eukaryotes. In a phylogenetic tree constructed from pairwise comparisons of eukaryotic small subunit rRNA sequences, the apicomplexan line branches at a rather early point in eukaryotic evolution before any multicellular kingdoms had yet appeared.     

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.     

Streptococcus suis serotypes characterized by analysis of chaperonin 60 gene sequences

Streptococcus suis is an important pathogen of swine which occasionally infects humans as well. There are 35 serotypes known for this organism, and it would be desirable to develop rapid methods methods to identify and differentiate the strains of this species. To that effect, partial chaperonin 60 gene sequences were determined for the 35 serotype reference strains of S. suis. Analysis of a pairwise distance matrix showed that the distances ranged from 0 to 0.275 when values were calculated by the maximum-likelihood method. For five of the strains the distances from serotype 1 were greater than 0.1, and for two of these strains the distances were were more than 0.25, suggesting that they belong to a different species. Most of the nucleotide differences were silent; alignment of protein sequences showed that there were only 11 distinct sequences for the 35 strains under study. The chaperonin 60 gene phylogenetic tree was similar to the previously published tree based on 16S rRNA sequences, and it was also observed that strains with identical chaperonin 60 gene sequences tended to have identical 16S rRNA sequences. The chaperonin 60 gene sequences provided a higher level of discrimination between serotypes than the 16S RNA sequences provided and could form the basis for a diagnostic protocol.     

Sequence of the small subunit ribosomal RNA gene expressed in the bloodstream stages of Plasmodium berghei: evolutionary implications

We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene expressed by bloodstream stages of the apicomplexan Plasmodium berghei. It is 2059 nucleotides long. Elements contributing to its relatively large size are all concentrated in regions known to be variable in length among eukaryotes. In a phylogenetic tree constructed from pairwise comparisons of eukaryotic small subunit rRNA sequences, the apicomplexan line branches at a rather early point in eukaryotic evolution before any multicellular kingdoms had yet appeared.     

Streptococcus suis Serotypes Characterized by Analysis of Chaperonin 60 Gene Sequences

Streptococcus suis is an important pathogen of swine which occasionally infects humans as well. There are 35 serotypes known for this organism, and it would be desirable to develop rapid methods methods to identify and differentiate the strains of this species. To that effect, partial chaperonin 60 gene sequences were determined for the 35 serotype reference strains of S. suis. Analysis of a pairwise distance matrix showed that the distances ranged from 0 to 0.275 when values were calculated by the maximum-likelihood method. For five of the strains the distances from serotype 1 were greater than 0.1, and for two of these strains the distances were were more than 0.25, suggesting that they belong to a different species. Most of the nucleotide differences were silent; alignment of protein sequences showed that there were only 11 distinct sequences for the 35 strains under study. The chaperonin 60 gene phylogenetic tree was similar to the previously published tree based on 16S rRNA sequences, and it was also observed that strains with identical chaperonin 60 gene sequences tended to have identical 16S rRNA sequences. The chaperonin 60 gene sequences provided a higher level of discrimination between serotypes than the 16S RNA sequences provided and could form the basis for a diagnostic protocol.     

Evolutionary processes,dispersal limitation and climatic history shape current diversity patterns of European dragonflies

We investigated the effects of contemporary and historical factors on the spatial variation of European dragonfly diversity. Specifically, we tested to what extent patterns of endemism and phylogenetic diversity of European dragonfly assemblages are structured by 1) phylogenetic conservatism of thermal adaptations and 2) differences in the ability of post‐glacial recolonization by species adapted to running waters (lotic) and still waters (lentic). We investigated patterns of dragonfly diversity using digital distribution maps and a phylogeny of 122 European dragonfly species, which we constructed by combining taxonomic and molecular data. We calculated total taxonomic distinctiveness and mean pairwise distances across 4192 50 × 50 km equal‐area grid cells as measures of phylogenetic diversity. We compared species richness with corrected weighted endemism and standardized effect sizes of mean pairwise distances or residuals of total taxonomic distinctiveness to identify areas with higher or lower phylogenetic diversity than expected by chance. Broken‐line regression was used to detect breakpoints in diversity–latitude relationships. Dragonfly species richness peaked in central Europe, whereas endemism and phylogenetic diversity decreased from warm areas in the south‐west to cold areas in the north‐east and with an increasing proportion of lentic species. Except for species richness, all measures of diversity were consistently higher in formerly unglaciated areas south of the 0°C isotherm during the Last Glacial Maximum than in formerly glaciated areas. These results indicate that the distributions of dragonfly species in Europe were shaped by both phylogenetic conservatism of thermal adaptations and differences between lentic and lotic species in the ability of post‐glacial recolonization/dispersal in concert with the climatic history of the continent. The complex diversity patterns of European dragonflies provide an example of how integrating climatic and evolutionary history with contemporary ecological data can improve our understanding of the processes driving the geographical variation of biological diversity.     

Identification,genetic variation,and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar

Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.     

RNA secondary structure, an important bioinformatics tool to enhance multiple sequence alignment: a case study (Sordariomycetes, Fungi)

In a case study of fungi of the class Sordariomycetes, we evaluated the effect of multiple sequence alignment (MSA) on the reliability of the phylogenetic trees, topology and confidence of major phylogenetic clades. We compared two main approaches for constructing MSA based on (1) the knowledge of the secondary (2D) structure of ribosomal RNA (rRNA) genes, and (2) automatic construction of MSA by four alignment programs characterized by different algorithms and evaluation methods, CLUSTAL, MAFFT, MUSCLE, and SAM. In the primary fungal sequences of the two functional rRNA genes, the nuclear small and large ribosomal subunits (18 S and 28 S), we identified four and six, respectively, highly variable regions, which correspond mainly to hairpin loops in the 2D structure. These loops are often positioned in expansion segments, which are missing or are not completely developed in the Archaeal and Eubacterial kingdoms. Proper sorting of these sites was a key for constructing an accurate MSA. We utilized DNA sequences from 28 S as an example for one-gene analysis. Five different MSAs were created and analyzed with maximum parsimony and maximum likelihood methods. The phylogenies inferred from the alignments improved with 2D structure with identified homologous segments, and those constructed using the MAFFT alignment program, with all highly variable regions included, provided the most reliable phylograms with higher bootstrap support for the majority of clades. We illustrate and provide examples demonstrating that re-evaluating ambiguous positions in the consensus sequences using 2D structure and covariance is a promising means in order to improve the quality and reliability of sequence alignments.     

Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (P > 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.     

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (P > 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.     

A secondary structural model of the 28S rRNA expansion segments D2 and D3 for Chalcidoid wasps (Hymenoptera: Chalcidoidea)

We analyze the secondary structure of two expansion segments (D2, D3) of the 28S ribosomal (rRNA)-encoding gene region from 527 chalcidoid wasp taxa (Hymenoptera: Chalcidoidea) representing 18 of the 19 extant families. The sequences are compared in a multiple sequence alignment, with secondary structure inferred primarily from the evidence of compensatory base changes in conserved helices of the rRNA molecules. This covariation analysis yielded 36 helices that are composed of base pairs exhibiting positional covariation. Several additional regions are also involved in hydrogen bonding, and they form highly variable base-pairing patterns across the alignment. These are identified as regions of expansion and contraction or regions of slipped-strand compensation. Additionally, 31 single-stranded locales are characterized as regions of ambiguous alignment based on the difficulty in assigning positional homology in the presence of multiple adjacent indels. Based on comparative analysis of these sequences, the largest genetic study on any hymenopteran group to date, we report an annotated secondary structural model for the D2, D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related apocritan taxa.     

Genome-Wide Comparisons of Phylogenetic Similarities between Partial Genomic Regions and the Full-Length Genome in Hepatitis E Virus Genotyping

Besides the complete genome, different partial genomic sequences of Hepatitis E virus (HEV) have been used in genotyping studies, making it difficult to compare the results based on them. No commonly agreed partial region for HEV genotyping has been determined. In this study, we used a statistical method to evaluate the phylogenetic performance of each partial genomic sequence from a genome wide, by comparisons of evolutionary distances between genomic regions and the full-length genomes of 101 HEV isolates to identify short genomic regions that can reproduce HEV genotype assignments based on full-length genomes. Several genomic regions, especially one genomic region at the 3′-terminal of the papain-like cysteine protease domain, were detected to have relatively high phylogenetic correlations with the full-length genome. Phylogenetic analyses confirmed the identical performances between these regions and the full-length genome in genotyping, in which the HEV isolates involved could be divided into reasonable genotypes. This analysis may be of value in developing a partial sequence-based consensus classification of HEV species.