DNA barcoding the ichthyofauna of the Yangtze River: Insights from the molecular inventory of a mega‐diverse temperate fauna

Intensification of inland fisheries and aquatic landscape conversion led to a drastic decline of fish populations in the Yangtze River (YR) during the last decades. This situation urges for the development of a large‐scale molecular assessment of YR ichthyofauna to further develop standardized methods of molecular identification for conservation and fisheries management purposes. We present here the results of a large‐scale campaign to DNA barcode YR freshwater fishes that succeeded in producing 1,424 new DNA barcodes for 123 species. Together with 1,406 sequences mined from BOLD and GenBank, a reference library including 2,830 DNA barcodes for 238 species was compiled. By using four DNA‐based species delimitation methods, RESL, ABGD, mPTP and mGMYC, 230 operational taxonomic units (OTUs) were identified and 195 species displayed OTUs that tightly match species boundaries. No barcoding gap was observed; however, and conflicting cases of species and OTU delimitation were identified. A total of 23 species with maximum intraspecific distances above 2% were detected and null genetic distances to the nearest phylogenetic relatives were detected in 11 species. Among those 23 species, 16 were represented by multiple OTUs amounting to 40 OTUs delineated. Several cases of multiple OTUs confined to species boundaries were detected suggesting the presence of overlooked species. A total of 18 OTUs, however, were shared by several species and particularly so for the Qinghai‐Tibet plateau endemic species. These results are discussed with reference to previous large‐scale DNA barcoding campaign and compared to previous phylogeographic studies in the YR.     

Algorithmic single‐locus species delimitation: effects of sampling effort,variation and nonmonophyly in four methods and 1870 species of beetles

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode‐based OTU‐picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.     

DNA barcode‐based delineation of putative species: efficient start for taxonomic workflows

The analysis of DNA barcode sequences with varying techniques for cluster recognition provides an efficient approach for recognizing putative species (operational taxonomic units, OTUs). This approach accelerates and improves taxonomic workflows by exposing cryptic species and decreasing the risk of synonymy. This study tested the congruence of OTUs resulting from the application of three analytical methods (ABGD, BIN, GMYC) to sequence data for Australian hypertrophine moths. OTUs supported by all three approaches were viewed as robust, but 20% of the OTUs were only recognized by one or two of the methods. These OTUs were examined for three criteria to clarify their status. Monophyly and diagnostic nucleotides were both uninformative, but information on ranges was useful as sympatric sister OTUs were viewed as distinct, while allopatric OTUs were merged. This approach revealed 124 OTUs of Hypertrophinae, a more than twofold increase from the currently recognized 51 species. Because this analytical protocol is both fast and repeatable, it provides a valuable tool for establishing a basic understanding of species boundaries that can be validated with subsequent studies.     

One species in eight: DNA barcodes from type specimens resolve a taxonomic quagmire

Each holotype specimen provides the only objective link to a particular Linnean binomen. Sequence information from them is increasingly valuable due to the growing usage of DNA barcodes in taxonomy. As type specimens are often old, it may only be possible to recover fragmentary sequence information from them. We tested the efficacy of short sequences from type specimens in the resolution of a challenging taxonomic puzzle: the Elachista dispunctella complex which includes 64 described species with minuscule morphological differences. We applied a multistep procedure to resolve the taxonomy of this species complex. First, we sequenced a large number of newly collected specimens and as many holotypes as possible. Second, we used all >400 bp examine species boundaries. We employed three unsupervised methods (BIN, ABGD, GMYC) with specified criteria on how to handle discordant results and examined diagnostic bases from each delineated putative species (operational taxonomic units, OTUs). Third, we evaluated the morphological characters of each OTU. Finally, we associated short barcodes from types with the delineated OTUs. In this step, we employed various supervised methods, including distance‐based, tree‐based and character‐based. We recovered 658 bp barcode sequences from 194 of 215 fresh specimens and recovered an average of 141 bp from 33 of 42 holotypes. We observed strong congruence among all methods and good correspondence with morphology. We demonstrate potential pitfalls with tree‐, distance‐ and character‐based approaches when associating sequences of varied length. Our results suggest that sequences as short as 56 bp can often provide valuable taxonomic information. The results support significant taxonomic oversplitting of species in the Elachista dispunctella complex.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTClust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets — even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.     

基于16S rRNA基因序列分析1例健康人龈上菌群

目的构建细菌16S rRNA基因文库分析健康人龈上菌群的组成。方法取1例健康成年女性龈上菌斑并构建细菌16S rRNA基因文库,分析其龈上菌群组成。结果 1例健康人龈上菌斑细菌的种水平分类有62种,其中可以培养的细菌有34种而尚无法培养的细菌有28种(45.1%);新发现的细菌物种有17种(27.4%);缓症链球菌是克隆子数最多的优势菌种;链球菌属、奈氏菌属和嗜血杆菌属占文库的60%,为主要菌群;构建的细菌16S rRNA基因文库的覆盖率为95%,文库的均匀度值为0.016。结论 1例健康人龈上菌群中45.1%的细菌尚无法分离培养、27.4%的物种尚不清楚,未培养菌及尚不清楚的菌种中可能藏匿着与口腔疾病密切相关的致病菌,全面地了解健康人龈上菌群的组成有助于研究龋病的发病机制。     

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

Influence of antimicrobial feed additives on broiler commensal posthatch gut microbiota development and performance

The effects of avilamycin, zinc bacitracin, and flavophospholipol on broiler gut microbial community colonization and bird performance in the first 17 days posthatch were investigated. Significant differences in gut microbiota associated with gut section, dietary treatment, and age were identified by terminal restriction fragment length polymorphism (T-RFLP), although no performance-related differences between dietary treatments were detected. Similar age-related shifts in the gut microbiota were identified regardless of diet but varied between the ilea and ceca. Interbird variabilities in ileal bacterial communities were reduced (3 to 7 days posthatch) in chicks fed with feed containing antimicrobial agents. Avilamycin and flavophospholipol had the most consistent effect on gut microbial communities. Operational taxonomic units (OTU) linked to changes in gut microbiota in birds on antimicrobial-supplemented diets were characterized and identified. Some OTUs could be identified to the species level; however, the majority could be only tentatively classified to the genus, family, order, or domain level. OTUs 140 to 146 (Lachnospiraceae), OTU 186/188 (Lactobacillus johnsonii), OTU 220 (Lachnospiraceae), OTUs 284 to 288 (unclassified bacterial spp. or Ruminococcaceae), OTU 296/298 (unclassified bacterium or Clostridiales), and OTU 480/482 (Oxalobacteraceae) were less prevalent in the guts of chicks fed antimicrobial-supplemented diets. OTU 178/180 (Lactobacillus crispatus), OTU 152 (Lactobacillus reuteri or unclassified Clostridiales), OTU 198/200 (Subdoligranulum spp.), and OTU 490/492 (unclassified bacterium or Enterobacteriaceae) were less prevalent in the gut of chicks raised on the antimicrobial-free diet. The identification of key bacterial species influenced by antimicrobial-supplemented feed immediately posthatch may assist in the formulation of diets that facilitate beneficial gut microbial colonization and, hence, the development of alternatives to current antimicrobial agents in feed for sustainable poultry production.     

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly 62% of all DNA sequences analyzed. Other phylogenetic groups identified included Proteobacteria (20%), Actinobacteria (9%), Cyanobacteria (4%), and Bacteroidetes (2%). The composition of RNA-based libraries (1122 sequences) was similar to the DNA-based libraries with a few notable exceptions: Proteobacteria were more dominant in the RNA clone libraries (i.e., 35% RNA; 20% DNA). Differences in the Proteobacteria composition were also observed; alpha-Proteobacteria was 22 times more abundant in the RNA-based clones while beta-Proteobacteria was eight times more abundant in the DNA libraries. Nearly twice as many DNA operational taxonomic units (OTUs) than RNA OTUs were observed at distance 0.03 (101 DNA; 53 RNA). Twenty-four OTUs were shared between all RNA- and DNA-based libraries (OTU_0.03) representing only 18% of the total OTUs, but 81% (1527/1883) of all sequences. Such differences between clone libraries demonstrate the necessity of generating both RNA- and DNA-derived clone libraries to compare these two different molecular approaches for community analyses.     

Fungal community composition in the gut of rove beetles (Coleoptera: Staphylinidae) from the Canadian boreal forest reveals possible endosymbiotic interactions for dietary needs

The goal of this study was to investigate the fungal community composition in the gut of Staphylinidae from boreal forest in order to better understand the diversity and the complexity of fungus-insect relationships. DNA gut content analyses of nine abundant rove beetle species (Coleoptera, Staphylinidae) living in the boreal balsam fir forest ecosystem (Montmorency Forest, Quebec, Canada) were performed to identify the fungal taxa present either as endosymbiotic taxa or as a source of nutrition. A total of 42 fungal operational taxonomic units (OTUs) were recorded from the analysis of 441 fungal ITS rDNA sequences recovered from gut extracts. The OTU richness per species ranged between four in Tachinus quebecensis and 16 in Atheta ventricosa. The fungal mycobiota in posterior gut extracts was dominated by Saccharomycetales (12 OTUs), followed by Sordariomycetes (nine OTUs). No significant difference was observed between the OTU richness recorded within each of the three subfamilies of rove beetles investigated. The core mycobiome of the posterior gut extracts was dominated by three OTUs related to yeasts, with ITS sequences having pairwise similarities equal to or greater than 99% with Candida mesenterica, Debaryomyces spp. and Ophiostoma pluriannulatum. These results provide some evidence of the consumer-resource relationships of these beetles. Predominance of yeast and fungal spores in the posterior gut of rove beetles suggests that they may play an important role in their dietary requirements and as endosymbionts.     

Molecular identification of ectomycorrhizal mycelium in soil horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (> or = 99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had > or = 98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods

Metabarcoding has the potential to become a rapid, sensitive, and effective approach for identifying species in complex environmental samples. Accurate molecular identification of species depends on the ability to generate operational taxonomic units (OTUs) that correspond to biological species. Due to the sometimes enormous estimates of biodiversity using this method, there is a great need to test the efficacy of data analysis methods used to derive OTUs. Here, we evaluate the performance of various methods for clustering length variable 18S amplicons from complex samples into OTUs using a mock community and a natural community of zooplankton species. We compare analytic procedures consisting of a combination of (1) stringent and relaxed data filtering, (2) singleton sequences included and removed, (3) three commonly used clustering algorithms (mothur, UCLUST, and UPARSE), and (4) three methods of treating alignment gaps when calculating sequence divergence. Depending on the combination of methods used, the number of OTUs varied by nearly two orders of magnitude for the mock community (60–5068 OTUs) and three orders of magnitude for the natural community (22–22191 OTUs). The use of relaxed filtering and the inclusion of singletons greatly inflated OTU numbers without increasing the ability to recover species. Our results also suggest that the method used to treat gaps when calculating sequence divergence can have a great impact on the number of OTUs. Our findings are particularly relevant to studies that cover taxonomically diverse species and employ markers such as rRNA genes in which length variation is extensive.     

Disparities in second‐generation DNA metabarcoding results exposed with accessible and repeatable workflows

Different second‐generation sequencing technologies may have taxon‐specific biases when DNA metabarcoding prey in predator faeces. Our major objective was to examine differences in prey recovery from bat guano across two different sequencing workflows using the same faecal DNA extracts. We compared results between the Ion Torrent PGM and the Illumina MiSeq with similar library preparations and the same analysis pipeline. We focus on repeatability and provide an R Notebook in an effort towards transparency for future methodological improvements. Full documentation of each step enhances the accessibility of our analysis pipeline. We tagged DNA from insectivorous bat faecal samples, targeted the arthropod cytochrome c oxidase I minibarcode region and sequenced the product on both second‐generation sequencing platforms. We developed an analysis pipeline with a high operational taxonomic unit (OTU) clustering threshold (i.e., ≥98.5%) followed by copy number filtering to avoid merging rare but genetically similar prey into the same OTUs. With this workflow, we detected 297 unique prey taxa, of which 74% were identified at the species level. Of these, 104 (35%) prey OTUs were detected by both platforms, 176 (59%) OTUs were detected by the Illumina MiSeq system only, and 17 (6%) OTUs were detected using the Ion Torrent system only. Costs were similar between platforms but the Illumina MiSeq recovered six times more reads and four additional insect orders than did Ion Torrent. The considerations we outline are particularly important for long‐term ecological monitoring; a more standardized approach will facilitate comparisons between studies and allow faster recognition of changes within ecological communities.     

Characterization and identification of productivity-associated rhizobacteria in wheat

The rhizosphere is populated by a numerous and diverse array of rhizobacteria, and many impact productivity in largely unknown ways. Here we characterize the rhizobacterial community in a wheat variety categorized according to shoot biomass using 16S rRNA pyrosequencing abundance data. Plants were grown in homogenized field soil under greenhouse conditions, and DNA was extracted and pyrosequenced, resulting in 29,007 quality sequences. Operational taxonomic units (OTUs) that were significantly associated with biomass productivity were identified using an exact test adjusted for the false-discovery rate. The productivity deviation expressed as a percentage of the total mean square for regression (PMSR) was determined for each OTU. Out of 719 OTUs, 42 showed significant positive associations and 39 showed significant negative associations (q value, ≤0.05). OTUs with the greatest net positive associations, by genus, were as follows: Duganella, OTU 43 and OTU 3; Janthinobacterium, OTU 278; Pseudomonas, OTU 588; and Cellvibrio, OTU 1847. Those with negative associations were as follows: Bacteria, OTU 273; Chryseobacterium, OTU 508; Proteobacteria, OTU 249; and Enterobacter, OTU 357. Shoot biomass productivity was strongly correlated with the balance between the overall abundances of positive- and negative-productivity-associated OTUs. High-productivity rhizospheres contained 9.2 significant positives for every negatively associated rhizobacterium, while low-productivity rhizospheres showed 2.3 significant negatives for every positively associated rhizobacterium. Overall rhizobacterial community diversity as measured by the Chao1, Shannon, and Simpson indexes was nonlinearly related to productivity, closely fitting a wavelike cubic equation. We conclude that shoot biomass productivity is strongly related to the ratio of positive- to negative-productivity-associated rhizobacteria in the rhizosphere. This study identifies significant OTUs composing the productive and unproductive rhizobacterial communities.     

Amplicon –Based Metagenomic Analysis of Mixed Fungal Samples Using Proton Release Amplicon Sequencing

Next generation sequencing technology has revolutionised microbiology by allowing concurrent analysis of whole microbial communities. Here we developed and verified similar methods for the analysis of fungal communities using a proton release sequencing platform with the ability to sequence reads of up to 400 bp in length at significant depth. This read length permits the sequencing of amplicons from commonly used fungal identification regions and thereby taxonomic classification. Using the 400 bp sequencing capability, we have sequenced amplicons from the ITS1, ITS2 and LSU fungal regions to a depth of approximately 700,000 raw reads per sample. Representative operational taxonomic units (OTUs) were chosen by the USEARCH algorithm, and identified taxonomically through nucleotide blast (BLASTn). Combination of this sequencing technology with the bioinformatics pipeline allowed species recognition in two controlled fungal spore populations containing members of known identity and concentration. Each species included within the two controlled populations was found to correspond to a representative OTU, and these OTUs were found to be highly accurate representations of true biological sequences. However, the absolute number of reads attributed to each OTU differed among species. The majority of species were represented by an OTU derived from all three genomic regions although in some cases, species were only represented in two of the regions due to the absence of conserved primer binding sites or due to sequence composition. It is apparent from our data that proton release sequencing technologies can deliver a qualitative assessment of the fungal members comprising a sample. The fact that some fungi cannot be amplified by specific “conserved” primer pairs confirms our recommendation that a multi-region approach be taken for other amplicon-based metagenomic studies.     

How stable is stable? Function versus community composition.

The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.     

Could nested PCR be applicable for the study of microbial diversity?

PCR-based methods for rRNA gene analysis have been widely used to study diversity of microbiology. However, the analysis would be difficult when the DNA content in samples is too low to be amplified by conventional PCR. Nested PCR comes up with the advantage of higher sensitivity. It can detect target DNA at several-fold lower concentrations than conventional PCR. However, the amplification bias and factors that potentially affect measurement of sample diversity associated with nested PCR method has received little attention. Here, nested PCR was compared to reconditioning PCR which is based on conventional PCR and it would reduce the formation of heteroduplex. We investigated the use of both nested and reconditioning PCR methods to construct clone libraries of 16S rRNA genes from four swimming pool water samples. Abundances of OTUs (operational taxonomic units) were correlated between the libraries (r ² = 0.88, P < 0.0005), and some OTUs had equivalent abundances in the two libraries using the Chi-square test. Differences in taxonomic groups, as well as diversity and richness estimators, were compared by paired t-test and the Wilcoxon test, respectively. There were no significant differences between clone libraries using these two PCR methods. The results of ∫-Libshuff analysis suggested that nested PCR have no particular biases in revealing OTU diversity of a bacterial community. Thus, nested PCR produce congruent pictures with reconditioning PCR in the microbial community analysis.