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Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data.

ABSTRACT: By far the greatest challenge for diversity studies is to characterize the diversity of prokaryotes, which probably encompasses billions of species, most of which are unculturable. Recent advances in theory and analysis have focused on multi-locus approaches and on combined analysis of molecular and ecological data. However, broad environmental surveys of bacterial diversity still rely on single-locus data, notably 16S ribosomal DNA, and little other detailed information. Evolutionary methods of delimiting species from single-locus data alone need to consider population genetic and macroevolutionary theories for the expected levels of interspecific and intraspecific variation. We discuss the use of a recent evolutionary method, based on the theory of coalescence within independently evolving populations, compared with a traditional approach that uses a fixed threshold divergence to delimit species.

SUBMITTER: Barraclough TG

PROVIDER: S-EPMC2679934 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data.

Barraclough Timothy G TG Hughes Martin M Ashford-Hodges Natalie N Fujisawa Tomochika T

Biology letters 20090415 3

By far the greatest challenge for diversity studies is to characterize the diversity of prokaryotes, which probably encompasses billions of species, most of which are unculturable. Recent advances in theory and analysis have focused on multi-locus approaches and on combined analysis of molecular and ecological data. However, broad environmental surveys of bacterial diversity still rely on single-locus data, notably 16S ribosomal DNA, and little other detailed information. Evolutionary methods of ...[more]

PMID: 19369225

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Project description:The most common practice in studying and cataloguing prokaryotic diversity involves the grouping of sequences into operational taxonomic units (OTUs) at the 97% 16S rRNA gene sequence identity level, often using partial gene sequences, such as PCR-generated amplicons. Due to the high sequence conservation of rRNA genes, organisms belonging to closely related yet distinct species may be grouped under the same OTU. However, it remains unclear how much diversity has been underestimated by this practice. To address this question, we compared the OTUs of genomes defined at the 97% or 98.5% 16S rRNA gene identity level against OTUs of the same genomes defined at the 95% whole-genome average nucleotide identity (ANI), which is a much more accurate proxy for species. Our results show that OTUs resulting from a 98.5% 16S rRNA gene identity cutoff are more accurate than 97% compared to 95% ANI (90.5% versus 89.9% accuracy) but indistinguishable from any other threshold in the 98.29 to 98.78% range. Even with the more stringent thresholds, however, the 16S rRNA gene-based approach commonly underestimates the number of OTUs by ∼12%, on average, compared to the ANI-based approach (∼14% underestimation when using the 97% identity threshold). More importantly, the degree of underestimation can become 50% or more for certain taxa, such as the genera Pseudomonas, Burkholderia, Escherichia, Campylobacter, and Citrobacter These results provide a quantitative view of the degree of underestimation of extant prokaryotic diversity by 16S rRNA gene-defined OTUs and suggest that genomic resolution is often necessary.IMPORTANCE Species diversity is one of the most fundamental pieces of information for community ecology and conservational biology. Therefore, employing accurate proxies for what a species or the unit of diversity is are cornerstones for a large set of microbial ecology and diversity studies. The most common proxies currently used rely on the clustering of 16S rRNA gene sequences at some threshold of nucleotide identity, typically 97% or 98.5%. Here, we explore how well this strategy reflects the more accurate whole-genome-based proxies and determine the frequency with which the high conservation of 16S rRNA sequences masks substantial species-level diversity.

Project description:While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.

Project description:Mongolia's salmonids are suffering extensive population declines; thus, more comprehensive fisheries management and conservation strategies are required. To assist with their development, a better understanding of the genetic structure and diversity of these threatened species would allow a more targeted approach for preserving genetic variation and ultimately improve long-term species recoveries. It is hypothesized that the unfragmented river basins that have persisted across Mongolia provide unobstructed connectivity for resident salmonid species. Thus, genetic structure is expected to be primarily segregated between major river basins. We tested this hypothesis by investigating the population structure for three salmonid genera (Hucho, Brachymystax and Thymallus) using different genetic markers to identify evolutionarily significant units (ESUs) and priority rivers to focus conservation efforts. Fish were assigned to separate ESUs when the combined evidence of mitochondrial and nuclear data indicated genetic isolation. Hucho taimen exhibited a dichotomous population structure forming two ESUs, with five priority rivers. Within the Brachymystax genus, there were three B. lenokESUs and one B. tumensisESU, along with six priority rivers. While B. tumensiswas confirmed to display divergent mtDNA haplotypes, haplotype sharing between these two congeneric species was also identified. For T. baicalensis,only a single ESU was assigned, with five priority rivers identified plus Lake Hovsgol. Additionally, we confirmed that T. nigrescens from Lake Hovsgol is a synonym of T. baicalensis. Across all species, the most prominent pattern was strong differentiation among major river basins with low differentiation and weak patterns of isolation by distance within river basins, which corroborated our hypothesis of high within-basin connectivity across Mongolia. This new genetic information provides authorities the opportunity to distribute resources for management between ESUs while assigning additional protection for the more genetically valuable salmonid rivers so that the greatest adaptive potential within each species can be preserved.

Dataset Information

Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data.

Publications

Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data.

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