DNA barcoding - taxon identification using a standardized DNA region - has received much attention recently, and is being further developed through an international initiative. We anticipate that DNA barcoding techniques will be increasingly used by ecologists. They will be able to not only identify a single species from a specimen or an organism's remains but also determine the species composition of environmental samples. Short DNA fragments persist in the environment and might allow an assessment of local biodiversity from soil or water. Even DNA-based diet composition can be estimated using fecal samples. Here we review the new avenues offered to ecologists by DNA barcoding, particularly in the context of new sequencing technologies.

https://www.cell.com/trends/ecology-evolution/pdf/S0169-5347(08)00344-3.pdf

DNA barcoding for ecologists

Despite recognition of the importance of soil bacteria to terrestrial ecosystem functioning there is little consensus on the factors regulating belowground biodiversity. Here we present a multi-scale spatial assessment of soil bacterial community profiles across Great Britain (> 1000 soil cores), and show the first landscape scale map of bacterial distributions across a nation. Bacterial diversity and community dissimilarities, assessed using terminal restriction fragment length polymorphism, were most strongly related to soil pH providing a large-scale confirmation of the role of pH in structuring bacterial taxa. However, while α diversity was positively related to pH, the converse was true for β diversity (between sample variance in α diversity). β diversity was found to be greatest in acidic soils, corresponding with greater environmental heterogeneity. Analyses of clone libraries revealed the pH effects were predominantly manifest at the level of broad bacterial taxonomic groups, with acidic soils being dominated by few taxa (notably the group 1 Acidobacteria and Alphaproteobacteria). We also noted significant correlations between bacterial communities and most other measured environmental variables (soil chemistry, aboveground features and climatic variables), together with significant spatial correlations at close distances. In particular, bacterial and plant communities were closely related signifying no strong evidence that soil bacteria are driven by different ecological processes to those governing higher organisms. We conclude that broad scale surveys are useful in identifying distinct soil biomes comprising reproducible communities of dominant taxa. Together these results provide a baseline ecological framework with which to pursue future research on both soil microbial function, and more explicit biome based assessments of the local ecological drivers of bacterial biodiversity.

The bacterial biogeography of British soils.

Biochar has been shown to aid soil fertility and crop production in some circumstances. We investigated effects of the addition of Jarrah (Eucalyptus marginata) biochar to a coarse textured soil on soil carbon and nitrogen dynamics. Wheat was grown for 10 weeks, in soil treated with biochar (0, 5, or 25 t ha−1) in full factorial combination with nitrogen (N) treatments (organic N, inorganic N, or control). Samples were analysed for plant biomass, soil microbial biomass carbon (MBC) and nitrogen (MBN), N mineralisation, CO2 evolution, community level physiological profiles (CLPP) and ammonia oxidising bacterial community structure. MBC significantly decreased with biochar addition while MBN was unaltered. Net N mineralisation was highest in control soil and significantly decreased with increasing addition of biochar. These findings could not be attributed to sorption of inorganic N to biochar. CO2 evolution decreased with 5 t ha−1 biochar but not 25 t ha−1. Biochar addition at 25 t ha−1 changed the CLPP, while the ammonia oxidising bacterial community structure changed only when biochar was added with a N source. We conclude that the activity of the microbial community decreased in the presence of biochar, through decreased soil organic matter decomposition and N mineralisation which may have been caused by the decreased MBC.

Decreased soil microbial biomass and nitrogen mineralisation with Eucalyptus biochar addition to a coarse textured soil

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.

/pdf/advances-in-the-use-of-terminal-restriction-fragment-length-5e61av5uot.pdf

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities.

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.

/pdf/assessment-of-bias-associated-with-incomplete-extraction-of-2m2wx9m0bz.pdf

Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil

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

/pdf/interpreting-ecological-diversity-indices-applied-to-4a2etxnidc.pdf

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

Deborah Hudleston

Papers

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