@article {KNZ001615, title = {High-throughput amplicon sequencing of rRNA genes requires a copy number correction to accurately reflect the effects of management practices on soil nematode community structure}, journal = {Molecular Ecology}, volume = {22}, year = {2013}, pages = {5456 -5471}, abstract = {

Nematodes are abundant consumers in grassland soils, but more sensitive and specific methods of enumeration are needed to improve our understanding of how different nematode species affect, and are affected by, ecosystem processes. High-throughput amplicon sequencing is used to enumerate microbial and invertebrate communities at a high level of taxonomic resolution, but the method requires validation against traditional specimen-based morphological identifications. To investigate the consistency between these approaches, we enumerated nematodes from a 25-year field experiment using both morphological and molecular identification techniques in order to determine the long-term effects of annual burning and nitrogen enrichment on soil nematode communities. Family-level frequencies based on amplicon sequencing were not initially consistent with specimen-based counts, but correction for differences in rRNA gene copy number using a genetic algorithm improved quantitative accuracy. Multivariate analysis of corrected sequence-based abundances of nematode families was consistent with, but not identical to, analysis of specimen-based counts. In both cases, herbivores, fungivores and predator/omnivores generally were more abundant in burned than nonburned plots, while bacterivores generally were more abundant in nonburned or nitrogen-enriched plots. Discriminate analysis of sequence-based abundances identified putative indicator species representing each trophic group. We conclude that high-throughput amplicon sequencing can be a valuable method for characterizing nematode communities at high taxonomic resolution as long as rRNA gene copy number variation is accounted for and accurate sequence databases are available.

}, keywords = {LTER-KNZ}, doi = {10.1111/mec.12480}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/mec.12480}, author = {Darby, D.J. and Todd, T.C. and Herman, M.A.} } @article {KNZ001598, title = {Long-term nitrogen amendment alters the diversity and assemblage of soil bacterial communities in tallgrass prairie}, journal = {PLoS ONE}, volume = {8}, year = {2013}, pages = {67884 -}, abstract = {

Anthropogenic changes are altering the environmental conditions and the biota of ecosystems worldwide. In many temperate grasslands, such as North American tallgrass prairie, these changes include alteration in historically important disturbance regimes (e.g., frequency of fires) and enhanced availability of potentially limiting nutrients, particularly nitrogen. Such anthropogenically-driven changes in the environment are known to elicit substantial changes in plant and consumer communities aboveground, but much less is known about their effects on soil microbial communities. Due to the high diversity of soil microbes and methodological challenges associated with assessing microbial community composition, relatively few studies have addressed specific taxonomic changes underlying microbial community-level responses to different fire regimes or nutrient amendments in tallgrass prairie. We used deep sequencing of the V3 region of the 16S rRNA gene to explore the effects of contrasting fire regimes and nutrient enrichment on soil bacterial communities in a long-term (20 yrs) experiment in native tallgrass prairie in the eastern Central Plains. We focused on responses to nutrient amendments coupled with two extreme fire regimes (annual prescribed spring burning and complete fire exclusion). The dominant bacterial phyla identified were Proteobacteria, Verrucomicrobia, Bacteriodetes, Acidobacteria, Firmicutes, and Actinobacteria and made up 80\% of all taxa quantified. Chronic nitrogen enrichment significantly impacted bacterial community diversity and community structure varied according to nitrogen treatment, but not phosphorus enrichment or fire regime. We also found significant responses of individual bacterial groups including Nitrospira and Gammaproteobacteria to long-term nitrogen enrichment. Our results show that soil nitrogen enrichment can significantly alter bacterial community diversity, structure, and individual taxa abundance, which have important implications for both managed and natural grassland ecosystems.

}, keywords = {LTER-KNZ, Bacteria, Biodiversity, Ecosystem functioning, Grasslands, Microbial ecosystems, Plant communities, Ribosomal RNA, Sequence analysis}, doi = {10.1371/journal.pone.0067884}, url = {https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067884}, author = {Coolon, J.D. and Jones, K.L. and Todd, T.C. and John M. Blair and Herman, M.A.} } @article {KNZ001483, title = {Several grassland soil nematodes species are insensitive to RNA-mediated interference}, journal = {Journal of Nematology}, volume = {44}, year = {2012}, pages = {92 -101}, abstract = {

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.

}, keywords = {LTER-KNZ}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593264/}, author = {Wheeler, D. and Darby, B.J. and Todd, T.C. and Herman, M.A.} } @article {KNZ001482, title = {Normalization and centering of array-based heterologous genome hybridization based on divergent control probes}, journal = {BMC Bioinformatics}, volume = {12}, year = {2011}, abstract = {

Background Hybridization of heterologous (non-specific) nucleic acids onto arrays designed for model-organisms has been proposed as a viable genomic resource for estimating sequence variation and gene expression in non-model organisms. However, conventional methods of normalization that assume equivalent distributions (such as quantile normalization) are inappropriate when applied to non-specific (heterologous) hybridization. We propose an algorithm for normalizing and centering intensity data from heterologous hybridization that makes no prior assumptions of distribution, reduces the false appearance of homology, and provides a way for researchers to confirm whether heterologous hybridization is suitable. Results Data are normalized by adjusting for Gibbs free energy binding, and centered by adjusting for the median of a common set of control probes assumed to be equivalently dissimilar for all species. This procedure was compared to existing approaches and found to be as successful as Loess normalization at detecting sequence variations (deletions) and even more successful than quantile normalization at reducing the accumulation of false positive probe matches between two related nematode species, Caenorhabditis elegans and C. briggsae. Despite the improvements, we still found that probe fluorescence intensity was too poorly correlated with sequence similarity to result in reliable detection of matching probe sequence. Conclusions Cross-species hybridizations can be a way to adapt genome-enabled tools for closely related non-model organisms, but data must be appropriately normalized and centered in a way that accommodates hybridization of nucleic acids with diverged sequence. For short, 25-mer probes, hybridization intensity alone may be insufficiently correlated with sequence similarity to allow reliable inference of homology at the probe level.

}, keywords = {LTER-KNZ}, doi = {10.1186/1471-2105-12-183}, url = {https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-183}, author = {Darby, B.J. and Jones, K.L. and Wheeler, D. and Herman, M.A.} } @article {KNZ001101, title = {Development of taxon-specific markers for high-throughput screening of microbial-feeding nematodes}, journal = {Molecular Ecology Notes}, volume = {6}, year = {2006}, pages = {712 -714}, abstract = {In an effort to assess the taxonomic identity of large-scale samplings of nematodes from the Konza Tallgrass Prairie, we sequenced a portion of the 18S rRNA gene and its associated internally transcribed spacer (ITS1) from 74 nematodes encompassing four taxonomic families. From these sequences, we have developed a series of molecular probes to distinguish 16 distinct microbivorous nematode taxa. Using a combination of low power microscopy and taxon-specific real-time probes, the 74 nematodes were correctly assigned to their respective taxonomic groups. This optimized method provides a high-throughput assay to determine nematode identities across larger data sets.}, keywords = {LTER-KNZ, 18S rRNA, 5' nuclease assaym, Nematoda, real-time PCR, Taqman}, doi = {10.1111/j.1471-8286.2006.01319.x}, author = {Jones, K.L. and Todd, T.C. and Herman, M.A.} } @article {KNZ001054, title = {Molecular approach for assessing responses of microbial-feeding nematodes to burning and chronic nitrogen enrichment in a native grassland}, journal = {Molecular Ecology}, volume = {15}, year = {2006}, pages = {2601 -2609}, abstract = {A substantial proportion of the primary productivity in grassland ecosystems is allocated belowground, sustaining an abundant and diverse community of microbes and soil invertebrates. These belowground communities drive many important ecosystem functions and are responsive to a variety of environmental changes. Nematodes, an abundant and diverse component of grassland soil communities, are particularly responsive to altered environmental conditions, such as those associated with reduced fire frequency and nitrogen enrichment, with the most consistent responses displayed by microbial-feeding nematodes. However, much of the available research characterizing nematode responses to environmental change has been carried out at the taxonomic level of family or by broad trophic categories (e.g. fungivores, bacterivores). The extent to which differential responses to environmental change occurs at the genus level or below is unclear. Therefore, the objective of this study was to use molecular methods to quantify the response of microbial-feeding nematodes, at the lowest levels of taxonomic resolution, to nitrogen enrichment and changes in fire frequency. Using sequencing and quantitative polymerase chain reaction (PCR) probes for the 18S ribosomal RNA gene and the ITS1 region, we identified 19 microbial-feeding nematode taxa across four families. When nematodes were sampled across treatments, we found that some nematode taxa within a family responded similarly to nitrogen and burning treatments, while other taxa within the same family respond quite differently. Additionally, although nematodes from different families on average responded differently to nitrogen enrichment and burning, similar responses were seen in nematode taxa that span three taxonomic families. Thus, if nematodes are to be used as indicators of environmental change, care should be taken to assess the response at the lowest taxonomic level possible.}, keywords = {LTER-KNZ}, doi = {10.1111/j.1365-294X.2006.02971.x}, author = {Jones, K.L. and Todd, T.C. and Wall-Beam, J.L. and Coolon, J.D. and John M. Blair and Herman, M.A.} }