High-throughput amplicon sequencing of tallgrass prairie soil nematodes
Nematodes are abundant consumers in the grassland soil ecosystem but many groups are difficult, and in some cases impossible, to identify at the level of species. We need more sensitive and specific methods of enumeration in order to understand how different nematode species affect, and are affected by, the soil ecosystem. High-throughput amplicon sequencing is commonly used to enumerate microbial communities, but the method needs "ground truthing" to confirm its qualitative and quantitative accuracy. This study has both an ecological and a methodological objective. The ecological objective of this study was to determine the long-term effects of annual burning and elevated nitrogen inputs on soil nematode community composition. We enumerated nematodes from long-term plots that were burned or enriched with inorganic nitrogen each year since they were established 25 years ago. The methodological objective of this study was to test the qualitative and quantitative accuracy of high-throughput amplicon sequencing. To achieve this, we enumerated nematodes from the same samples with both traditional morphological-based methods and by molecular methods from high-throughput amplicon sequencing. Finally, we used qPCR to independently confirm variation in rRNA copy number among cultured specimens.
Herbivores, fungivores, and predator/omnivores were more abundant in burned plots than non-burned plots. In contrast, bacterivores were more abundant in nitrogen supplemented plots than non-amended plots while predator/omnivores were more abundant in non-amended plots than in nitrogen supplemented plots. Amplicon-sequencing of the same samples correctly identified the major families and genera that are common in tallgrass prairies, but not in the correct proportional representation. We found that some species, especially those in the family Rhabditidae, can have rRNA copy number values up to several orders of magnitude more than others. We conclude that high-throughput amplicon sequencing cannot reflect accurately the proportional abundance of specimens in a sample if copy number variation is not taken into account. Despite the methodological inconvenience, rRNA copy number variation is still an interesting biological phenomenon that could prove to be ecologically informative because it is correlated with genome size, colonizer-type life history traits, and the stoichiometry of phosphorous limitation in rapidly growing species. High-throughput amplicon sequencing can still be a valuable method for characterizing nematode communities at fine taxonomic resolution if we can improve our understanding of copy number variation and maintain accurate and complete sequence databases.