Distinct community structures of soil nematodes from three ecologically different sites revealed by high-throughput amplicon sequencing of four 18S ribosomal RNA gene regions.

Harutaro Kenmotsu,Tomokazu Nitta,Masahiro Ishikawa,Yuu Hirose,Toshihiko Eki,Ebrahim Shokoohi

doi:10.1371/journal.pone.0249571

Abstract

Quantitative taxonomic compositions of nematode communities help to assess soil environments due to their rich abundance and various feeding habitats. DNA metabarcoding by the 18S ribosomal RNA gene (SSU) regions were preferentially used for analyses of soil nematode communities, but the optimal regions for high-throughput amplicon sequencing have not previously been well investigated. In this work, we performed Illumina-based amplicon sequencing of four SSU regions (regions 1–4) to identify suitable regions for nematode metabarcoding using the taxonomic structures of nematodes from uncultivated field, copse, and cultivated house garden soils. The fewest nematode-derived sequence variants (SVs) were detected in region 3, and the total nematode-derived SVs were comparable in regions 1 and 4. The relative abundances of reads in regions 1 and 4 were consistent in both orders and feeding groups with prior studies, thus suggesting that region 4 is a suitable target for the DNA barcoding of nematode communities. Distinct community structures of nematodes were detected in the taxon, feeding habitat, and life-history strategy of each sample; i.e., Dorylamida- and Rhabditida-derived plant feeders were most abundant in the copse soil, Rhabditida-derived bacteria feeders in the house garden soil, and Mononchida- and Dorylamida-derived omnivores and predators and Rhabditida-derived bacteria feeders in the field soil. Additionally, low- and high-colonizer–persister (cp) groups of nematodes dominated in the house garden and copse soils, respectively, whereas both groups were found in the field soil, suggesting bacteria-rich garden soil, undisturbed and plant-rich copse soil, and a transient status of nematode communities in the field soil. These results were also supported by the maturity indices of the three sampling sites. Finally, the influence of the primer tail sequences was demonstrated to be insignificant on amplification. These findings will be useful for DNA metabarcoding of soil nematode communities by amplicon sequencing.

Highlights

Many soil-dwelling organisms, invisible to the naked eye, form stable ecosystems and play important roles in nutrient recycling in the pedosphere [1]
During DNA barcoding of nematodes, the nucleotide sequences of polymerase chain reaction (PCR)-amplified barcode DNAs from individual nematodes were determined by DNA sequencing; the nematodes were classified by their sequences into taxonomic groups sharing identical DNA barcode sequences, known as operational taxonomic units (OTUs)
We investigated the suitable regions for DNA metabarcoding for the complex nematode populations among the four small subunit ribosomal RNA (SSU) regions

Summary

Introduction

Many soil-dwelling organisms, invisible to the naked eye, form stable ecosystems and play important roles in nutrient recycling in the pedosphere [1]. Nematode taxonomic compositions vary by ecosystem type [8, 9] and are influenced by various factors such as food availability and abundance, physical and chemical parameters (e.g., pH, temperature) [10, 11], soil properties [11, 12], and agricultural conditions (e.g., tillage, cultivated plants, fertilizers) [13,14,15,16,17,18,19,20,21,22]. There have been several studies on the DNA metabarcoding of terrestrial nematodes using high-throughput sequencing of SSU gene-derived amplicons [20, 29,30,31,32,33], the regions most suitable for complex nematode communities have yet not been identified

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Conclusion