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

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.

SUBMITTER: Kenmotsu H

PROVIDER: S-EPMC8049254 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nematodes are representative soil metazoans with diverged species that play crucial roles in nutrient recycling in the pedosphere. Qualitative and quantitative information on nematode communities is useful for assessing soil quality, and DNA barcode-mediated taxonomic analysis is a powerful tool to investigate taxonomic compositions and changes in nematode communities. Here, we investigated four regions (regions 1-4) of the 18S small subunit ribosomal RNA (SSU) gene as PCR targets of deep amplicon sequencing for the taxonomic profiling of individual soil nematodes. We determined the sequence variants (SVs) of 4 SSU regions for 96 nematodes (total 384 amplicons) isolated from copse soils and assigned their taxonomy using the QIIME2 software with dada2 or deblur algorithm and the SILVA database. Dada2 detected approximately 2-fold more nematode-derived SVs than deblur, and a larger number of SVs were obtained in regions 1 and 4 than those in other regions. These results and sufficient reference sequence coverage in region 4 indicated that DNA barcoding using a primer set for region 4 followed by dada2-based analysis would be most suitable for soil nematode taxonomic analysis. Eighteen SSU-derived operational taxonomic units (rOTUs) were obtained from 68 isolates, and their orders were determined based on the phylogenetic trees built by four regional sequences of rOTUs and 116 nematode reference species as well as the BLASTN search. The majority of the isolates were derived from three major orders Dorylaimida (6 rOTUs, 51.5% in 68 isolates), Rhabditida (4 rOTUs, 29.4%), and Triplonchida (7 rOTUs, 17.6%). The predicted feeding types of the isolates were fungivores (38.2% in total nematodes), plant feeders (32.4%), and 14.7% for both bacterivores and omnivores/predators. Additionally, we attempted to improve the branch structure of phylogenetic trees by using long nucleotide sequences artificially prepared by connecting regional sequences, but the effect was limited.

Project description:Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis ((13)C/(12)C and (15)N/(14)N, expressed as ? (13)C and ? (15)N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis-isotopic ratio mass spectrometry (?EA-IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in ? (15)N (p = 0.290) or ? (13)C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (? (15)N = 1.08 to 3.22 mUr‰, ? (13)C = -29.58 to -27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in ? (15)N between the plant feeder and the predator group (? (15)N = 9.89 to 12.79 mUr, ? (13)C = -27.04 to -25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr(2)) and the predators (1.73 mUr(2)), but largest for omnivores (3.83 mUr(2)). These data may reflect more preferential feeding by the plant feeder and predators, as assumed by classical morphology-based feeding groups, and indicate that omnivory may be more widespread across detritivore groups i.e. bacterial feeders (3.81 mUr(2)). Trophic information for soil nematodes derived from stable isotope analysis, scaled as finely as species level in some cases, will complement existing indices for soil biological assessment and monitoring, and can potentially be used to identify new trophic interactions in soils. The isotopic technique used here, to compare nematode feeding group members largely confirm their trophic relations based on morphological studies.

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

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