Fig 1.
Soil sampling site, PCR target regions of 18S rRNA gene, and experimental scheme of this study.
Sample soils for nematode isolation were obtained from the copse (picture) at the campus of Toyohashi University of Technology, Aichi Prefecture, Japan (A). Four PCR target regions (regions 1–4, with amplicon sizes shown) used in this study are indicated by double-headed arrows (B). The numbers indicating the nucleotide positions of the 5’-end of forward primers are shown on the entire SSU gene prepared from the nucleotide sequence of the C. elegans ribosomal RNA gene cluster (X03680). Orange boxes correspond to the hypervariable regions of the eukaryotic SSU genes reported by Hugerth et al. [38] and Hadziavdic et al. [45]. The regions that were amplified by the four indicated published primer sets (SSUF04-SSUR22 [29], EcoF-EcoR [24], NF1-18Sr2b [20], and NemF-18Sr2b [21]), as well as the amplified region from our previous study [19], are indicated by double-headed arrows with amplicon sizes shown. The experimental scheme of the amplicon sequencing that was prepared from the four SSU regions using 96 nematode isolates is shown (C). Fig 1 was produced by the authors specifically for this manuscript.
Table 1.
PCR primers used for amplification of four regions of 18S rRNA gene.
Fig 2.
Images of 96 nematode isolates.
Ninety-six images of nematodes are shown with their sample IDs. The sample ID numbers increase from left to right (as indicated by an arrow) and from top to bottom. Typical images for two morphologically distinct nematodes (sample ID_15 and ID_70) are shown below. Bars: 0.5 mm.
Fig 3.
Histograms of sequence variants (SVs) at the phylum level in the samples.
Relative abundance and phylum of SVs obtained from regions 1 (A), 2 (B), 3 (C), and 4 (D) in each sample are indicated in the histogram by color. The sample ID numbers are aligned from left to right, and the colors corresponding to phylum are shown in a legend box. Red arrow heads at the top of the histograms indicate sample IDs exhibiting poor PCR amplifications. NA: Not assigned.
Table 2.
Number of nematode- and non-nematode-derived SVs detected by dada2 and deblur.
Fig 4.
Histograms of nematode-derived SVs at the order level in the samples.
Relative abundance and orders of nematode-derived SVs obtained from regions 1 (A), 2 (B), 3 (C), and 4 (D) in each sample are shown in the histogram by color. Classification of the nematode orders in each SV was based on the SILVA database. The sample ID numbers are aligned from left to right, and the colors corresponding to order are shown in a legend box. Black arrow heads and red circles at the top of the histograms indicate sample IDs containing possible polymorphic nematode-derived SVs and two different nematodes, respectively. NA: Not assigned.
Table 3.
Summary of nematode-derived rOTUs identified from deep amplicon sequencing in four SSU gene regions.
Fig 5.
A cladogram of phylogenetic tree generated by regional SSU nucleotide sequences from reference nematode species and regional rOTUs corresponding to region 1.
The phylogenetic tree was prepared using regional sequences of the reference nematode species and the regional rOTUs corresponding to region 1 (R1) as described in Materials and methods. Orders of the reference species are indicated by colored dots, as shown in the legend box. Each regional rOTU in the cladograms is indicated by colored letters corresponding to the order’s color.
Fig 6.
A cladogram of phylogenetic tree generated by regional SSU nucleotide sequences from reference nematode species and regional rOTUs corresponding to region 2.
The phylogenetic trees were prepared using regional sequences of the reference nematode species and the regional rOTUs corresponding to region 2 (R2) as described in Materials and methods. Orders of the reference species are indicated by colored dots, as shown in the legend for Fig 5.
Fig 7.
A cladogram of phylogenetic tree generated by regional (region 3) SSU nucleotide sequences from reference nematode species and regional rOTUs.
The phylogenetic trees were prepared using regional sequences of the reference nematode species and the regional rOTUs corresponding to region 3 (R3) as described in Materials and methods. Orders of the reference species are indicated by colored dots, as shown in the legend for Fig 5.
Fig 8.
A cladogram of phylogenetic tree generated by regional (region 4) SSU nucleotide sequences from reference nematode species and regional rOTUs.
The phylogenetic trees were prepared using regional sequences of the reference nematode species and the regional rOTUs corresponding to region 4 (R4) as described in Materials and methods. Orders of the reference species are indicated by colored dots, as shown in the legend for Fig 5.
Fig 9.
A cladogram of phylogenetic tree generated by combined SSU nucleotide sequences from reference nematode species and rOTUs.
The phylogenetic tree was prepared using regional sequences of the reference nematode species and the regional rOTUs corresponding to the combined sequences of the four regions in numerical order (R1_2_3_4), as described in Materials and methods. Orders of the reference species are indicated by colored dots, as shown in the legend for Fig 5.
Table 4.
Numbers of clusters in the phylogenetic trees built by regional and artificially combined SSU sequences.