Fig 1.
Sampling sites and relative abundances of sequence variants (SVs) at the phylum level and the nematode order level.
(A) Soil sampling sites. Photographs of uncultivated field and copse are reprinted from prior PLoS One papers [26, 28]. (B) Relative abundance and phylum of SVs obtained from regions 1 (R1), 2 (R2), 3 (R3), and 4 (R4) of the 18S ribosomal RNA (SSU) gene in each sample. (C) Relative abundance and order of nematode-derived SVs from each region (R1–R4). Each sample was derived from the sampling site in (A) on top of histograms in (B) and (C). Boxes in the histogram indicate the SVs and their abundances; phylum and order levels are indicated by color. Taxonomic classification of SVs was based on the SILVA database [46]. NA: Not assigned.
Table 1.
Polymerase chain reaction (PCR) primers used for amplifying the small subunit ribosomal RNA (SSU) gene regions.
Table 2.
Numbers of SILVA-based total and nematode-derived SVs in each SSU region.
Fig 2.
Numbers of regional nematode SVs and relative abundance of nematode-derived sequence reads at the order level in each region.
Numbers of nematode SVs assigned to 10 orders are indicated at the bottom of (D) and unassigned SVs identified from the field (blue), copse (green), and house garden (red) samples are indicated in the histograms in regions (A) 1, (B) 2, (C) 3, and (D) 4. The number on top of the histogram corresponds to the numbers of SVs. The percentage of the abundance of sequence reads corresponding to nematode order obtained from regions (E) 1, (F) 2, (G) 3, and (H) 4 are indicated in the field (H), copse (Z), and house garden (S) samples in the horizontal histogram by color. The colors corresponding to orders are indicated at the bottom of (H). Orders of regional nematode SVs were assigned based on the BLASTN search.
Fig 3.
A cladogram of nematode SVs in region 1 with the corresponding orders, feeding types, and relative read abundances.
The cladogram was prepared using nematode SVs from region 1 and the corresponding SSU gene sequence of H. crispae as the outgroup as described in the Materials and methods section. Bootstrap numbers of over 500 per 1000 are indicated at the nodes of the cladogram. The name of the nematode-derived SV in region 1 was indicated as R1_SV_number at the branch end of the cladogram. Orders and feeding types (FT) of the nematode SVs are indicated in the corresponding columns at the right of the horizontally compressed cladogram by colored bars and abbreviations, as shown in the legend boxes. Relative read abundance of nematode SVs in the samples from the field (H02), copse (Z02), and house garden (S01) soils are indicated by density bars. Orders, feeding types, and relative abundance (i.e., percentage of the number of nematodes in the SSU gene-derived operational taxonomic units (rOTU)) of the rOTUs that were previously identified also indicated, where H01 represents the rOTUs from the soybean-cultivated field and K01 represents those from the unmanaged flowerbed soils sampled in 2010 [26] and Z01 represents those from the copse soils sampled in 2018 [28] (see S2 Fig).
Fig 4.
A cladogram of nematode SVs in region 2 with the corresponding orders, feeding types, and relative read abundances.
The cladogram was prepared using nematode SVs of region 2 as described in the Materials and methods section. Orders, feeding types, and relative read abundances of the nematode SVs are indicated in the corresponding columns at the right of the horizontally compressed cladogram by colored bars, colored abbreviations, and density bars, respectively, as detailed in the caption of Fig 3 above.
Fig 5.
A cladogram of nematode SVs in region 3 with the corresponding orders, feeding types, and relative read abundances.
The cladogram was prepared using nematode SVs of region 3 as described in the Materials and methods section. Orders, feeding types, and relative read abundances of the nematode SVs are indicated in the corresponding columns at the right of the horizontally compressed cladogram by colored bars, colored abbreviations, and density bars, respectively, as detailed in the caption of Fig 3 above.
Fig 6.
A cladogram of nematode SVs in region 4 with the corresponding orders, feeding types, and relative read abundances.
The cladogram was prepared using nematode SVs of region 4 as described in the Materials and methods section. Orders, feeding types, and relative read abundances of the nematode SVs are indicated in the corresponding columns at the right of the horizontally compressed cladogram by colored bars, colored abbreviations, and density bars, respectively, as detailed in the caption of Fig 3 above.
Table 3.
Maturity indices for three sampling sites in four SSU regions.
Fig 7.
Relative abundances of sequence reads by feeding group across all samples.
Nematode SVs identified from the field (H), copse (Z), and house garden (S) samples were assigned to one of seven feeding types (bacteria feeder, fungus feeder, plant feeder, omnivore, predator, eukaryote feeder, and animal parasite) as described in the Materials and methods section. Percentages of the sequence reads of nematode SVs in feeding groups are shown by colored fractions of horizontal histograms in regions (A) 1, (B) 2, (C) 3, and (D) 4, where the colors corresponding to each feeding type are shown in the legend box. The fractions of the SVs with multiple feeding types are classified as “not assigned” and indicated in gray.
Table 4.
Total and nematode-derived SVs from the high-throughput sequencing of amplicons prepared from one-step PCR with tailed primers and two-step PCR with tailless and tailed primers.
Fig 8.
Relative read abundance of nematode-derived SVs in each order from massive sequencing of amplicons prepared by one- and two-step PCRs.
The amplicons of the four SSU gene regions indicated on top of the histograms were prepared via a one-step PCR with tailed primers (Tail) and a two-step PCR using tailless and tailed primers (No_tail) with the field sample DNA as a template. Nematode-derived SVs were identified from the SVs as described in the Materials and methods sections and their orders were determined based on the SILVA database. The boxes in each order’s column indicate the SVs and their relative abundance, whereas the colors corresponding to order are shown in the legend. NA: Not assigned.