Figure 1:
Millipedes (Parafontaria laminata) collected for this study.
Table 1.
Isolation of Pristionchus spp. in the preliminary study.
| Matsubara Lake population | Nobeyama population | ||||
|---|---|---|---|---|---|
| Individual number | Gender | Nematode species | Individual number | Gender | Nematode species |
| Mat_1 | M | P. degawai | Nob_1 | F | P. degawai |
| Mat_2 | F | P. degawai | Nob_2 | M | P. degawai, P. laevicollis |
| Mat_3 | M | P. degawai | Nob_3 | M | P. degawai |
| Mat_4 | F | P. degawai | Nob_4 | M | P. degawai |
| Mat_5 | F | P. degawai | Nob_5 | M | P. degawai |
| Mat_6 | M | P. degawai | Nob_6 | M | P. degawai |
| Mat_7 | F | P. degawai | Nob_7 | F | P. degawai, P. laevicollis |
| Mat_8 | F | P. degawai | Nob_8 | F | P. degawai |
| Mat_9 | F | P. degawai | Nob_9 | M | P. degawai |
| Mat_10 | F | P. degawai | Nob_10 | F | P. degawai |
| Mat_11 | F | P. degawai | Nob_11 | F | P. degawai |
| Mat_12 | F | P. degawai | Nob_12 | F | P. degawai, P. laevicollis |
| Mat_13 | F | P. degawai | Nob_13 | F | P. degawai |
| Mat_14 | F | P. degawai | Nob_14 | M | P. degawai |
| Mat_15 | F | P. degawai | Nob_15 | F | P. degawai |
| Mat_16 | F | P. fukushimae | Nob_16 | F | P. degawai |
| Mat_17 | F | P. fukushimae | Nob_17 | M | P. degawai |
| Mat_18 | M | P. degawai | Nob_18 | M | P. degawai |
| Mat_19 | F | P. degawai | Nob_19 | F | P. laevicollis |
| Mat_20 | F | P. degawai | Nob_20 | F | P. degawai |
| Mat_21 | M | P. degawai | Nob_21 | M | P. degawai |
| Mat_22 | M | P. degawai | Nob_22 | M | P. degawai |
| Mat_23 | F | P. degawai | Nob_23 | F | P. degawai |
| Mat_24 | F | P. degawai | Nob_24 | M | P. degawai |
| Mat_25 | F | P. degawai | Nob_25 | F | P. laevicollis |
| Mat_26 | F | P. degawai | Nob_26 | F | P. laevicollis |
| Mat_27 | F | P. degawai | Nob_27 | F | P. laevicollis |
| Mat_28 | M | P. degawai | Nob_28 | M | P. laevicollis |
| Mat_29 | M | P. degawai | Nob_29 | M | P. degawai |
| Mat_30 | F | P. degawai | Nob_30 | M | P. degawai |
| Mat_31 | M | P. degawai | Nob_31 | F | P. laevicollis |
| Mat_32 | F | P. degawai | Nob_32 | M | P. degawai |
| Mat_33 | F | P. degawai | Nob_33 | M | P. laevicollis |
| Nob_34 | F | P. laevicollis | |||
| Nob_35 | F | P. degawai | |||
| Nob_36 | F | P. degawai | |||
| Nob_37 | F | P. degawai | |||
| Nob_38 | M | P. degawai | |||
Table 2.
Mitochondrial cytochrome oxidase subunit I haplotypes isolated in the extensive study.
| Matsubara Lake population | Nobeyama population | ||||
|---|---|---|---|---|---|
| Individual number | Gender | Haplotype | Individual number | Gender | Nematode species |
| Mat101 | M | D1, D3, D12, F8, N | Nob101 | F | F11, La, Ma |
| Mat102 | F | D1, D2, F17 | Nob102 | M | D8, F2, Oa |
| Mat103 | M | D3, D5, F1, F14, F15 | Nob103 | M | F2, D14 |
| Mat104 | F | D1, D8, D16 | Nob104 | M | D8, D14, F2 |
| Mat105 | F | D1, D12, F1 | Nob105 | M | D8, D14 |
| Mat106 | M | C, D1, D6, D16 | Nob106 | M | D8, F2, F6, E3, E6 |
| Mat107 | F | D3, D8, D17 | Nob107 | F | D8 |
| Mat108 | F | D1, D5, D6, F15, K | Nob108 | F | D14, F12, F13 |
| Mat109 | F | D5, D8, D13, E1, E6, F1, K | Nob109 | M | D1, E6, E7, La |
| Mat110 | F | D2, D3, D12, F1, K, Xa | Nob110 | F | F2, E4 |
| Mat111 | F | D1, D2, D11, E6, F2, F12 | Nob111 | F | D8, D14 |
| Mat112 | F | D1, D3, D7, F1, F8 | Nob112 | F | E1, I1 |
| Mat113 | F | D1, D3, D6, D12, F3 | Nob113 | F | D1, F4, F5, I2 |
| Mat114 | F | D3, D9, E6, F1, F2, F9, K, Ga | Nob114 | M | D1, F6, D8 |
| Mat115 | F | D1, D2, D3, D6, D13, F1, F7, F14 | Nob115 | F | F6 |
| Mat116 | F | D1, F1, F10 | Nob116 | F | D1, D8, D14 |
| Mat117 | F | D1, D6, D11, F8 | Nob117 | M | F2, F12 |
| Mat118 | M | D1, D2, D3, D4, D5, D6, D12 | Nob118 | M | D1, D11, E1, E5 |
| Mat119 | F | D1, D2, D3, F2, H | Nob119 | F | D1, D3, D10, E2, J, Oa |
| Mat120 | F | D1, D2, D7, E6, F3 | Nob120 | F | D15, Ma |
1 Notes: aHaplotype G is P. entomophagus (Mat114); L, M, O are P. laevicollis (Nob 101, 102, 109, 119 and 120); and X which was not amplified with universal primer set is P. fukushimae (Mat110). In addition to these strains, haplotypes A and B (P. fukushimae strains established in the first isolation) were included the phylogenetic analysis.
Table 3.
GenBank accession numbers for the mtCOI haplotypes.
| Type | Species | Accession number | Type | Species | Accession number |
|---|---|---|---|---|---|
| A | P. fukushimae | LC589007 | E4 | P. degawai | LC589034 |
| B | LC589008 | E5 | LC589035 | ||
| G | P. entomophagus | LC589009 | E6 | LC589036 | |
| L | P. laevicollis | LC589010 | E7 | LC589037 | |
| M | LC589011 | F1 | LC589038 | ||
| O | LC589012 | F2 | LC589039 | ||
| C | P. degawai | LC589013 | F3 | LC589040 | |
| D1 | LC589014 | F4 | LC589041 | ||
| D2 | LC589015 | F5 | LC589042 | ||
| D3 | LC589016 | F6 | LC589043 | ||
| D4 | LC589017 | F7 | LC589044 | ||
| D5 | LC589018 | F8 | LC589045 | ||
| D6 | LC589019 | F9 | LC589046 | ||
| D7 | LC589020 | F10 | LC589047 | ||
| D8 | LC589021 | F11 | LC589048 | ||
| D9 | LC589022 | F12 | LC589049 | ||
| D10 | LC589023 | F13 | LC589050 | ||
| D11 | LC589024 | F14 | LC589051 | ||
| D12 | LC589025 | F15 | LC589052 | ||
| D13 | LC589026 | F16 | LC589053 | ||
| D14 | LC589027 | F17 | LC589054 | ||
| D15 | LC589028 | H | LC589055 | ||
| D16 | LC589029 | I1 | LC589056 | ||
| D17 | LC589030 | I2 | LC589057 | ||
| E1 | LC589031 | J | LC589058 | ||
| E2 | LC589032 | K | LC589059 | ||
| E3 | LC589033 | N | LC589060 |
Figure 2:
Phylogenetic relationships among the 54 haplotypes of four Pristionchus species found in this study. A: Unrooted tree showing the relationships among the four species; B: Phylogenetic relationships among the 47 genotypes of P. degawai. The Maximum Likelihood tree was inferred from partial sequences of the mtCOI gene. The GTR+G model was applied, and the parameters were as follows: lnL = –45,723.93825, freqA = 0.21, freqC = 0.10, freqG = 0.16, freqT = 0.43, R(a) = 3.9, R(b) = 100.0, R(c) = 15.8, R(d) = 14.0, R(e) = 100.0, R(f) = 1.0, and Shape = 0.12. Bootstrap values exceeding 50% are given on the appropriate clades. Some P. fukushimae strains did not amplify with the universal mtCOI primers (Kanzaki and Futai, 2002), probably because of a mutation in the primer region, and these strains were treated as a genotype (they do not appear in the tree). Symbols after haplotype codes indicate the haplotype found only from Nob (open circle), only from Mat (closed circle) and both Nob and Mat (arrow).
Figure 3:
Previous isolation record of Pristionchus spp. associated with millipedes in Japan. The collection localities for the present study are suggested by solid line box, previous record of the species mentioned in the present study are suggested by dotted line box, and Hachijojima Isl. (Carta et al., 2018) is suggested by double line box. N. Kanzaki’s unpubl. obs. is indicated with an asterisk.