Ring nematodes of the genus
The specimens were recovered from two localities in Dehdasht and Basht (Kohgiluyeh and Boyer-Ahmad province, Southern Iran). The nematodes were extracted from the soil around roots of a grass (
Morphometric and morphological characters of the nematode populations were studied by a light microscope, equipped with a Dino-eye microscope eyepiece camera in conjunction with its Dino Capture version 2.0 software. The nematode species identified by using data documented by Brzeski et al. (2002) and Geraert (2010), as well as by comparison with recently published descriptions (Cid del Prado Vera, 2009; Cordero et al., 2012; Powers et al., 2016; Olson et al., 2017).
For molecular analysis, DNA was extracted from a single specimen, and three amplifications were conducted on that single specimen. A single female nematode was transferred into a drop of distilled water on a microscopic slide and examined under a light microscope. The nematode specimen was transferred into deionized water, washed three times and then put into an Eppendorf tube with 8 µl distilled water. Then, 12 µl lysis buffer (500 mM KCl, 100 mM Tris-HCL pH 8, 15 mM MgCl2, 10 mM DTT, 4.5% Tween 20) and 2 µl proteinase K were added to the Eppendorf tube. Nematode specimen was crushed with a microhomogenizer during 2 min. The tubes were incubated at 65°C for an hour and then at 95°C for 15 min (Tanha Maafi et al., 2003).
For DNA amplification the protocol described by Tanha Maafi et al. (2003) was used. The D2 to D3 expansion regions of the 28S rRNA gene was amplified with the forward D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and the reverse D3B (5´-TCGGAAGGAACCAGCTACTA-3´) primers (Nunn, 1992). The 18S rRNA was amplified as two partially overlapping fragments, using three universal and one nematode-specific primer (1912R). First 18S fragment forward primer 988F (5´-CTCAAAGATTAAGCCATGC-3´) and reverse primer 1912R (5´-TTTACGGTCAGAACTAGGG-3´) and the second fragment forward primer 1813F (5´-CTGCGTGAGAGGTGAAAT-3´) and reverse 2646R (5´-GCTACCTTGTTACGACTTTT-3´) were used in the PCR reactions for the amplification of the 18S rRNA gene (Holterman et al., 2006). The ITS1-5.8S-ITS2 regions were amplified with the forward TW81 (5´-GTTTCCGTAGGTGAACCTGC-3´) and reverse AB28 (5´-ATATGCTTAAGTTCAGCGGGT-3´) primers (Joyce et al., 1994).
The PCR products were purified using the QIAquick Gel Extraction Kit (Takapozist, Iran) according to the manufacturer’s instruction and used for direct sequencing. The PCR products were sequenced in both directions (BioNeer Inc., Korea). The newly obtained sequences of the new species were submitted to GenBank database under accession numbers MN334221 for the 18S, MN334222 for the 28S D2-D3, and MN334228 for the ITS sequences.
The sequences of the studied specimens were compared with sequences of other taxa in GenBank, and then, the closest sequences were selected for phylogenetic analyses. The sequences of 18S rRNA and D2 to D3 segments of 28S rRNA were aligned with ClustalX 1.83 (Thompson et al., 1997), using default parameter values and were manually edited if necessary. The best fitted model of DNA evolution was obtained using jModelTest v. 2 (Darriba et al., 2012) with the Akaike information criterion (AIC). The best-fit nucleotide substitution models were considered to be GTR + I + G for 18S and 28S, and SYM + G for ITS. The phylogenetic tree of sequences was inferred by the Bayesian method using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Four MCMC chains for 1,000,000 generations were run. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given for appropriate clades. Pairwise divergences between taxa were computed as absolute distance values and as percentage mean distance values based on whole alignment, with adjustment for missing data with PAUP* 4.0b 10 (Swofford, 2002). Trees were visualized using TreeView (Page, 1996).
Morphometric characters of
Dehdasht population | Basht population | |||
---|---|---|---|---|
Characters | Holotype | 10 paratype females | 3 paratype males | 5 females |
L | 540 | 540 ± 65 (402–612) | 471 ± 33.6 (435–502) | 519 ± 65 (425–605) |
a | 14.9 | 14 ± 1.5 (10.3–15.3) | 24.6 ± 3.2 (20.9–27.1) | 12.6 ± 1.2 (10.9–14.4) |
b | 4.8 | 4.8 ± 0.6 (3.2–5.7) | 24.6 | 4.3 ± 0.5 (3.5–5.0) |
c | 14.5 | 15.8 ± 2.4 (12.1–19.5) | 10.5 ± 0.5 (9.9–11) | 19.1 ± 2.4 (16.3–21.9) |
c´ | 1.4 | 1.3 ± 0 (1.3–1.4) | 3.3 ± 0.5 (2.9–3.9) | 1.3 ± 0 (1.1–1.4) |
V | 90.5 | 90.3 ± 1.1 (87.8–91.9) | – | 90.1 ± 0.4 (89.8–90.8) |
Stylet | 56.9 | 55.2 ± 1.3 (52.8–57.2) | – | 56.8 ± 2.3 (54–60) |
Conus | 42.3 | 44 ± 6.3 (38.6–55.0) | – | 41.9 ± 1.6 (40.0–44.5) |
m (conus/stylet %) | 74.4 | 80.1 ± 11.2 (73.1–100.0) | – | 73.7 ± 1.7 (71.9–76.3) |
Pharynx | 110.7 | 113 ± 8 (103–124) | 59.3 | 119 ± 8 (105–126) |
Post-vulval body length (VL) | 50.7 | 51.8 ± 4.9 (44.6–58.9) | – | 51.2 ± 6.7 (43–60) |
Secretory-excretory pore | 109 | 108 ± 7 (99–118) | 106 ± 12 (92–113) | 108 ± 8 (97–118) |
Lip region-vulva | 485.2 | 488 ± 62 (353–553) | 468 ± 58.6 (382–545) | |
Lip region-anus | 498.9 | 506 ± 64.1 (372–575) | 426 ± 29.5 (396–455) | 492 ± 63.8 (399–577) |
Vulva-anus | 24.0 | 22 ± 2.9 (17.1–26.0) | – | 24 ± 5.3 (17–32) |
Tail length | 37.0 | 34.3 ± 3.6 (28.0–39.3) | 44.8 ± 4.5 (39.5–47.5) | 27.2 ± 2.9 (23–31) |
Body width | 36.1 | 38.3 ± 2.4 (34.1–42.0) | 19.2 ± 1.6 (17.5–20.7) | 41.2 ± 4.3 (34–46) |
Vulval body width (VB) | 30.4 | 30.1 ± 1.6 (27.2–32.3) | – | 30.7 ± 1.9 (27.5–32.5) |
VL/VB | 1.7 | 1.7 ± 0.1 (1.6–1.8) | – | 1.6 ± 0.1 (1.5–1.8) |
Annulus width | 5.2 | 5.4 ± 0.7 (4.1–6.5) | 2.8 ± 0.4 (2.3–3.2) | 5.7 ± 0.6 (4.9–6.8) |
R | 104.0 | 104.2 ± 4 (97–113) | 132 | 97 ± 4.3 (90–101) |
Rst | 16.0 | 15.5 ± 0.8 (14–17) | – | 14.5 ± 0.5 (14–15) |
Rph | 26.0 | 25 ± 1.4 (23–28) | – | 24.8 ± 1.9 (23–28) |
Rexp | 29 | 23.2 ± 4.2 (19–30) | 49.6 ± 2 (48–52) | 27.1 ± 4.2 (24–29) |
RV | 12.0 | 12 ± 0.8 (11–14) | – | 12.8 ± 0.8 (12–14) |
Ran | 8.0 | 9 ± 0.9 (8–10) | – | 7.4 ± 0.5 (7–8) |
RVan | 3.0 | 3.6 ± 0.4 (3–4) | – | 3.6 ± 0.8 (3–5) |
St/L × 100 | 10.6 | 10.3 ± 1.3 (9.2–13.6) | – | 11 ± 1.1 (9.4–12.7) |
Spicules | – | – | 34.2 ± 0.7 (33.6–35.0) | – |
Gubernaculum | – | – | 6.2 ± 0.9 (5.3–7.2) | – |
Body is slightly curved ventrally when relaxed by heat, assuming an open C-shaped. Cuticular annuli are retrorse, and margins are smooth to irregular across the entire body, without any hint of crenation. Not more than one anastomosis is observed. Cephalic region is not offset, tapering and slightly conical. Oral disc has rounded edges, and slit-like amphidial apertures are located laterally on the disc. Four small flattened submedian lobes are visible at the same level with a labial plate. Submedian lobes in SEM resemble a tongue with a central, longitudinal crease, not fused with labial plates. First body annulus is slightly smaller than the second annulus, not retrorse. Stylet is robust, with well-developed knobs that possess moderate anterior projections and 9 to 11 µm in width. Secretory–excretory pore is located at 27 to 30 annuli from the anterior end, almost at 4 to 5 annuli behind the pharyngeal basal bulb, which is small, pyriform, 10 to 13.5 µm in width and 18 to 20 µm in length. Female genital gonad is outstretched and spermatheca is slightly oval, offset from gonad, filled with globular sperm cells about 1 µm in diameter. Vagina is straight, occasionally slightly curved. Vulva is open with simple anterior lip. The post-vulval region of the body tapers gradually, ending in a pointed terminus or a small bilobed end annulus. SEM reveals that the anal opening is located usually three annuli posterior to the vulva.
Body is vermiform and curved ventrally when fixed by heat treatment. Lateral field has four distinct longitudinal incisures. Lip region has distinct transverse striation, 8 to 9 µm wide and 5 to 7 µm high. Stylet and pharynx are degenerated. Spicules (33.6-35.0 µm in length) are slender and tylenchoid, ending to a distinct penial tube (4-5 µm in length). Gubernaculum is simple and slightly curved. Bursa not is observed. Tail is elongated-conical, ending to a pointed terminus.
Similar to female in general characteristics. Body is straight or slightly curved ventrally after fixation. Annuli are retrorse, lacking any crenation and ornamentation. Total number of annuli approximately equals to that of females, but annuli are narrower than of females (average 4.4 vs 5.5 µm).
Regarding general morphometric characters and tail shape, our populations can also come similar to
The males recovered in the type population have a unique elongated tail with pointed terminus, which only could be observed in
The type population was found from a canebrake in Dehdasht, Kohgiloyeh and Boyer-Ahmad province (30°49.42´N, 51°28.91´E). The other population was recovered from the rhizosphere of dog-rose shrubs (
Holotype, 10 paratype females and three paratype males, as well as five female specimens from the other recovered population were deposited in the nematode collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran.
The species epithet refers to the name of Dr. Joaquín Abolafia, the well-known nematologist from University of Jaén, Spain, who works on nematode systematics.
The 28S alignment was 738 bp long and consisted of 58 sequences as ingroups and three sequences, including
List of species, collection localities and GenBank accession numbers of individual specimens used in this study for phylogenetic analysis based on 28S rRNA gene.
Species name | GeneBank accession no. | Locality | Species name | GeneBank accession no. | Locality |
---|---|---|---|---|---|
|
AY780979 | Belgium |
|
KF430515 | South Africa |
|
GU989627 | USA |
|
KC329574 | USA |
|
MH828126 | Russia |
|
KF430497 | USA |
|
MH828128 | Russia |
|
KP313844 | Iran |
|
MK170079 | South Africa |
|
MN334222 | Iran |
|
FN433874 | USA |
|
AY780968 | Venezuela |
|
JQ231183 | South Africa |
|
AY780969 | Russia |
|
JQ231184 | South Africa |
|
AY780967 | Italy |
|
KC937033 | China |
|
AB933464 | Japan |
|
KU722386 | Iran |
|
AB933465 | Japan |
|
MH444641 | China |
|
AY780951 | Italy |
|
JQ231186 | South Africa |
|
AB933468 | Japan |
|
JQ231187 | South Africa |
|
AY780961 | Germany |
|
AY780980 | Germany |
|
AY780963 | USA |
|
KT381016 | China |
|
AY780965 | Italy |
|
KT381017 | China |
|
FN433855 | USA |
|
JQ246422 | USA |
|
FN433858 | USA |
|
MK050500 | China |
|
FN433859 | USA |
|
MK371811 | India |
|
KC538862 | USA |
|
MK371813 | India |
|
MG680454 | Portugal |
|
KF856531 | Japan |
|
AY780955 | Venezuela |
|
MH142613 | China |
|
MF683230 | South Africa |
|
KF856521 | USA |
|
KU291239 | Iran |
|
FN433875 | Belgium |
|
AY780978 | Germany |
|
KF430512 | Italy |
|
AY780971 | Italy |
|
KF430482 | USA |
|
KM598334 | Iran |
|
KF430444 | New Zealand |
|
KM598335 | Iran |
|
GQ406240 | South Africa |
|
MH156801 | China |
|
GQ406241 | South Africa |
|
MH156802 | China |
|
MG019824 | Mozambique |
|
AY780960 | Italy |
|
MG701275 | China |
The 18S alignment was 1538 bp long and consisted of 60 sequences as ingroups and
List of species, collection localities and GenBank accession numbers of individual specimens used in this study for phylogenetic analysis based on 18S rRNA gene.
Species name | GeneBank accession no. | Locality | Species name | GeneBank accession no. | Locality |
---|---|---|---|---|---|
|
MF094908 | USA |
|
AJ966471 | GenBank |
|
MF094914 | USA |
|
KJ934172 | USA |
|
MF094899 | USA |
|
KJ934173 | USA |
|
MF094927 | USA |
|
MG701280 | China |
|
MF094968 | USA |
|
MF094981 | USA |
|
MF095015 | USA |
|
MF094928 | USA |
|
MF095024 | USA |
|
MF094996 | USA |
|
MF094902 | USA |
|
FJ969128 | GenBank |
|
MF095025 | USA |
|
MN334221 | Iran |
|
MF795587 | China |
|
MF094891 | USA |
|
MF095026 | USA |
|
MF094892 | USA |
|
MF094960 | USA |
|
MF094903 | USA |
|
MF094937 | USA |
|
MF094909 | USA |
|
MF795591 | China |
|
MF094893 | USA |
|
MF095031 | Costa Rica |
|
MF094965 | USA |
|
MH200615 | China |
|
MF094967 | USA |
|
MF095028 | Canada |
|
MF095012 | USA |
|
JQ446376 | USA |
|
KJ934182 | USA |
|
KJ934162 | USA |
|
KJ934180 | USA |
|
MH444626 | China |
|
KJ934177 | USA |
|
MG029558 | China |
|
MF095021 | USA |
|
MG029559 | China |
|
MF094992 | USA |
|
MH444635 | China |
|
MF094952 | USA |
|
MG029556 | China |
|
EU669919 | GenBank |
|
MG029557 | China |
|
MF094933 | USA |
|
AY284622 | GenBank |
|
KJ934175 | USA |
|
MF095013 | Thailand |
|
AY284631 | GenBank |
|
KJ934163 | USA |
|
MH136626 | China |
|
KJ934166 | USA |
|
AJ966511 | UK |
|
MF094911 | USA |
|
MF095001 | USA |
|
EU669914 | GenBank |
The ITS alignment was 401 bp long and consisted of 70 sequences as ingroups and
List of species, collection localities and GenBank accession numbers of individual specimens used in this study for phylogenetic analysis based on ITS rRNA gene.
Species name | GeneBank accession no. | Locality | Species name | GeneBank accession no. | Locality |
---|---|---|---|---|---|
|
MK874505 | South Africa |
|
MF094891 | USA |
|
MF683236 | South Africa |
|
HM116070 | USA |
|
MF683237 | South Africa |
|
HM116069 | USA |
|
KC937032 | China |
|
HM116058 | USA |
|
JQ231188 | South Africa |
|
HM116055 | USA |
|
MH444640 | China |
|
HM116052 | USA |
|
MH444639 | China |
|
HM116051 | USA |
|
JQ231189 | South Africa |
|
MH013431 | USA |
|
JQ231190 | South Africa |
|
KY574844 | USA |
|
MK292124 | USA |
|
KY574860 | USA |
|
KF856558 | USA |
|
KY574861 | USA |
|
KF856560 | USA |
|
KY574862 | USA |
|
KF856543 | USA |
|
KY574863 | USA |
|
MH444616 | China |
|
KY574864 | USA |
|
EF126179 | Taiwan |
|
KY574865 | USA |
|
MG029566 | China |
|
JQ708120 | USA |
|
MG029568 | China |
|
JQ708124 | USA |
|
KF856552 | Spain |
|
JQ708122 | USA |
|
MG029560 | China |
|
KY574858 | USA |
|
KF856555 | Spain |
|
KY574857 | USA |
|
MK371815 | India |
|
KY574856 | USA |
|
KM516185 | USA |
|
JQ708112 | USA |
|
KF856565 | South Africa |
|
HM116073 | USA |
|
MH142617 | China |
|
HM116057 | USA |
|
KM516186 | USA |
|
MF095021 | USA |
|
KM516190 | USA |
|
MF094992 | USA |
|
KM516191 | USA |
|
MF094915 | USA |
|
KF430576 | USA |
|
MF094916 | USA |
|
MG019827 | USA |
|
MF683239 | USA |
|
KF430577 | USA |
|
MF683238 | USA |
|
KF430569 | Russia |
|
MF683235 | USA |
|
MN334228 | Iran |
|
KF242257 | USA |
|
HM116062 | USA |
|
JN112274 | USA |
|
HM116064 | USA |
|
FJ588909 | China |
|
HM116066 | USA |
|
MH124562 | China |
|
HM116067 | USA |
|
MH124561 | China |
|
HM116068 | USA |
The genus
Summary of the diagnostic characteristics of 12 species of
Species | St | R | Rex | RV | RVan | Ran | V | VL/VB | Anas. | A. M. | Vagina | A. V. lip | Tail shape | L | S. M. L. | Original des. |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
52–60 | 90–113 | 19–30 | 11–14 | 3–5 | 7–10 | 87–90 | 1.5–1.8 | 1 | smo.–irr. | straight | simple | con–acute | 0.4–0.61 | flat | Present study |
|
48–52 | 140–147 | 36–38 | 9–12 | 1–2 | 8–9 | 93–95 | – | 0 | smo. | straight | lobulated, without projections | con.–rounded | 0.38–0.45 | rounded | Crozzoli and Lamberti (2001) |
|
58–62 | 102–108 | 25–28 | 11–15 | – | 5–7 | 89–93 | 1.2–1.8 | rare | smo.–ser. | straight | serrated | con.–pointed | 0.37–0.45 | – | Wouts (2006) |
|
79–86 | 89–95 | 26 | 8–9 | 1–2 | 5–6 | 91–92 | – | few | smo. | straight | with two projections | conical | 0.46–0.51 | flat | Crozzoli and Lamberti (2001) |
|
65–70 | 89–98 | 17–31 | 6–8 | 0–2 | 4–8 | 91–95 | rare | smo. | slightly curved | with two projections | con.–truncate | 0.40–0.49 | rounded | Cid del Prado Vera (2009) | |
|
51–60 | 85–92 | 25 | 6–7 | 2–3 | 3–4 | 92–93 | 0.9–1.2 | rare | – | straight | simple | rounded | 0.41–0.53 | rounded | Pramodini et al. (2007) |
|
45–49 | 108–114 | 33–35 | 8–9 | 2 | 4–6 | 93–94 | – | many | smo. | straight | lobulated, without projections | con.–truncate | 0.34–0.40 | rounded | Crozzoli and Lamberti (2001) |
|
45–59 | 84–113 | 24–31 | 6–11 | 2–3 | 3–7 | 90–96 | 0.7–1.6 | 1–4 | smo. | straight | with two projections | rounded | 0.39–0.60 | rounded | Olson et al. (2017) |
|
82 | 109 | 38 | 9 | 3 | 6 | 93 | – | 0 | finely crenate | straight | ? | conical | 0.22 | ? | Mohilal and Dhanachand (1998) |
|
49–61 | 107–119 | 27–34 | 10–14 | 2–4 | 6–10 | 89–93 | 1.0–1.4 | 0–1 | som.–irr. | straight | simple | conical | 0.38–0.51 | flat | Cordero et al. (2012) |
|
47–51 | 73–74 | 24–26 | 7 | 1 | 5 | 93–94 | – | many | som. | straight | lobulated, without projections | conical | 0.27–0.32 | rounded | Crozzoli and Lamberti (2001) |
|
66–78 | 117-140 | 26–31 | 9–10 | 4–5 | 4–6 | 92–94 | 0.9–1.0 | many | som.-irr. | sigmoid | simple | rounded | 0.43-0.53 | flat? | Pramodini et al. (2006) |