The genus
The type species of the genus,
Soil samples were collected from the rhizosphere of different plants and localities in Iran. Nematodes were extracted by the tray method (Whitehead and Hemming, 1965), killed and fixed by hot FPG (4: 1: 1, formaldehyde: propionic acid: glycerol) and processed to anhydrous glycerol (de Grisse, 1969). Nematodes were mounted in glycerol on permanent slides using paraffin wax and studied using a light microscope, equipped with a Dino-eye microscope eye-piece camera in conjunction with its Dino Capture version 2.0 software. Specimens were identified at species level using available identification keys (Geraert, 2008).
Nematode DNA was extracted from single individuals and stored at −20°C until used as PCR template. DNA extraction was performed using the protocols described by Tanha Maafi et al. (2003). The D2–D3 expansion fragments of 28S rRNA were amplified using the forward D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and reverse D3B (5'-TCGGAAGGAACCAGCTACTA-3') primers (Subbotin et al., 2006). The 30 μ l PCR contained 15 μ l 2× Taq DNA polymerase mix (Ampliqon, Denmark), 1 μ l (10 pmol μ l−1) each of forward and reverse primers, 2 μ l of DNA template and 11 μ l deionized water. PCR cycling conditions were as follows: denaturation at 95 °C for 4 min, then 33 cycles of denaturation at 94 °C for 30 sec, annealing at 57°C for 30 sec, and extension at 72°C for 90 sec, with a final extension was performed at 72°C for 10 min. The quality of PCR was checked by electrophoresis of 4 μ l of the PCR reaction in 1% agarose gel containing ethidium bromide and products were visualized and photographed under UV light. The length and concentration of each PCR product were measured by comparison with a low DNA mass ladder (Invitrogen, Carlsbad, CA). The PCR product was purified and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer (Bioneer, Seoul, South Korea). The newly obtained sequences were submitted to GenBank database under accession numbers MK542004 for
For phylogenetic relationships, analyses were based on D2–D3 expansion fragments of the 28S rRNA gene sequences. The newly obtained sequences were edited and aligned with another segments of 28S rRNA gene sequences available in GenBank using MUSCLE alignment tool implemented in the MEGA7 (Kumar et al., 2016). The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al., 2012) and phylogenetic tree was generated with the Bayesian inference method using MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001; Ronquist et al., 2012). The analysis under GTR+I+G model was initiated with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) for 1 × 106 generations. The tree was visualized and saved with FigTree 1.4.3 (Rambaut, 2014) and edited with Adobe® Acrobat® XI Pro 11.0.1. A sequence of
Morphometric characters of
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Character\Species | Holotype | Females | Males | Females | |||
n | – | 10 | CV | 8 | CV | 12 | CV |
L | 519 | 537 ± 22.3 (508–572) | 4.2 | 529 ± 31.4 (474–571) | 5.9 | 446 ± 29.9 (388–481) | 6.7 |
a | 26.6 | 27.3 ± 3 (22.5–33.6) | 11.1 | 36 ± 1.6 (33.7–38.2) | 4.6 | 25.6 ± 1.8 (23.2–28.1) | 7.3 |
b | 5.9 | 5.4 ± 0.3 (5.1–6.1) | 5.9 | 14.8 ± 0.4 (14–15.3) | 2.9 | 4.7 ± 0.4 (3.4–5.1) | 10.6 |
c | 4.5 | 4.7 ± 0.4 (4.3–5.7) | 9.6 | 4.6 ± 0.1 (4.3–4.9) | 4.0 | 5.8 ± 0.5 (4.9–6.5) | 9.1 |
c′ | 11.9 | 10.7 ± 1.2 (7.8–11.8) | 11.5 | 11.3 ± 0.9 (10.1–13) | 8.6 | 7.5 ± 0.7 (6.5–8.4) | 9.6 |
V or T | 62.6 | 63.8 ± 2.3 (61.8–69.6) | 3.7 | 34 ± 4.1 (28.6–39.4) | 12.1 | 66.4 ± 1.5 (64.6–69.9) | 2.3 |
V' | 80.4 | 80.5 ± 1.9 (76.7–83.2) | 2.4 | – | – | 80.2 ± 1.8 (77–82.9) | 2.3 |
Stylet | 13.5 | 12.8 ± 0.6 (12–13.9) | 5.2 | 12.4 ± 0.2 (12–12.9) | 2.3 | 10.9 ± 0.3 (10.5–11.4) | 2.8 |
m (conus/stylet %) | 45.3 | 46.1 ± 1.8 (43.2–48.3) | 3.9 | 47.8 ± 1.3 (45.8–49.5) | 2.7 | 44.8 ± 2 (42.2–48.5) | 4.7 |
Pharynx | 90 | 97.9 ± 5.8 (85–107) | 6.0 | 97.1 ± 4.4 (92–103) | 4.6 | 95.4 ± 9 (85–112) | 9.5 |
Median bulb | 44.5 | 44.6 ± 2.1 (41–47) | 4.9 | 44.6 ± 2.2 (42–49) | 5.1 | 41.9 ± 3.8 (38–50) | 9.3 |
MB | 47 | 45.6 ± 2.5 (42.4–51.7) | 5.6 | 45.9 ± 1.1 (44.5–47.5) | 2.4 | 43.9 ± 1.4 (41.8–46.6) | 3.2 |
Excretory pore | 79 | 81 ± 3.3 (76–86) | 4.2 | 79.9 ± 3 (74–83) | 3.8 | 75.6 ± 12 (59–103) | 15.9 |
Head-vulva | 325 | 340 ± 17.6 (314–370) | 5.2 | – | – | 296 ± 18.2 (257–319) | 6.2 |
Head-anus | 404 | 423 ± 22.7 (394–459) | 5.4 | 414 ± 24.1 (373–442) | 5.8 | 369 ± 28 (310–401) | 7.6 |
Vulva–anus | 89 | 82.6 ± 10.5 (71–104) | 12.8 | – | – | 73 ± 11.5 (53–92) | 15.8 |
Body width | 19 | 19.6 ± 1.6 (17–22) | 8.6 | 14.7 ± 1.1 (12.5–15.8) | 7.5 | 17.4 ± 2.1 (14.2–20.7) | 12.5 |
Vulval body width | 16 | 17.1 ± 1 (15.5–19) | 6.3 | – | – | 15.2 ± 1.5 (13–17) | 10.3 |
Anal body width | 9.7 | 10.6 ± 0.6 (9.7–12) | 5.8 | 10.1 ± 0.2 (10–10.6) | 2.3 | 10.2 ± 0.6 (9–11) | 6.5 |
Tail/Vulva–Anus | 1.3 | 1.4 ± 0.2 (0.9–1.8) | 17.4 | – | – | 1 ± 0.1 (0.7–1.4) | 17.6 |
Annulus width | 2.8 | 2.8 ± 0.1 (2.6–3) | 5.1 | 2.8 ± 0.2 (2.4–3) | 7.2 | 2.9 ± 0.2 (2.4–3.3) | 7.6 |
Tail length | 115 | 114 ± 9.9 (94–128) | 8.7 | 115 ± 8.9 (101–130) | 7.7 | 76.8 ± 6.9 (65–87) | 9.0 |
R | 216 | 222.4 ± 10 (210–240) | 4.5 | 214.6 ± 10.7 (201–233) | 5.0 | 188.5 ± 13.8 (170–210) | 7.3 |
Rv | 85 | 86 ± 5.8 (76–96) | 6.7 | – | – | 65.6 ± 7.5 (58–83) | 11.5 |
Ran | 54 | 53.2 ± 6.4 (41–59) | 12.2 | 53.8 ± 4 (48–60) | 7.5 | 31.7 ± 2.8 (28–36) | 9.0 |
Rvan | 30 | 32.8 ± 4.5 (26–40) | 13.9 | – | – | 33.9 ± 6.3 (27–47) | 18.7 |
Gubernaculum | – | – | – | 5.1 ± 0.3 (4.5–5.5) | 6.7 | – | – |
Spicules | – | – | – | 14.3 ± 0.8 (13–15.5) | 6.3 | – | – |
Figure 1:
Figure 2:
Body almost straight to slightly ventrally curved. Body annuli distinct, 2.6 to 3.0 µm wide at mid-body, and 2.3 to 2.7 µm at the Esophageal region. Lateral field protruded, longitudinal incisures begin 5 to 8 annuli posterior to head and continue until 15 to 22 annuli to anus, with two prominence ridges and four incisures, 5.3 to 7.5 µm wide, covering 26 to 40% of body. Cuticle with further 18 longitudinal ridges excluding lateral lines. Head continuous to the body contour or slightly offset by a depression, 2.8 to 4.0 µm high and 5.8 to 6.9 µm wide, narrower than the rest of body. Head with distinct transverse striae separating four narrow annuli, cephalic framework weakly developed. Amphidial apertures indistinct. Stylet short and delicate, with distinct knobs 2.0 to 3.2 µm wide, conus less than half of the total stylet length, 5.2 to 6.6 µm long; dorsal gland orifice 1.0 to 2.0 µm behind stylet knobs. Esophageal median bulb oval, 9.8 to 12.3 µm in length and 5.9 to 7.7 µm in width, filling 39 to 54% of the body diameter with small valve apparatus. Terminal bulb pear-shaped, 14 to 19 µm long and 7.7 to 9.5 µm wide. Excretory pore 76 to 86 µm from anterior end, or anterior to terminal bulb, at level or one annulus posterior to hemizonid; Nerve ring encircling middle of isthmus, 62 to 73 µm from anterior end. Deirids located at the same level, 76 to 87 µm from anterior end. Esophageal-intestinal valve distinct. Rectum small, curved. Anus usually indistinct. Female reproductive system monodelphic, anteriorly directed; vulva with lateral flaps about two annuli long. Vagina with thick walls, slightly bent anteriorly; post-vulval uterine sac short, 8 to 12 µm, about half of the vulval body width (45 to 60%). Spermatheca offset, usually bilobed, filled with globular to slightly oval sperm, about 1 µm in diameter. Tail elongated, with sharply pointed to filiform terminus.
Body straight to ventrally curved. Cuticle annuli 2.4 to 3.0 µm apart at mid-body and 2.3 to 3.0 µm at the Esophageal region. Lateral field 4.5 to 5.0 µm wide, occupying 30 to 36% of the body diameter, with two prominent separate ridges, hence four distinct incisures can be observed on mostly body length, without areolation. The cephalic region truncated, continuous or slightly set off from body with four annuli, 6.1 to 6.8 µm wide and 3.6 to 4.1 µm high. Cephalic framework not refractive. Stylet knobs well developed and globular, 2.1 to 2.5 µm in diameter. Esophageal median bulb oval, (5.8–6.5) × (9.8–12.0) µm. Bursa limited to the cloacal region, with crenate margins. Spicules slightly curved ventrally, with pointed tip. Gubernaculum simple, about one-third of spicule length. Tail elongated ending to a pointed to filiform terminus.
The new species,
The new species differs from
Soil around of wild fig (
Holotype, 10 paratype females and 7 males were deposited in the collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran.
The species epithet refers to the proximity of the new species with the other known species,
Figure 3:
Figure 4:
Body almost straight to slightly ventrally curved. Body annuli pronounce and wide, 2.4 to 3.3 µm at mid-body, and 2.3 to 2.9 µm at the posterior region of esophagus. Lateral field protruded, with two prominence ridges, 4.0 to 7.5 µm wide, occupying 28 to 39% of the body diameter. Head slightly offset by a depression, 2.5 to 3.3 µm high and 6.3 to 7.1 µm wide, narrower than its adjacent body, cephalic framework weakly developed. Amphidial apertures indistinct. Stylet short and delicate, with distinct knobs 2.2 to 2.7 µm wide; conus less than half of stylet length, 4.6 to 5.3 µm long, dorsal gland orifice 1.0 to 1.8 µm behind stylet knobs. Esophagus median bulb oval, 7.8 to 10.3 µm wide, filling 52 to 66% of the corresponding body diameter. Posterior bulb pear-shaped, 15 to 20 µm long and 8 to10 µm wide. Excretory pore from anterior end 59 to 103 µm, i.e. at the level of terminal bulb, at the same level with hemizonid or posterior to it; deirids at the same level or slightly posterior. Nerve ring encircling middle of isthmus, 55 to 78 µm from anterior end. Deirids at the level of excretory pore, 68 to 110 µm from anterior end. Esophageal-intestinal valve distinct. Rectum small, curved. Anus usually indistinct. Female reproductive system monodelphic-prodelphic. Lateral vulval flaps about two annuli or 4.2 to 6.8 µm long; vagina bent anteriad with thick walls, post-vulval uterine sac absent. Spermatheca offset, devoid of sperm. Tail conical with finely rounded to pointed terminus.
Not found.
This species was first described by Cobb (1925) from USA as
Our population was recovered from the rhizosphere of Alpine Milkvetch (
The amplification of D2-D3 expansion fragments of 28S rRNA gene sequences of
Figure 5: The 50% majority rule consensus trees from Bayesian analysis generated from the D2–D3 expansion fragments of 28 S rRNA gene dataset under GTR+I+G model. Posterior probabilities for BI analysis more than 50% are given for appropriate clades. New sequences are indicated in bold.
In our phylogeny tree, the new species formed a sister clade with three unknown populations of