The cosmopolitan genus
Some nematologists place
During a survey on nematodes of the Karkheh protected area in Khuzestan province, southwest Iran,
Several soil samples were collected from the rhizosphere of euphrates poplar (
For molecular analyses, single female specimens were picked out, examined in a drop of distilled water on a temporary slide under the light microscope, transferred to 3 μl of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) on a clean slide, and then crushed using a cover slip. The suspension was collected by adding 20 μl TE buffer. The DNA samples were stored at –20°C until used as a PCR template. Primers for LSU rDNA D2-D3 amplification were forward primer D2A (5′-ACAAGTACCGTGAGGGAAAGT-3′) and reverse primer D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (Nunn, 1992). Primers for amplification of ITS rDNA were forward primer rDNA1 (5′-TTGATTACGTCCCTGCCCTTT-3′) and reverse primer rDNA1.58S (5′-ACGAGCCGAGTGATCCACCG-3′) (Subbotin et al., 2000). The 30 μl PCR mixture contained 10 μl of distilled water, 15 μl of Master Mix (2X), 1 μl of each primer (10 pmol/μl), and 3 μl of DNA template. The thermal cycling program for amplification of both markers was as follows: denaturation at 95°C for 6 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52.5°C (LSU D2-D3 primers)/54.7°C (ITS rDNA primers) for 30 s, and extension at 72°C for 60 s. A final extension was performed at 72°C for 10 min. Amplification success was evaluated by electrophoresis on 1% agarose gel. The PCR products were purified using the QIAquick PCR purification kit (Qiagen®) following the manufacturer’s protocol and sequenced directly using the PCR primers with an ABI 3730XL sequencer (Bioneer Corporation, South Korea). The newly obtained sequences of the studied species were deposited into the GenBank database (accession numbers MZ725030/MZ725031 for LSU D2-D3 and MZ725020 for ITS rDNA).
The newly obtained sequences of the D2-D3 fragments of LSU rDNA and ITS rDNA and additional sequences of relevant species were selected after a BlastN search. The sequences were aligned by Clustal X version 2 using the default parameters (Larkin et al., 2007). The editing of both alignments was performed manually in MEGA7 program (Kumar et al., 2016). The base substitution model was selected using MrModeltest 2 (Nylander, 2004) based on the Akaike information criteria. A general time reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR + G + I), was selected for the both phylogenies.
The Bayesian analysis was performed to infer the phylogenetic trees using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), running the chains for four million generations. After to discard burn-in samples and to evaluate convergence, the remaining samples were retained for further analyses. The Markov chain Monte Carlo (MCMC) method within the Bayesian framework were used to determine equilibrium distribution and help estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using the 50% majority rule. Bayesian posterior probability (BPP) values higher than 0.50 are given on appropriate clades. The output files of the phylogenetic program was visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and re-drawn in CorelDRAW software version 17.
Morphometrics of
Khuzestan province, Iran | Handoo et al. (2014) Córdoba, Córdoba, Spain | Tzortzakakis et al. (2018) Crete, Greece | |||
---|---|---|---|---|---|
Character | Females | Males | Females | Males | Females |
n | 14 | 9 | 7 | 10 | 3 |
L | 622.1 ± 41.0 (570–704) | 661.7 ± 58.0 (591–763) | 662 ± 38.4 (612–732) | 640 ± 60.9 (548–760) | 722 ± 47 (670–760) |
a | 25.6 ± 3.5 (21.8–2.8) | 29.5 ± 0.1 (27.6–32.8) | 30.4 ± 1.7 (27.8–32.5) | 31.0 ± 3.9 (27.0–39.1) | 32.3 ± 0.6 (31.9–33.0) |
b | 5.5 ± 0.4 (5.0–6.3) | 5.9 ± 0.4 (5.3–6.5) | 6.0 ± 0.4 (5.6–6.8) | 6.1 ± 0.7 (5.4–7.8) | 6.3 ± 0.4 (6.1–6.8) |
c | 13.3 ± 0.7 (12.8–15.7) | 14.6 ± 0.7 (13.5–15.7) | 14.2 ± 1.7 (11.8–16.8) | 17.7 ± 1.4 (15.2–20.7) | 14.9 ± 0.6 (14.4–15.5) |
c′ | 2.8 ± 0.4 (2.5–3.2) | 2.9 ± 0.2 (2.6–3.2) | 2.9 ± 0.3 (2.4–3.3) | 2.7 ± 0.2 (2.5–3.0) | 3.0 ± 0.2 (2.7–3.2) |
V | 54.3 ± 1.5 (52.7–57.5) | - | 54.6 ± 1.5 (52.5–57.0) | - | 55.0 ± 1.0 (54–56) |
Lip region height | 3.7 ± 0.9 (3.2–4.1) | 3.4 ± 0.3 (3.2–4.1) | 3.6 ± 0.3 (3–4) | - | - |
Lip region width | 6.7 ± 0.2 (6.5–7.0) | 6.4 ± 0.1 (6.0–6.5) | 7.2 ± 0.3 (6.5–7.5) | - | - |
Stylet length | 17.5 ± 0.3 (17–18) | 17.1 ± 0.4 (16.2–17.5) | 16.1 ± 0.5 (15.5–17.0) | 15.9 ± 0.8 (15.0–18.0) | 17.3 ± 0.3 (17.0–17.5) |
m | 58.0 ± 2.7 (55.7–63.1) | 54.2 ± 2.7 (48.2–57.3) | - | - | - |
DGO | 2.9 ± 0.2 (2.6–3.0) | 2.8 ± 0.2 (2.5–3.0) | 2.5 | - | - |
MB | 49.2 ± 2.1 (46.8–52.6) | 51.2 ± 4.0 (45.2–57.3) | - | - | - |
Maximum body width | 24.7 ± 7.4 (20.8–29.2) | 20.8 ± 1.3 (18.8–23.4) | 21.8 ± 1.0 (20.5–23.5) | 20.7 ± 1.3 (18.0–23.0) | 22.3 ± 1.2 (21–23) |
Pharynx length | 115.3 ± 6.7 (105–120) | 116.0 ± 6.8 (106–124) | 110.0 ± 6.5 (100–120) | 105.4 ± 8.2 (90–120) | 114.0 ± 5.3 (110–120) |
Anterior end to median bulb | 56.7 ± 3.8 (50.0–61.7) | 56.9 ± 2.8 (52–58) | - | - | - |
Anterior end to nerve ring | 77.8 ± 2.9 (73.0–84.5) | 80.0 ± 4.2 (75.0–87.7) | 83.1 ± 5.6 (73–95) | - | - |
Anterior end to excretory pore | 93.3 ± 6.1 (85.5–104.0) | 87.6 ± 9.0 (87–104) | 89.7 ± 3.9 (85–95) | - | - |
Body width at vulva | 24.5 ± 2.7 (21.5–27.9) | - | - | - | |
Anal body width | 16.5 ± 1.5 (15.0–19.5) | 15.4 ± 1.5 (13.6–17.5) | 16.1 ± 1.4 (13.5–17.5) | 12.8 ± 0.8 (12–15) | 16.5 ± 2.2 (14–18) |
Tail length | 47.4 ± 6.5 (37.0–55.2) | 43.9 ± 3.5 (39.5–48.0) | 47.4 ± 6.3 (37.5–57.0) | 36.6 ± 5.6 (30–50) | 48.3 ± 3.1 (45–51) |
Tail annuli | 51.8 ± 5.3 (40–58) | - | 55.1 ± 8.1 (40–66) | - | - |
Spicule length | - | 25.5 ± 0.8 (24.0–26.5) | - | 25.3 ± 1.8 (23–30) | - |
Gubernaculum length | - | 12.5 ± 0.4 (12.0–13.3) | - | 11.4 ± 1.6 (10–15) | - |
Body arcuate ventrally to open C shape after heat fixation. Cuticle annuli 1–1.4 µm wide at mid-body. Lateral field with four incisures along the body, including the tail region, outer two incisures areolated. Lip region rounded, bearing 5–6 fine annuli, continuous to slightly offset from the body, cephalic framework slightly sclerotized. Stylet knobs rounded, laterally to posteriorly directed, 3–4 µm across. Median esophageal bulb elliptical to slightly oblong, 14.2 ± 1.2 (13.0–16.2) µm long and 12.0 ± 1.1 (11.7–13.1) µm wide, hemizonid usually two to three annuli anterior to excretory pore, almost 1.5 annuli wide, basal bulb pyriform, 25.0 ± 2.8 (20.0–29.2) µm long and 14.8 ± 1.3 (14.0–16.5) µm wide. Cardia well developed. Intestinal fasciculi present in the intestinal region. Reproductive system didelphic-amphidelphic, vagina 9–11 µm long, epiptygma absent, vulva a transverse slit, spermatheca rounded, filled with rounded sperm. Tail rounded hemispherical, tail terminus annulated, hyaline portion 6–8 µm. Phasmids located almost at the middle of the tail, at 22.4 ± 0.5 (21.5–23.1) µm distance behind the anus. Post-anal intestinal sac absent.
General morphology is similar to that of female except for character states associated with sexual differences. Tail conoid and pointed, enveloped by bursa. Spicules slightly curved ventrally. Gubernaculum well developed, half of the spicule length. The bursa is 61.5 ± 3.0 (52–65) µm long.
Among the
From
The general morphology of the recovered population of the species closely resembles the characters given for the type population (Handoo et al., 2014) and another population from Greece (Tzortzakakis et al., 2018). The type population from Spain and another population from Greece were extracted from the rhizosphere of olive. The presently studied species was recovered from the rhizosphere of euphrates poplar tree in the Karkheh protected area (GPS coordinates: 31°53ʹ49.308ʺN, 48°15ʹ33.3ʺE), Khuzestan province, southwest Iran.
Two 713 nt long D2-D3 expansion segments of LSU rDNA (MZ725030, MZ725031) were obtained for the Iranian population of this species. The BLAST search using these sequences revealed they have 99.85% identity with other sequences of the same species (MG770479, KJ461545 and KJ461547). A total of 74 sequences of Tylenchoidea Örley, 1880 and two sequences of Aphelenchoidea (Fuchs, 1937) Thorne, 1949 as outgroup taxa (LC583316 and DQ328683), were selected for the LSU phylogeny. This dataset comprised 796 total characters. The phylogenetic tree inferred using this dataset is presented in Figure 3. The newly generated sequences of the Iranian population of
The amplification and sequencing of the ITS rDNA of the Iranian population of
The objectives of this study were the morphological and molecular characterization of the Iranian population of
In the present study based on the 28S rRNA gene and ITS rRNA gene,