According to Geraert (2008), the subfamily Tylenchinae (Örley, 1880), in the family Tylenchidae (Örley, 1880), currently includes 15 genera. The genus
During recent years, several taxonomic studies were performed on Tylenchidae in Iran (e.g. Gharahkhani et al., 2020; Hosseinvand et al., 2020; Panahandeh et al., 2019a, b). In the present study, two populations of the family Tylenchidae were recovered from natural forests of Golestan province, north Iran. The first species appeared as being a new member of
A total of 50 soil, and 36 rotten wood samples were collected from the natural forests in Golestan province, north Iran, during 2017 and 2018. The samples were placed in plastic bags, transferred to the nematology laboratory of Tarbiat Modares University and maintained at cool temperature condition. Nematodes were extracted from samples using the tray method (Whitehead and Hemming, 1965), heat killed by adding boiling 4% formalin solution and transferred to anhydrous glycerin according to De Grisse (1969). Drawings and morphological studies were performed using a drawing tube attached to a Nikon E600 light microscope; and were redrawn using CorelDraw software version 2012. The light microphotographs of the fresh individuals and mounted specimens were prepared using an Olympus BX51 microscope, equipped with a digital DP72 camera (Olympus) and differential interference contrast (DIC) optics.
Four mounted female specimens of the new species were selected for observation under SEM following the protocol of Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol and acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin scanning electron microscope (Carl Zeiss, Germany).
DNA was extracted from four female specimens of the both recovered populations by squashing each specimen in 15 µl TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0, Qiagen) (four DNA samples were prepared for each species) after their examination on temporary slides. DNA samples were stored at ‒20°C until used as PCR templates. Partial sequence of the SSU rDNA gene was amplified using primers 988F (5′-CTCAAAGATTAAGCCATGC-3′), 1912R (5′-TTTACGGTCAGAACTAGGG-3′), 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and 2646R (5′-GCTACCTTGTTACGACTTTT-3′) with resulting PCR products ranging from 890 to 930 and 970 to 1,017 bp, respectively (Holterman et al., 2006). The forward primer D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and reverse primer D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (Nunn, 1992) were used for amplification of D2-D3 expansion segments of LSU rDNA. The thermocycling program for amplification of both loci was as follows: denaturation at 95°C for 4 min, followed by 32 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 40 sec, and extension at 72°C for 80 sec. A final extension was performed at 72°C for 10 min. The PCR products were sequenced using the same primers used for their amplification. The newly obtained sequences were deposited into the GenBank database (accession numbers: MW346650 for the SSU sequence of the new species, MW346649 for the LSU of the new species; MW346646, MW346647, MW346648 for the LSU sequences of
The newly obtained SSU and LSU sequences were compared with those of other nematode species available in GenBank using the BLAST homology search program. The selected DNA sequences (for species and accession numbers, see SSU and LSU trees) for inferring the SSU and LSU phylogenies were aligned using ClustalX2 (
(Table 1; Figs. 1-3; Supplementary Fig. 1).
Morphometrics of
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Holotype | Paratypes | Males | Females | |
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1 female | 10 females | 2 | 8 |
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593 | 586 ± 22.3 (555-618) | 268, 290 | 318 ± 16.8 (296-341) |
|
39.5 | 37.0 ± 2.4 (30.8-39.5) | 26.8, 36.3 | 28.7 ± 3.2 (25-34) |
|
6.1 | 5.8 ± 0.3 (5.5-6.3) | 4.1, 3.6 | 4.0 ± 0.3 (3.6-4.3) |
|
4.7 | 4.7 ± 0.1 (4.5-4.8) | 5.7, 5.9 | 6.9 ± 0.5 (6.0-7.7) |
|
12.6 | 11.8 ± 0.7 (10.9-12.8) | 4.7, 8.9 | 7.2 ± 1.1 (6-9) |
|
63 | 63.1 ± 0.8 (62-64) | 33.6, 29.3 | 66.8 ± 4.8 (64-76) |
Cephalic region height | 2 | 2.4 ± 0.2 (2-3) | 2.5, 3.0 | 2.0 ± 0.5 (2-3) |
Cephalic region width | 5 | 5.1 ± 0.3 (5-6) | 5, 5 | 5.0 ± 0.5 (4-5) |
Stylet length | 10 | 9.6 ± 0.4 (9-10) | 7.2, 8.0 | 7.5 ± 0.5 (7-8) |
Conus length | 3 | 3.3 ± 0.3 (3-4) | 3.5, 4.0 | 2.5 ± 0.6 (3-4) |
m | 34 | 34.3 ± 1.6 (32-36) | 36.8, 42.5 | 37.8 ± 2.0 (35.5-41.9) |
Dorsal gland opening | 1 | 1.4 ± 0.3 (1-2) | – | – |
Median bulb from ant. end | 42 | 43.0 ± 2.3 (40-46) | – | – |
MB | 43 | 42.5 ± 2.7 (39-47) | – | – |
Excretory pore to ant. end | 80 | 80.3 ± 6.0 (72-94) | 54, 60 | 60.8 ± 3.8 (56-65) |
Pharynx length | 98 | 101.2 ± 3.3 (97-107) | 65, 80 | 81.5 ± 3.9 (75-86) |
Cephalic region to vulva | 375 | 370.3 ± 14.8 (350-389) | – | 211 ± 11 (197-226) |
Body width | 15 | 15.9 ± 1.3 (15-19) | 15, 18 | 11.0 ± 1.4 (9-13) |
Tail length | 126 | 125 ± 5 (115-133) | 47, 49 | 45 ± 5 (42-55) |
Tail/vulva-anus | 1 | 1.4 ± 0.1 (1.2-1.5) | – | – |
Anal body width | 10 | 10.6 ± 0.5 (10-11) | – | 7.4 ± 1.0 (6-9) |
Post-vulval uterine sac | 8 | 10.0 ± 1.5 (8-12) | – | 6.3 ± 0.8 (5-7) |
Line drawings of
Light microphotographs of
Scanning electron microphotographs of
Body slightly ventrally curved after heat relaxation. Annuli fine but distinct. Lateral fields with four incisures in cross section (Fig. 2E), lateral view under LM (Supplementary Fig. 1G) and SEM photos (Fig. 3E, H). In some specimens, there is a slightly wider portion on lateral field at 62 to 80 μm distance from vulva toward anterior body with irregular arrangement of lateral lines on only one side of the body. This portion is characterized by irregular lines forming about eight differently sized blocks (Fig. 3F and Supplementary Fig. 1E, F). Cephalic region continuous with the body, 2 to 3 μm high and 5 to 6 μm broad at base, having a disc-like structure at apex with 3 to 4 diameter under LM. SEM images showing a high, smooth cephalic region, lacking a true disc at apex, having one narrow annulus behind cephalic plate, the smooth region behind this annulus about twice body annuli wide, the amphidial apertures as elongated slits, starting behind cephalic plate, extending into anterior portion of the smooth region, the cephalic plate is four-lobed, includes four vestigial cephalic sensilla in the shape of shallow pits at corners of each lobe, and a small rounded oral aperture encircled by six sensilla. Stylet moderately developed, conus
Unknown.
The new species was recovered from a soil sample collected from the rhizosphere of
Holotype female and ten paratype females were deposited at the Nematology Collection of Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
The specific epithet refers to the disc-like differentiation in frontal end of cephalic region of the new species under LM.
The new species is mainly characterized by its disc-like differentiation at frontal end under LM, smooth cephalic region under SEM and a narrow annulus behind the cephalic plate (Fig. 3C). It is further characterized by having a squarish four lobed cephalic plate including vestigial cephalic papillae at corners, short longitudinal amphidial slits under SEM, lateral fields with four incisures, offset elongate-ellipsoid spermatheca filled with spheroid sperm, short PUS and elongate uniformly tapering tail with pointed tip. By lacking a true disc in frontal end (also see Discussion), and having a squarish cephalic plate with rounded corners, the new species was assigned to the genus
In comparison with the relevant species of
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By having a disc-like differentiation under LM, the new species was compared with three known relevant species of
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Light microphotographs of Golestan province population
The presently studied population of
The amplification and sequencing of SSU and LSU rDNA (D2-D3 region) fragments of the new species yielded single fragments of 788 and 726 nt long (accession numbers MW346650 and MW346649, respectively). The BLAST search using the SSU sequence, revealed that its highest identity (99.37%) with currently available SSU sequences of Tylenchidae belonged to
The SSU dataset included 69 sequences (including newly generated sequence of the new species and two aphelenchoidid sequences as outgroups). Figure 5 represents the Bayesian phylogenetic tree inferred using this dataset. In this tree, several species of
Bayesian 50% majority rule consensus tree inferred from SSU rDNA of
The LSU dataset was composed of 98 sequences (including newly generated sequence of the new species, three newly generated sequences for
Bayesian 50% majority rule consensus tree inferred from LSU rDNA D2-D3 of
In the present study, one new species of the family Tylenchidae was described and illustrated based upon morphological, morphometric, and molecular characters. It was assigned to the genus
Currently there are six species under the genus
There are now two well-accepted subgenera under
Light microphotographs of