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Systematic position of the genus Atetylenchus Khan, 1973 (Nematoda: Tylenchidae) with description of two new species

In: Nematology
Authors:
Manouchehr HosseinvandDepartment of Plant Protection, Faculty of Agriculture, University of Zanjan, 45371-38791, Zanjan, Iran

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Ali EskandariDepartment of Plant Protection, Faculty of Agriculture, University of Zanjan, 45371-38791, Zanjan, Iran

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Pablo CastilloInstituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Excelencia Internacional Agroalimentario (ceiA3), Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain

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Juan E. Palomares-RiusInstituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Excelencia Internacional Agroalimentario (ceiA3), Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain

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Reza GhaderiDepartment of Plant Protection, School of Agriculture, Shiraz University, 71441-65186, Shiraz, Iran

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Summary

Two new and one known species of Atetylenchus recovered from Dezful, Khuzestan Province, south-western Iran, are illustrated and described using morphological, morphometric and molecular data. Atetylenchus dezfuliensis n. sp. is characterised by medium-sized body 700-998 μm long, stylet 10.0-11.5 μm long, characteristic tail shape, first tapering regularly but then cylindrical to slightly clavate and 90-136 μm long, phasmids located at one-third anterior part of tail and short spicules 18.7 μm long. Atetylenchus longilabiatus n. sp. is characterised by small-sized body 593-720 μm long, prominent lip region, stylet 9.5-12.5 μm long, conical tail with hook-shaped terminus and 42-63 μm long, phasmids located near mid-tail and short spicules 17.5 μm long. Atetylenchus cf. graminus is characterised by small-sized body 643-787 μm long, stylet 10.7-11.7 μm long, vulva located at 51.7-55.1% of the body length, and conical tail with finely rounded end and 63-69 μm long. Molecular data are provided for the characterisation of this genus from these two new and one known species using ribosomal genes (18S and 28S rRNA). This genus is molecularly separated from Psilenchus, but closely related in both genes. These molecular markers showed different phylogenies for both genera; partial 18S showed their relationship with Tylenchidae, while 28S rRNA showed their relationship with Merliniidae. A key to the genus is provided for species identification.

Summary

Two new and one known species of Atetylenchus recovered from Dezful, Khuzestan Province, south-western Iran, are illustrated and described using morphological, morphometric and molecular data. Atetylenchus dezfuliensis n. sp. is characterised by medium-sized body 700-998 μm long, stylet 10.0-11.5 μm long, characteristic tail shape, first tapering regularly but then cylindrical to slightly clavate and 90-136 μm long, phasmids located at one-third anterior part of tail and short spicules 18.7 μm long. Atetylenchus longilabiatus n. sp. is characterised by small-sized body 593-720 μm long, prominent lip region, stylet 9.5-12.5 μm long, conical tail with hook-shaped terminus and 42-63 μm long, phasmids located near mid-tail and short spicules 17.5 μm long. Atetylenchus cf. graminus is characterised by small-sized body 643-787 μm long, stylet 10.7-11.7 μm long, vulva located at 51.7-55.1% of the body length, and conical tail with finely rounded end and 63-69 μm long. Molecular data are provided for the characterisation of this genus from these two new and one known species using ribosomal genes (18S and 28S rRNA). This genus is molecularly separated from Psilenchus, but closely related in both genes. These molecular markers showed different phylogenies for both genera; partial 18S showed their relationship with Tylenchidae, while 28S rRNA showed their relationship with Merliniidae. A key to the genus is provided for species identification.

Khan (1973) proposed the genus Atetylenchus for Tylenchus abulbosus Thorne, 1949, a species of Tylenchus lacking stylet knobs. Sher (1974) differentiated it from Psilenchus de Man, 1921 by the absence of slit-like amphidial apertures on the lip region and the low, flat lip region, as well as differences in the position of the median bulb; in A. abulbosus (Thorne, 1949) Khan, 1973, the median bulb is slightly anterior to middle of pharynx while in Psilenchus species it is distinctly posterior to the middle. A second species was added by Siddiqi (1986) after studying a paratype of Psilenchus graminus Bajaj, Kaushik & Bhatti, 1982. Geraert & Raski (1987) transferred Leipotylenchus amiri Maqbool & Shahina, 1984 to Atetylenchus. In this paper, the authors, using SEM, restudied the material used by Sher (1974) and found that large slit-like amphidial apertures are present together with six papillae around the oral opening. The fourth and fifth members of Atetylenchus were described as A. metaporus Erum & Shahina, 2008 and A. minor Yaghoubi, Pourjam, Atighi & Pedram, 2015 from Pakistan and Iran, respectively (Erum & Shahina, 2008; Yaghoubi et al., 2015).

Siddiqi (1986, 2000) observing the didelphic nature of the female reproductive system and the presence of phasmids, separated Atetylenchus from Tylenchidae Örley, 1880, and placed it in the Psilenchidae Paramonov, 1967 under the Dolichodoroidea Chitwood in Chitwood & Chitwood, 1950 (Siddiqi, 1986). Geraert & Raski (1987) did not consider the paired female genital system a character of sufficient value to sustain a separate subfamily or family and synonymised Psilenchinae Paramonov, 1967 with Boleodorinae Khan, 1964. Holterman et al. (2006, 2009) and Bert et al. (2008), studying the molecular phylogeny of Tylenchida Thorne, 1949, found unresolved support for the position of Psilenchus, which is treated as a sister-group to the Tylenchidae-Anguinidae complex or as a sister-group to all derived plant parasites, depending on the method used. Geraert (2008) considered Atetylenchus and Psilenchus under Boleodorinae in Tylenchidae. However, close relationships of Psilenchus (Subbotin et al., 2006; Ghaderi et al., 2014) and Atetylenchus (Yaghoubi et al., 2015) species with the members of Merliniidae Siddiqi, 1971 (Ryss, 1993) in the inferred phylogenetic trees support treating Psilenchidae as a separate family from Tylenchidae. However, more sequences from Atetylenchus are needed from both molecular markers (18S and 28S rRNA) in order to validate this hypothesis.

The objectives of this study were: i) to describe two new and one known species of Atetylenchus by morphological and molecular approaches using the D2-D3 segments and partial 18S rRNA gene sequences; ii) to provide a key for species identification in this genus; and iii) to determine the phylogenetic relationships of Atetylenchus with the representatives of Psilenchinae, Boleodorinae and Merliniidae.

Materials and methods

Nematode sampling and morphological identification

Soil samples were collected from the rhizosphere of different wild plants in Dezful, Khuzestan Province, south-western Iran. Nematodes were extracted by the tray method (Whitehead & Hemming, 1965), then killed and fixed by hot FPG (4:1:1, formaldehyde:propionic acid:glycerin), and processed to anhydrous glycerin (De Grisse, 1969). The nematodes were transferred to a drop of glycerin surrounded by a support ring of paraffin wax on permanent slides 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. Drawings were made through a drawing tube attached to a light microscope and redrawn using Adobe Photoshop 7.0 ME software. Specimens were identified at species level using available identification keys (Geraert, 2008).

Nematode molecular identification

For molecular analyses, and in order to avoid mistakes in case of mixed populations in the same sample, two specimens from each sample were temporarily mounted in a drop of 1 M NaCl containing glass beads (to avoid crushing/damaging the nematodes) to ensure specimens conformed with the unidentified populations. All necessary morphological and morphometric data were recorded by taking pictures and measurements using the above camera-equipped microscope. This was followed by DNA extraction from single individuals as described by Subbotin et al. (2000). The D2-D3 segments were amplified using the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers (De Ley et al., 1999). Finally, the portion of 18S rRNA was amplified using primers 988F (5′-CTCAAAGATTAAGCCATGC-3′), 1912R (5′-TTTACGGTCAGAACTAGGG-3′), 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and 2646R (5′-GCTACCTTGTTACGACTTTT-3′) (Holterman et al., 2006).

All PCR assays were carried out according to the conditions described by Archidona-Yuste et al. (2016). Then, the amplified PCR products were purified using ExoSAP-IT (Affimetrix, USB products) and used for direct sequencing on a DNA multicapillary sequencer (Model 3130XL genetic analyser, Applied Biosystems), using the BigDye Terminator Sequencing Kit V.3.1 (Applied Biosystems), at the Stab Vida sequencing facilities. The newly obtained sequences were submitted to the GenBank database under the accession numbers indicated on the phylogenetic trees.

Phylogenetic analyses

D2-D3 segments and partial 18S rRNA sequences of different Atetylenchus and Psilenchinae, Boleodorinae, and Merliniidae species were obtained from GenBank and used for phylogenetic reconstruction. Outgroup taxa for each dataset were chosen following previous published studies (Yaghoubi et al., 2015; Qiao et al., 2019). Multiple sequence alignments of both genes were made using MAFFT V.7.205 (Katoh & Standley, 2013). Sequence alignments were manually visualised using BioEdit (Hall, 1999) and edited by Gblocks ver. 0.91b (Castresana, 2000) in the Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences + 1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half). Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al., 2012) with the Akaike Information Criterion (AIC). The best-fit model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses. The general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2-D3 segments of 28S rRNA and 18S rRNA gene were run with four chains for 2 × 106 generations, respectively. A combined analysis of the two genes was not undertaken due to some sequences not being available for all species. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each 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 on appropriate clades. Trees from all analyses were visualised using FigTree software V.1.42 (http://tree.bio.ed.ac.uk/software/figtree/).

Results

Atetylenchus dezfuliensis* n. sp. = A. graminus apud Husseinvand et al. (2016) nec Bajaj et al. (1982) (Fig. 1)

Fig. 1.
Fig. 1.

Atetylenchus dezfuliensis n. sp. Female (A, C, E, G, H) and male (B, D, F, I). A, B: Entire body; C, D: Anterior end and pharyngeal region; E, F: Stylet and lip region; G: Reproductive system; H, I: Posterior end.

Citation: Nematology 22, 10 (2020) ; 10.1163/15685411-bja10019

Measurements

See Table 1.

Description

Female

Body slightly curved to C-shaped after fixation. Cuticle thick and finely annulated caμm at mid-body. Lateral field with three equally distant bands and four incisures, 6.3 (6.0-7.3) μm wide at mid-body. Lip region truncate, with four or five transverse annuli, 3.4 (3.2-3.7) μm high and 6.8 (6.4-7.3) μm wide. Amphidial apertures short with longitudinal grooves anteriorly. Stylet weak, 1.5 (1.3-1.7) times lip region diam. long, conus forming 41 (38-45)% of total stylet length, lacking distinct basal knobs but with swollen arms at base of shaft. Orifice of dorsal pharyngeal gland (DGO) located slightly less than one stylet length posterior to stylet base. Pharyngeal procorpus cylindrical, median bulb well developed, 8.3 (8.0-9.2) μm diam. and 17.2 (17.0-18.0) μm long, occupying 48 (41-55)% of corresponding body diam. Basal pharyngeal bulb nearly cylindroid, 9.8 (9.0-11.0) μm diam. and 22.5 (22.0-24.0) μm long. Excretory pore located at level of beginning of basal bulb, 97 (88-114) μm from anterior end. Deirids at level of excretory pore, hemizonid situated 1-2 annuli anterior to pore. Nerve ring surrounding middle part of isthmus, located 77 (71-87) μm from anterior end. Reproductive system with two well-developed genital tracts of nearly equal length, anterior gonad 194 (121-249) μm long and posterior gonad 200 (160-250) μm long, ovaries outstretched without any flexure, oocytes arranged in a single row. Spermatheca rectangular, with globular to slightly oval sperm, sperm slightly less than 1 μm in diam. Vulva in form of a transverse slit, vagina perpendicular to body axis, ca 0.5 vulval body diam. long. Vulva to anus distance 2.2-2.9 times tail length. Tail elongated clavate, tapering regularly until near terminus but becoming slightly broader near terminus, appearing as cylindrical to slightly clavate and ending in a finely rounded terminus. Phasmids located at 38 (28-43) μm from anus level or at ca 32 (27-38)% of tail length.

Male

Similar to female in general characteristics. Body slightly curved. Lateral field with four incisures. Pharynx slightly shorter than that of female. Basal pharyngeal bulb pyriform to slightly cylindroid. Testis outstretched, sperm cells observed in a single row. Spicules slender, ventrally arcuate, bursa short and adcloacal, 30-31 μm long, gubernaculum short with a slight proximal curvature.

Type host and locality

The present population was recovered from the rhizosphere of Astragalus sp. and mosses in Dezful, Khuzestan Province, south-western Iran (GPS coordinates 32°60′51″ N, 48°56′68″ E; altitude 605 m a.s.l.).

Type material

Female holotype, three female and one male paratype deposited in the collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran; two female paratypes deposited in the collection of the Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz, Iran; and two female and one male paratype deposited in the nematode collection of the Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Spain.

Table 1.
Table 1.

Morphometric characters of Atetylenchus dezfuliensis n. sp., A. longilabiatus n. sp. and A. cf. graminus recovered from Iran. All measurements are in μm and in the form: mean ± s.d. (range) coefficient of variation (CV).

Citation: Nematology 22, 10 (2020) ; 10.1163/15685411-bja10019

Diagnosis and relationships

Atetylenchus dezfuliensis n. sp. is characterised by the medium-sized body 808 (700-998 μm), lip region truncate without pronounced lobes, median pharyngeal bulb located at 45.7 (43.5-47.0)% of total pharynx, tail 118 (90-136) μm long, elongated conical in anterior half and cylindrical to slightly clavate in posterior half, phasmids located at ca one-third of tail posterior to anus, and short spicules 18.7 μm long.

Atetylenchus dezfuliensis n. sp. can be differentiated from all species in the genus by its characteristic tail, which is at first conical but does not taper towards the terminus and has a cylindrical to slightly clavate shape in its posterior half (vs typically conical or filiform in other species). It can be distinguished from A. abulbosus by slightly shorter body length (808 (700-998) vs 1000-1460 μm), smaller stylet (10.7 (10.0-11.5) vs 13-16 μm), and considerably shorter spicules (18.5 vs 33-37 μm); from A. graminus by longer tail (118 (90-136) vs 63-75 μm), and slightly shorter stylet (10.7 (10.0-11.5) vs 13 μm); from A. amiri by stylet form (without vs with weak knobs), slightly longer tail (118 (90-136) vs 80-85 μm), the number of lateral incisures (four vs five), and slightly shorter stylet (10.7 (10.0-11.5) vs 12-13 μm); from A. metaporus by slightly shorter body (808 (700-998) vs 970-1240 μm), smaller stylet (10.7 (10.0-11.5) vs 12-19 μm), and much shorter tail (118 (90-136) vs 165-230 μm); from A. minor by distinctly longer tail (118 (90-136) vs 51-62 μm), well developed ovaries vs less developed, and slightly shorter stylet (10.7 (10.0-11.5) vs 11.5-12.5 μm); and from A. longilabiatus n. sp. by differences in tail shape (clavate vs conical), lip region (truncate without raised lobes vs with pronounced lobes at anterior end), tail length (118 (90-136) vs 42-63 μm), and slightly longer body size (808 (700-998) vs 593-720 μm).

Remarks

Previously, a population named as A. graminus was reported from Dezful, Iran (Husseinvand et al., 2016). It should be noted that morphological and morphometric characters of a single female specimen from that population fitted well with those of A. dezfuliensis n. sp. (see Fig. 4C, J in that study). Indeed, the upper limit of the recorded range of the tail length (125 μm) and V (50) for A. graminus belongs to that specimen, which can is considered as a synonym of A. dezfuliensis n. sp. herein.

Atetylenchus longilabiatus* n. sp. (Fig. 2)

Fig. 2.
Fig. 2.

Atetylenchus longilabiatus n. sp. Female (A, C, E-H, J-L) and male (B, D, I, M). A, B: Entire body; C, D: Anterior end and pharyngeal region; E-G: Lip region; H: Reproductive system; I-K: Posterior end; L, M: Stylet and lip region.

Citation: Nematology 22, 10 (2020) ; 10.1163/15685411-bja10019

Measurements

See Table 1.

Description

Female

Body slightly curved in posterior part of body after fixation. Cuticle thick and finely annulated, annuli caμm apart at mid-body. Lateral field with four incisures, central band somewhat narrower than outer bands, 6.1 (5.0-7.0) μm wide at mid-body. Cephalic region truncate, with four or five transverse annuli, 3.0 (2.5-3.5) μm high and 7.1 (7.0-7.5) μm diam., four distinct raised lobes at anterior end producing a disc-like structure on lip region. Amphidial apertures in form of slightly sinuate longitudinal slits. Stylet weak, 1.6 (1.3-1.8) times longer than labial region diam., conus forming 43 (38-45)% of total length, lacking distinct basal knobs but with swollen arms at base of shaft. DGO located ca one stylet length posterior to stylet base. Procorpus cylindrical, median bulb well developed, 9.6 (9.0-10.5) μm diam. and 18.2 (15.5-20.0) μm long, occupying 65.5 (57.0-75.0)% of corresponding body diam. Basal bulb nearly cylindroid, 11.3 (9.5-13.0) μm diam. and 24 (20-29) μm long. Excretory pore at level of anterior part of basal bulb, 83 (73-90) μm from anterior end. Deirids at same level as excretory pore. Hemizonid located one or two annuli anterior to pore. Nerve ring at mid-isthmus, located 71 (60-80) μm from anterior end. Reproductive system didelphic-amphidelphic, with two well-developed genital branches of unequal length, anterior branch usually with one or two flexures. Ovaries with a single row of oocytes; spermatheca axial with gonad, bearing globular to slightly oval sperm cells caμm in diam. Vulva in form of a transverse slit, vagina perpendicular to body axis, extending inwards for less than 0.5 corresponding body diam. Vulva-anus distance 3.3-5.6 times tail length long. Tail conical, relatively short, strongly bent ventrally, ending in a hook-shaped (beak-like) terminus. Phasmids located at about middle of tail.

Male

Similar to female in general characteristics. Body straight and slightly shorter than female. Lateral field with four incisures. Raised lobes on lip region more expanded and pronounced. Pharynx slightly shorter than that of female, basal bulb pyriform. Testis outstretched, sperm cells arranged in single row. Spicules slender, ventrally arcuate, bursa adcloacal and short, 28 μm long, gubernaculum simple and short.

Type host and locality

Recovered from the rhizosphere of Cynodon dactylon (L.) Pers. and mosses in Dezful, Khuzestan Province, south-western Iran (GPS coordinates 32°49′06″ N, 48°73′81″ E; altitude 473 m a.s.l.).

Type material

Female holotype, seven female and one male paratype deposited in the collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran; two female paratypes deposited in the collection of the Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz, Iran; three female paratypes deposited in the nematode collection of the Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC), Spain.

Diagnosis and relationships

Atetylenchus longilabiatus n. sp. is characterised by the short body length of 640 (593-720 μm), lip region with four distinct pronounced lobes giving a disc-like structure, median pharyngeal bulb located at 45.0 (42.0-47.5)% of the pharynx, tail short conical, 56 (42-63) μm long, strongly bent ventrally and hook-shaped, phasmids located near the mid-tail, and short spicules 17.5 μm long.

Atetylenchus longilabiatus n. sp. can be distinguished from all known species in the genus by the expanded and pronounced lobes at the anterior end of both sexes, which produce a disc-like structure on the lip region. The population studied here also differs from A. abulbosus by shorter body length (640 (593-720) vs 1000-1460 μm), shorter tail (56 (42-63) vs 120 μm), smaller stylet (11.0 (9.5-12.5) vs 13-16 μm), and shorter spicules (17.5 vs 33-37 μm), as well as in the position of the phasmids (46 (41-55)% of tail length vs posterior to centre of tail); from A. graminus by shorter body length (640 (593-720) vs 810-1007 μm), slightly shorter stylet (11.0 (9.5-12.5) vs 13 μm), and shorter tail (56 (42-63) vs 63-75 μm); from A. amiri by shorter body length (640 (593-720) vs 800-1000 μm), stylet without vs with weak knobs, shorter tail (56 (42-63) vs 80-85 μm), V = 54 (52-56) vs 49-51%, tail shape (strongly curved ventrally vs straight), and tail terminus shape (regularly tapering vs becoming broader just near the terminus and ending in a pointed projection); from A. metaporus by a shorter body length (640 (593-720) vs 970-1240 μm), smaller stylet (11.0 (9.5-12.5) vs 12-19 μm), different tail shape (conical vs filiform), and tail length (56 (42-63) vs 165-230 μm); and from A. minor, to which it is close in morphometric characters and tail shape, yet differs by the lip region (with prominent lobes vs simple), ovary length (very long vs short) and lateral field (simple vs with crenate outer incisures).

Atetylenchus cf. graminus (Bajaj, Kaushik & Bhatti, 1982) Siddiqi, 1986 = Psilenchus graminus Bajaj, Kaushik & Bhatti, 1982

Measurements

See Table 1.

Description

Female

Body straight to slightly curved after fixation. Cuticle thick and finely annulated, annuli caμm apart at mid-body. Lateral field with three equally spaced bands and four incisures, 5.3 (4.5-6.0) μm wide at mid-body. Lip region truncate, with four transverse annuli, 3.5 (3.0-3.5) μm high and 6.6 (6.5-7.0) μm diam. Cephalic framework weakly sclerotised. Amphidial apertures inconspicuous. Stylet weak, 1.7 (1.6-1.8) times lip region diam. long, conus forming 39 (38-40)% of total stylet length, lacking distinct basal knobs but with swollen arms at base of shaft. DGO located less than one stylet length posterior to stylet base. Pharyngeal procorpus cylindrical, median bulb well developed, 9.4 (8.5-10.5) μm diam. and 17.7 (16.0-19.0) μm long, occupying 61 (57-66)% of corresponding body diam. Basal pharyngeal bulb nearly cylindroid, 11 (10-14) μm diam. and 29 (24-35) μm long. Excretory pore located at level of beginning of basal bulb, 81 (70-102) μm from anterior end. Deirids at level of excretory pore, hemizonid situated one or two annuli anterior to excretory pore. Nerve ring surrounding mid-isthmus region, located 72 (66-87) μm from anterior end. Reproductive system with two well-developed genital tracts of nearly equal length, anterior gonad 178 (164-186) μm and posterior gonad 154 (149-161) μm long, ovaries outstretched without any flexure, oocytes arranged in a single row. Spermatheca rectangular, without sperm. Vulva in form of a transverse slit, vagina perpendicular to body axis, extending inwards for less than 0.5 vulval body diam. Tail conical, ending in a finely rounded terminus. Phasmids located 24 (22-26) μm posterior from anus level or at ca 36 (34-40)% of tail length.

Male

Not found.

Remarks

Our population is close to both A. graminus and A. minor. It differs from A. graminus by a shorter body length (711 (643-787) vs 810-1070 μm), smaller stylet (11.2 (10.5-11.5) vs 13 μm), V = 53.0 (51.7-55.1) vs 45-51% (but in original drawing about 55%) and MB ratio (40.0 (38.5-43.5) vs 43-45%); and from A. minor by slightly longer body length (711 (643-787) vs 558-691 μm), slightly smaller stylet (11.2 (10.5-11.5) vs 11.5-12.5), V = 53.0 (51.7-55.1) vs 54-59%, longer tail (64 (63-69) vs 51-62 μm), distance from vulva to anus (266 (229-303) vs 182-238 μm), and longer reproductive system. We believe that these differences may be due to the low number of specimens in our population and so we tentatively refer it to Atetylenchus cf. graminus.

Key to species of Atetylenchus

  1. 1. Tail very long (165-230 μm), filiform .................... A. metaporus Erum & Shahina, 2008
  2. Tail shorter (< 140 μm), conical or clavate .................... 2

  3. 2. Body length 1-1.5 mm; stylet 13-16 μm long .................... A. abulbosus (Thorne, 1949) Khan, 1973
  4. Body length under 1 mm; stylet ⩽ 13 μm long .................... 3

  5. 3. Stylet with weak knobs; tail becoming broader near terminus, ending in a pointed projection .................... A. amiri (Maqbool & Shahina, 1984) Geraert & Raski, 1987
  6. Stylet without knobs; tail regularly tapering or clavate .................... 4

  7. 4. Lip region with four pronounced lobes giving a disc-like structure; phasmids at mid-tail .................... A. longilabiatus n. sp.
  8. Lip region simple, without such prominent lobes; phasmids at anterior half of tail .................... 5

  9. 5. Tail first conical, but then cylindrical to slightly clavate, 90-136 μm long .................... A. dezfuliensis n. sp.
  10. Tail conical, regularly tapering towards terminus, 51-75 μm long .................... 6

  11. 6. Body length < 0.7 mm; tail = 51-62 μm long; V = 54-59 .................... A. minor Yaghoubi, Pourjam, Atighi & Pedram, 2015
  12. Body length > 0.7 mm; tail = 63-75 μm long; V = 45-51 .................... A. graminus (Bajaj, Kaushik & Bhatti, 1982) Siddiqi, 2000

Molecular characterisation and phylogenetic relationships of Atetylenchus with other Psilenchinae, Boleodorinae and Merliniidae genera

The amplification of the D2-D3 expansion segments of the 28S and partial 18S rRNA genes yielded single fragments of approximately 900 and 1800 bp, respectively, based on estimation using gel electrophoresis. Six new D2-D3 of 28S rRNA gene sequences were obtained in the present study (MN807619-MN807624); these sequences showed 84-86% similarity values to other, far related genera, such as Pratylenchoides Winslow, 1958, Scutylenchus Jairajpuri, 1971, or Amplimerlinius Siddiqi, 1976 and 82% similarity with the close related genus Psilenchus (MK639384) using BlastN search in NCBI. The partial 18S rRNA from Atetylenchus spp. (MN807625-MN807629) matched well with the closely related genus Psilenchus, similarities ranging from 97.2 to 97.5% and followed by other genera such as Coslenchus Siddiqi, 1978 (AY284583) or Aglenchus Andrássy, 1954 (MK639387) with 97% and 96.8% similarities, respectively. The only partial 18S rRNA sequence from Atetylenchus minor (KP730045) had important problems in some sections of the sequence in the alignment used for the phylogenetic analysis. For this reason this sequence showed no similarity with our sequences or others deposited in GenBank of related genera.

There was no intraspecific variability for D2-D3 segments of 28S rRNA markers for two specimens of A. longilabiatus n. sp. or for A. cf. graminus, whereas for A. dezfuliensis n. sp. there were four nucleotides different (99.4% similarity) in D2-D3 segments of 28S rRNA and two nucleotides different (99.8% similarity) in the partial 18S rRNA between the two specimens studied.

Fig. 3.
Fig. 3.

Phylogenetic relationships among Atetylenchus species with genera of Psilenchinae, Boleodorinae, and Merliniidae. Bayesian 50% majority rule consensus tree as inferred from partial 18S rRNA gene sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. (Scale bar = expected changes per site.)

Citation: Nematology 22, 10 (2020) ; 10.1163/15685411-bja10019

Fig. 4.
Fig. 4.

Phylogenetic relationships among Atetylenchus species with genera of Psilenchinae, Boleodorinae, and Merliniidae. Bayesian 50% majority rule consensus tree as inferred from D2-D3 expansion segments of 28S rRNA sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. (Scale bar = expected changes per site.)

Citation: Nematology 22, 10 (2020) ; 10.1163/15685411-bja10019

Atetylenchus is well supported as a genus in both markers (PP = 1.00) (Figs 3, 4). It is related phylogenetically to Psilenchus in both markers studied and with moderate and good support in the partial 18S rRNA (Fig. 3) and D2-D3 region of 28S rRNA (Fig. 4) phylogenetic trees, respectively (PP = 0.91 and PP = 0.98: clade III and II). Other members of the family Tylenchidae related to the subfamily Boleodorinae, and with data available in GenBank, are included in other clades as Basiria Siddiqi, 1959 and Boleodorus Thorne, 1941, clade I in partial 18S rRNA and clade III in D2-D3 region of 28S rRNA. The phylogenetic relationship among the species described is similar for both markers. Atetylenchus longilabiatus n. sp. is closely related to A. cf. graminus (PP = 1.00) and both species are related to the clade containing A. dezfuliensis n. sp. (PP = 1.00).

Discussion

This research increased the number of species and the molecular diversity in Atetylenchus. Our data from the partial 18S rRNA include this genus within the Tylenchidae, which is in agreement with their close morphology with Psilenchus and the position under Boleodorinae in Tylenchidae as suggested by Geraert (2008). This is in disagreement with the molecular position of A. minor (Yaghoubi et al., 2015), in which the same marker included this genus with the Merliniidae. We did not include this sequence in our analysis because it showed a very low similarity and many mismatches on one side of this sequence when compared to our sequences from three species of the Atetylenchus and other sequences in our dataset.

In our 28S rRNA phylogeny, the position of Atetylenchus is highly supported with Merliniidae. A similar result is shown for the closely related genus Psilenchus with a broader phylogeny in Qing et al. (2017) for 28S rRNA, and in Qing & Bert (2019) for a combined dataset of both ribosomal markers. We agree more with a position inside the Tylenchidae as the 18S rRNA marker is better suited for the understanding of genus evolution in the context of families – it is a highly conserved gene and shows a lower mutation rate in comparison to 28S rRNA. Even for some specific genera (Malenchus Andrássy, 1968 and Filenchus Andrássy, 1954) the 28S rRNA showed high substitution rates using the substitution saturation test (Qing et al., 2017).

Further research is required to elucidate the systematic position of Atetylenchus and the broad phylogeny of Tylenchidae using other markers or combination of them, as suggested by Qing & Bert (2019).

*

Corresponding author, e-mail: eskandari.a@znu.ac.ir

*

The species epithet refers to Dezful, the Iranian city from where the new species was collected.

*

The species epithet refers to the expanded and pronounced lip region in females and males of the species.

Acknowledgements

The authors thank anonymous reviewers and the editor for their valuable suggestions to improve the manuscript. J.E.P-R. acknowledges the Spanish Ministry of Economy and Competitiveness for the ‘Ramon y Cajal’ Fellowship RYC-2017-22228.

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