Morphological and molecular characterization of Pungentus sufiyanensis n. sp. and additional data on P. engadinensis (Altherr, 1950) Altherr, 1952 (Dorylaimida: Nordiidae) from northwest of Iran

Abstract Two species of the genus Pungentus, one new and one known, collected in natural vegetation and cultivated soils in northwest of Iran, are studied. Pungentus sufiyanensis n. sp. is characterized by its 1.22 to 1.57 mm long body, offset lip region by a constriction and 7 to 9 μm broad, 18 to 21 μm long odontostyle, 304 to 348 μm long neck, 133 to 161 μm long esophageal expansion, mono-opisthodelphic female genital system without anterior uterine sac, slightly backward directed vagina, absence of pars refringens vaginae, V = 47−54, rounded-conoid caudal region (17.5–23 μm, c = 65–84, c´ = 0.7–1) with saccate bodies, and the absence of male. Molecular analysis, based on D2-D3 expansion segments of the 28S rDNA (LSU), confirms the monophyly of the family Nordiidae and suggests the monophyly of the genus Pungentus, with the new species forming a clade with other Iranian species. New data are presented for six Iranian populations of P. engadinensis, and an updated key for the identification of Pungentus species is also provided.

The genus Pungentus is an interesting dorylaimid genus, often found in forest habitats of the Northern Hemisphere, and with very restricted presence in southern territories. Its taxonomy was updated by Álvarez-Ortega and Peña-Santiago (2014), who listed 16 valid species and other four inquirendae or incertae sedis and provided a key to their identification as well a compendium of their main morphometrics.
Several Pungentus populations were collected in the course of a nematological survey conducted in natural and cultivated soils of northwest Iran (East-West Azarbaijan and Kurdistan provinces) to explore the dorylaimid diversity of this region. Their study revealed that they belonged to one new and one known species. The objective of this work was to report Pungentus sufiyanensis n. sp. using morphology, morphometric, and molecular methods and provide new data about P. engadinensis.

Materials and methods
Extraction and processing of nematodes Soil samples were collected from the rhizosphere of several crops and orchards of East-West Azarbaijan and Kurdistan provinces, northwest Iran, during the period 2010-2017. Nematodes were extracted following the protocols by Jenkins (1964) and Whitehead and Heming (1965), transferred to anhydrous glycerin according to De Grisse (1969), and mounted on glass slides for handling.

Light microscopy
Mounted specimens were observed under an Olympus BX 41 light microscope equipped with a drawing tube and a DP50 digital camera attached to it. Morphometrics include Demanian indices and the usual measurements and ratios. Line illustrations were prepared using CorelDRAW ® software version 12. Microphotographs were edited using Adobe ® Photoshop ® CS software.

DNA extraction, PCR and sequencing
For the molecular study of the new species, DNA samples were extracted from a live adult nematode, hand-picked, and placed on a clean slide containing a drop of distilled water or worm lysis buffer (WLB) and crushed by a sterilized scalpel. Then, the suspension was transferred to an Eppendorf tube containing 25.65 μ l ddH2O, 2.85 μ l 10 × PCR buffer and 1.5 μ l proteinase K (600 μ g/ml) (Promega, Benelux, the Netherlands). The tubes were incubated at −80°C (1 h), 65°C (1 h) and 95°C (15 min). The extracted DNA was stored at −20°C until use. The D2-D3 domains of the 28S rDNA were amplified with forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and reverse primer D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Nunn, 1992). In total, 25 μ l PCR reaction mixture was prepared constituting of 10 μ l ddH 2 O, 12.5 μ l master mix (Ampliqon, Denmark), 0.75 μ l of each forward and reverse primers, and 1 μ l of DNA template. PCR was carried out using a BIO RAD thermocycler machine in accordance with Archidona-Yuste et al. (2016). PCR cycle conditions were as follows: denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 s, annealing of primers at 55°C for 45 s and extension at 72°C for 3 min followed by a final elongation step at 72°C for 10 min. The purified PCR products were sent for sequencing to Bioneer Company, South Korea. The newly obtained sequences of P. sufiyanensis n. sp. were deposited in the GenBank database under accession number MN855359 as indicated on the phylogenetic tree of Table 2.

Phylogenetic analyses
The newly generated sequences were aligned with the other segments of 28S rDNA gene sequences avai lable in GenBank using MEGA6 software (Tamura et al., 2013). Paravulvus hartingii (de Man, 1880) Heyns, 1968 (AY593062) as outgroup was chosen. Bayesian analysis (BI) was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The best fit model of DNA evolution was obtained using MrModeltest 2.3 (Nylander, 2004) with Akaike-supported model in conjunction with PAUP* v4.0b10 (Swofford, 2003). BI analysis under the general time-reversible model with invariable sites and a gamma-shaped distribution (SYM + I + G) model for the 28S rDNA gene was done. 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 and posterior probabilities (PP) were given on appropriate clades. The tree was visualized using the program Figtree 1.4.3 v.

Description
Female: slender (a = 40-50) nematodes of medium size, 1.22 to 1.57 mm long. The body cylindrical, tapering toward both ends but more so toward the anterior extreme as the caudal region is short and rounded. Upon fixation, habitus slightly curved ventrad, to an open C-shape. Cuticle three layered, especially distinguishable at caudal region, bearing fine transverse striations, 2 to 3.5 μ m thick at anterior region, 3 to 6 μ m at mid-body, and 7 to 10 μ m at tail. Lateral chords 8 to 11 μ m thick or occupying onefourth to one-third of mid-body diameter. The lip region is somewhat angular, offset by a weak but perceptible constriction, with nearly truncated anterior margin, 2.1 to 2.6 times as wide as height and 21 to 27% of body diameter at neck base; lips mostly amalgamated, with hardly protruding papillae. Amphidial fovea cupshaped, opening at the level of constriction, with the aperture 4 to 5 μ m long or 52 to 60% of lip region diameter. Cheilostom nearly cylindrical, 1.2 to 1.8 times as long as the lip region diameter, with visible sclerotised walls in its anterior half, and bearing four distinct, sclerotized, circumoral platelets. Odontostyle slightly arcuate dorsally, slender, well sclerotized, 2.0 to 2.5 times as long as the diameter of lip region, 1.1 to 1.4% of total body length, and aperture 2 to 3 μ m long or occupying 9 to 17% its length. Guiding ring double. Odontophore rod-like, 0.8 to 1.0 times the odontostyle length. Nerve ring situated at 109 to 121 μ m or 30 to 35% of the neck length from the anterior end. Pharynx entirely muscular, consisting of an anterior portion enlarging gradually into the basal expansion that is 8.4 to 13 times as long as width, 4.1 to 5.2 times as long as body diameter at neck base and occupies 40 to 45% of total neck length; gland nuclei located as follows: DN = 61−63, S 1 N 1 = 68−70, S 1 N 2 = 77−80, S 2 N = 89−92 according to Loof and Coomans (1970). Cardia hemispherical, almost as long as wide, 8-11 × 7-10 μ m. Genital system monoopisthodelphic, without anterior uterine sac. Genital branch well developed, 154 to 203 μ m long or 9 to 16% of total body length. Ovary reflexed, 61 to 97 μ m long, usually not reaching the sphincter level, with oocytes arranged first in several rows and then in a single row. Oviduct joins ovary subterminally, 48 to 62 μ m or 1.1 to 2.0 times the corresponding body diameter long, consisting of a slender portion made up of prismatic cells and developed pars dilatata with perceptible lumen. Oviduct-uterus junction marked by a sphincter. Uterus a simple tube-like structure, 37 to 51 μ m long or 0.9 to 1.5 times the corresponding body diameter long. Sperm in genital tract absent. Vagina slightly directed backward, extending 15 to 20 μ m inwards and occupying 39 to 45% of the corresponding body diameter; pars proximalis vaginae 9-12 × 11-15 μ m, with nearly sigmoid walls and surrounded by moderately developed, circular musculature and pars distalis 2 to 3.5 μ m and pars refringens vaginae obscure in specimens examined. Vulva a nearly equatorial, transverse slit, preceded by a V-shaped depression of body surface. Prerectum 2.1 to 3.2 and rectum 0.6 to 1.0 times as long as the anal body diameter. The caudal region short, rounded-conoid, slightly more straight at the ventral side, where it bears saccate bodies; two pairs of caudal pores are present.

Rhyssocolpus vinciguerrae Astara forests, north-western Iran Forest trees KP204547
Male: unknown. Molecular characterization: one sequence of the D2-D3 segment of 28S rDNA nearly 800 bp long from the new species was obtained. The results of its analysis are represented in the molecular tree of Figure 3.
P. sufiyanensis n. sp. is phylogenetically related to P. azarbaijanensis but can be differentiated by the female genital system (mono-opisthodelphic vs didelphic-amphidelphic).
A Nblast search of the D2-D3 sequence of P. sufiyanensis n. sp. showed 96, 96, 99, 96, and 95% of similarity with P. azarbaijanensis (MH346476), P. engadinensis (AY593050), P. monohystera (MF325343), P. silvestris (AY593052), and Enchodelus macrodorus (AY593054), respectively, with 27, 26, 2, 26, and 26 different nucleotides, respectively, too. As derived from the analysis of the new sequence herein obtained, the evolutionary relationships of the new species with other representatives of the order Dorylaimida are shown in Figure 3. The most remarkable achievement is that the new species comes close to P. azarbaijanensis, another Iranian species. These both species form a clade together with other Pungentus species, suggesting a low supported monophyly of this genus based upon currently available sequences. All the sequences of Nordiidae representatives constitute a highly supported (100%) clade, a fact that confirms the monophyly of this taxon. Leaving aside Pungentus sequences, the remaining ones form together a second clade, which is not well supported, within the family Nordiidae.
Other localities and habitats: samples were collected from two locations in Northwest Iran: East-Azarbaijan province, Marand district, Kondolaj village, from the rhizosphere of almond and walnut trees; West Azarbaijan province, Bokan district, Khorasaneh area (GPS coordinates: N 36°35´ 68˝, E 46° 00´ 90˝) from the rhizosphere of natural vegetation.
Type material: female holotype and paratypes were deposited with the Nematode Collection of the Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. The new species binomial has been registered in the Zoobank database (zoobank.org) under the identifier B1F2B3F6-558F-4688-BFFC-0F90BD101357.
Etymology: the species name refers to the type locality of the new species, Sufiyan, East-Azarbaijan province, northwest of Iran.
Pungentus engadinensis (Altherr, 1950) Altherr, 1952; Table 3) Remarks: the six populations of this species herein examined are, morphologically and morphometri cally, very similar to each other, but some minor differences have also been noted, which are regarded as intraspecific variations. Anterior uterine sac according to Andrássy (2009) and Peña-Santiago et al. (2013), varying from absent (as in our population) to present with different sizes. Thus, anterior uterine sac in Sufiyan population varied from absent to 8.5 μ m long, but in all the remaining populations it was of different sizes. Saccate bodies were occasionally present (Peña-Santiago et al., 2013), and according to Heydari et al. (2019) saccate bodies were not present in their own Belgian populations and not seen in Sufiyan and Urmia populations but they were present in Divandarreh, Bokan, Maragheh, and Basmenj populations. Pars refringens vaginae, consisting of two small sclerotized pieces, were distinguishable in Divandarreh and Maragheh populations, but they were more inconspicuous in other populations. Vagina orientation also displays some differences: backwards directed in Bokan and Sufiyan populations and near perpendicular to body axis in other populations. Present Iranian populations of P. engadinensis fit very well with those previously studied by other authors (for comparative purposes, see Coomans and Geraert, 1962;Andrássy, 2009;Peña-Santiago et al., 2013;Álvarez-Ortega and Peña-Santiago, 2014;Heydari et al., 2019).
Pungentus engadinensis is a widely distributed species, having been recorded in Asia, Europe, and North America, where it mostly inhabits moist soils (Andrássy, 2009). In Iran, it has previously been reported (Kazemi, 2016) from the rhizosphere of vineyards in Uremia, West-Azarbaijan province; rangelands in Divandarreh, Kurdistan province; natural vegetation in Bokan, West-Azarbaijan province and Maragheh, East-Azarbaijan province; common wheat from Sufiyan, East-Azarbaijan province and Basmenj, East-Azarbaijan province, but in the form of taxonomic papers from three locations of the country reported by Heydari et al. (2019) and Solouki et al. (2010).