Description of Nothotylenchus savadkoohensis n. sp. (Rhabditida, Anguinidae) from Iran based on morphological and molecular data

A number of 45 soil, wood, and bark samples were collected from the natural forests of the city of Savadkooh, Mazandaran province. Nematodes were extracted from these samples using the tray method (Whitehead and Hemming, 1965). A population of Nothotylenchus sp. was recovered from rotten wood samples of an unidentified forest tree. The collected specimens were killed by adding boiling 4% formalin solution, transferred to anhydrous glycerin, mounted on permanent microscopic slides according to De Grisse (1969), and studied with a Nikon Eclipse E600 optical microscope. Photos were taken using an Olympus DP72 digital camera connected to an Olympus BX51 light microscope powered with differential interference contrast (DIC) view. Drawings were made using a drawing tube attached to the microscope and digitally drawn using CorelDRAW® software version 2020.

Three females were selected for this purpose. The specimens were studied in temporary slides and photographed. Each specimen was transferred into a small drop of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0, Qiagen) on another clean slide and squashed using a cover slip with the aid of the pressure of a pipette tip. The suspension was collected by adding 15 μL of the TE buffer. Three DNA samples were made in this manner and stored at −20°C until used as PCR templates. Primers for LSU rDNA D2–D3 amplification were forward primer D2A (5′-ACAAGTACCGTGAGGGAAAGT-3′) and reverse primer D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (Nunn, 1992). The primers rDNA1 (5′-TTGATTACGTCCCTGCCCTTT-3′) (Subbotin et al., 2000) and TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) (Joyce et al., 1994) were used to amplify ITS rDNA. Forward 1096F (5′-GGTAATTCTGGAGCTAATAC-3′) and reverse 1912R (5′-TTTACGGTCAGAACTAGGG-3′) and forward 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and reverse 2646R (5′-GCTACCTTGTTACGACTTTT-3′) primers were used to amplify the SSU rDNA (Holterman et al., 2006). PCRs were performed according to Jahanshahi Afshar et al. (2019) and the successfully amplified products were directly sequenced in both directions using the same primers used in PCR with an ABI 3730XL sequencer (Pishgam corporation, Tehran, Iran). The newly generated sequences were deposited into the GenBank database under the accession numbers PP174320 for D2–D3 expansion segments of LSU and PP174321 for ITS rDNA.

Body slender, ventrally slightly arcuate after heat relaxation, slightly narrowing toward both extremities, more so towards posterior end by having an elongated conoid tail with narrow distal end. Cuticular annulation fine. Lateral field with four incisures. Lip region low, anteriorly almost flat, the cephalic framework weak. Stylet fine, its conus about 34% of the total stylet length, with fine knobs, sloping backward. Procorpus slender, widened at about middle of the pharynx, metacorpus not valvate, isthmus narrow, joining to pharyngeal bulb, slightly overlapping intestine dorsally. Nerve ring enveloping isthmus slightly anterior to pharyngeal basal bulb. Hemizonid close to tip of nerve ring. Intestine simple, rectum and anus functional. Reproductive system monodelphic-prodelphic, composed of outstretched ovary with oocytes arranged in single row, oviduct tubular, spermatheca elongate and including spheroid sperm, crustaformeria with no clearly seen cells in each row, uterus, vagina perpendicular to body axis, vulva a small transverse slit, and postvulval uterine sac (PUS) about 1.3 times corresponding body width long. The sphincter encircling vagina triangle-shape at cross section. The distance from vulva to anus is about four times vulval body width. Tail is elongated and conical, almost straight, with a sharply pointed tip.

General morphology similar to that of the females, except slightly shorter body and characters related with sexuality. Reproductive system monorchic, testis single, outstretched. Spicules tylenchoid, slightly sclerotized, ventrally arcuate, gubernaculum simple. Bursa cloacal. Tail elongate conoid, uniformly narrowing toward distal end, with sharp tip.

The specific epithet refers to the city of Savadkooh from where the new species was recovered.

New species was recovered from rotten wood samples of an unidentified forest tree, collected from forests of Mazandaran province, city of Savadkooh in northern Iran (GPS coordinates; 36°12.795N, 052°55.581E).

Holotype female, 12 paratype females, and four paratype males in 16 slides were deposited at WaNeCo collection, Wageningen, The Netherlands (http://www.waneco.eu/). The LSID code for this publication is: urn:lsid:zoobank.org:pub:C9E20B3E-D502-4A31-957D-F5B81C3B68FD.

Nothotylenchus savadkoohensis n. sp. is mainly characterized by having four lines in lateral fields and an elongated conoid tail with sharply pointed tip. It is further characterized by 379–662 μm long females, having 5.8–6.9 μm long stylet with fine posteriorly sloping knobs, no valvate metacorpus, pharyngeal bulb slightly overlapping intestine dorsally, vulva at 76.5–84.0% of body length and males with cloacal bursa. By having an elongated conoid tail and four lines in the lateral fields, the new species comes close to four known species of the genus namely Nothotylenchus acris, N. acutus Khan, 1965, N. antricolus Andrássy, 1961 and N. truncatus Eliashvili & Vacheishvili, 1980. The new species could be separated from them as follow:

From N. acris by a shorter body (379–662 vs 900 μm) and tail shape (elongated conoid narrowing toward the distal region, sharply pointed at tip vs elongate conoid, thicker at distal region).

From N. acutus by greater V (76.5–84.0 vs 70–76.3), shorter stylet (5.8–6.9 vs 7–9 μm), greater a (27.1–47.3 vs 22–28) and tail shape (elongate conoid narrowing toward distal region, sharply pointed at tip vs elongate conoid, thicker at distal region).

From N. antricolus by greater c (8.4–13.9 vs 6.7–6.9), greater V (76.5–84.0 vs 70.7–72.4), smaller c′ (4.9–7.3 vs 9.6–10.0), longer PUS (longer than corresponding body with vs shorter), and difference in tip shape (elongate conoid, narrowing toward distal region, sharply pointed at tip vs elongate conoid, thicker at distal region).

From N. truncatus by greater V (76.5–84.0 vs 57–66), greater c (8.4–13.9 vs 6.3–7.4), shorter stylet (5.8–6.9 vs 11.0–12.5 μm), and difference in tail tip shape (sharply pointed vs rounded).

Several efforts to get sequences of SSU rDNA for the new species using formerly cited primer pairs failed. The PCRs for amplification of D2–D3 region of LSU rDNA of two female specimens were successful. The two LSU sequences obtained from two female specimens were identical, and as the result, one sequence was deposited into the GenBank database under the accession number PP174320. This sequence is 558 nt long. The BLAST search using this sequence revealed its identity with all currently available sequences as less than 91%. Fig. 3 represents the Bayesian phylogenetic tree reconstructed using the LSU dataset. In this tree, currently available D2–D3 sequences of the genus Nothotylenchus have occupied distant placements, and the D2–D3 sequence of the new species has formed a clade with a sequence assigned to Neotylenchus sp. (DQ328725). The morphological data of this isolate are, however, not available. Based on the currently resolved topology, the genus Nothotylenchus is polyphyletic according to currently available data.

Figure 3:

The partial ITS sequence of the new species (PP174321) was 394 nt long. The BLAST search using this sequence revealed the identity of this sequence with sequences having high (above 75%) coverage ranges from 91–94%. Fig. 4 represents the phylogenetic tree reconstructed using the ITS dataset. In this tree, the newly generated sequence of the new species has formed a clade with a clade that includes sequences of a Ditylenchus sp. (MW042918) and two sequences of Neomisticius platypi Masuya & Hamaguchi, 2021 and N. variabilis Kanzaki, Masuya & Hamaguchi, 2021 (LC631547 and LC631548).

Figure 4: