Description of Deladenus longicaudatus n. sp. (Sphaerularioidea: Neotylenchidae) from Mazandaran province, northern Iran: A morphological and molecular phylogenetic study

Decaying wood and bark samples were collected in natural forests of Javaherdeh, Ramsar county, Mazandaran province, northern Iran. Nematodes were extracted using the tray method (Whitehead and Hemming, 1965). Live specimens were handpicked under a Nikon SMZ1000 stereomicroscope, heat-killed in hot 4% formaldehyde solution, and processed to anhydrous glycerin following De Grisse (1969). Permanent slides were prepared and examined with a Nikon Eclipse E600 microscope. Photomicrographs were obtained using an Olympus DP72 digital camera attached to an Olympus BX51 microscope equipped with differential interference contrast optics. Line drawings were made with a drawing tube and digitally drawn in CorelDRAW^® software version 2020.

Deladenus in its broad sense was accepted in this research (Chitambar, 1991). The new species was compared with the mycetophagous phases of three genera, Deladenus, Hexatylus, and Rubzovinema, according to Rezaei et al. (2025) [valid species of the latter two genera according to Siddiqi (2000)].

Three live nematode specimens of the new species were picked out and transferred individually to a small drop of tris-ethylenediaminetetraacetic acid (TE) buffer (10 mM Tris-Cl; 0.5 mM ethylenediaminetetraacetic acid; pH 9.0; Qiagen) on a clean slide and gently squashed using a clean cover slip after photomicrographic documentation. The suspension from each specimen was collected by adding 35 μL of TE buffer. DNA samples were stored at –20°C until used as PCR templates. The partial SSU rDNA was amplified using forward primer 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and reverse primer 2646R (5′-GCTACCTTGTTACGACTTTT-3′) (Holterman et al., 2006). The LSU rDNA D2–D3 expansion domains were amplified using the forward primer D2Ab (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) (De Ley et al., 1999) and reverse primer 1006R (5′-GTTCGATTAGTCTTTCGCCCCT-3′) (Holterman et al., 2008). PCR reactions were performed in 35 μl volumes with an annealing temperature of 52°C. The successfully amplified PCR products were sequenced with the same primers used in PCR by Pishgam Corporation (Tehran, Iran), using an ABI 3730XL sequencer. The newly obtained sequences were deposited in the GenBank database under the accession numbers PX106384 and PX106385 for SSU rDNA; and PX108876, PX108877, and PX108878 for LSU rDNA.

The newly obtained SSU and LSU sequences of the new species were compared with those of other nematode species available in the GenBank database using the BLAST homology search program. For phylogenetic reconstruction, two independent datasets were prepared: the SSU and LSU. Sequences of both loci were aligned using MUSCLE implemented in MEGA7 (Kumar et al., 2016) and manually edited to remove poorly aligned or ambiguous positions. Model selection for nucleotide substitution was performed with MrModeltest v2 (Nylander, 2004), applying the Akaike information criterion. The analysis indicated that the general time reversible model, incorporating both a gamma distribution of rate heterogeneity among sites and a proportion of invariant sites (GTR + G + I), was the most suitable, and was therefore used for further analyses. Bayesian inference was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) for 5,000,000 generations for each dataset. Twenty-five percent of the samples were discarded as burn-in. The Markov chain Monte Carlo method within a Bayesian framework was used to estimate posterior probabilities of phylogenetic trees using the 50% majority rule. For the SSU phylogeny, sequences of Acrobeloides maximus Thorne, 1925 and Acrobeles ciliatus von Linstow, 1877 were used as outgroups, whereas for the LSU phylogeny, sequences of Poikilolaimus oxycerca de Man, 1895 and P. piniperdae Fuchs, 1930 served as outgroups. The resulting phylogenetic trees were visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and subsequently digitally drawn in CorelDRAW^® software version 2020.

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Decaying wood and bark of a dead broadleaf tree in forests of Javaherdeh, Ramsar county, Mazandaran province, northern Iran. GPS coordinates: 36°52′70″N, 50°33′39.5″E.

Holotype female and 11 paratype females were deposited at the WaNeCo nematode collection of Wageningen University, The Netherlands. The LSID code of this publication is: urn:lsid:zoobank.org:pub:800CA2DA-5C2C-47F8-9DED-95445BBDBCB0

The specific epithet refers to the elongate conical tail of the new species.

The mycetophagous females of the new species are mainly characterized by an elongate conical tail gradually tapering to a finely rounded terminus (51–63 µm long, c = 9.3–11.0, c′ = 3.9–5.3), a relatively anterior vulval position (V = 84.5–89.0%), and a tail length 2–3 times the vulva–anus distance long. Deladenus longicaudatus n. sp. is further characterized by 523–682 µm long females having the pharyngo-intestinal junction at the level with the nerve ring, the S–E pore posterior to the nerve ring and at the level with the hemizonid, seven lines in the lateral fields, a short stylet (5.0–6.5 µm), small lateral vulval flaps, and absence of a PUS.

By having similarities in tail shape, c′ ratio greater than 3 and position of S–E pore and hemizonid posterior to nerve ring as well as no PUS, the new species is comparable to four known species of the genus, namely, D. gilanica Jalalinasab, Esmaeili, Ye and Heydari, 2020, D. hyrcanus, D. parvus Zell, 1985, and D. zyzyphus Bajaj, 2015. The new species differs from D. gilanica by a longer body (523–682 vs 314–422 µm), shorter stylet (5.0–6.5 vs 7.5–8.0 µm), S–E pore 86–112 µm from anterior end (vs 61–76 µm), number of lateral field lines (seven vs eight), presence of lateral vulval flaps (vs absence), and longer tail (51–63 vs 27–32 µm); from D. hyrcanus by a shorter body (523–682 vs 718–806 µm), shorter stylet (5.0–6.5 vs 7.5–10.0 µm), shorter pharynx (51–69 vs 71–86 µm), vulva-anus distance (21–31 vs 28.5–46.0 µm), and presence of lateral vulval flaps (vs absence); from D. parvus by number of lateral field lines (seven vs four), a posterior vulval position (V = 84.5–89.0 vs 79.8–85.4%), presence of lateral vulval flaps (vs absence), and a finely rounded tail tip (vs having a short hairy process); from D. zyzyphus by shorter body (523–682 vs 790–1,110 μm), shorter stylet (5.0–6.5 vs 7–9 μm), S–E pore at the level with hemizonid (vs posterior to hemizonid), number of lateral lines (seven vs four), lower a ratio (19.5–34.0 vs 45–51), lower c′ ratio (3.9–5.3 vs 6.1–7.0), presence of lateral vulval flaps (vs absence), shorter tail (51–63 vs 69–90 μm), and absence of males (vs presence).

Among the mycetophagous phases of the species under the genera Hexatylus and Rubzovinema, the new species is typologically comparable to Hexatylus vigissi Skarbilovich, 1952 (a poorly known species), but differs from it by a shorter body (523–682 vs 986–1,326 μm), shorter stylet (5.0–6.5 vs 6–8 µm), S–E pore 86–112 µm from anterior end (vs 112–125 µm), lower c ratio (9.3–11.0 vs 12.7–16.5), and shorter tail (51–63 vs 60–100 µm).

Description of Hexatylus vigissi includes no data on the rectum acting as a feeding pump. This feature is regarded as one of the main generic characters of Hexatylus, as defined by Siddiqi (2000).