Description of Deladenus uljinensis n. sp., and additional DNA barcode data for Deladenus posteroporus (Nematoda: Neotylenchidae) from Korea

The nematode populations were recovered from the sapwood layers of a dead (wildfire-induced mortality) red pine (Pinus densiflora for. erecta) tree sampled from the Geumgang pine forest in Uljin, Gyeongsangbuk-do Province, Republic of Korea. The wood had signs of insect infestations in the form of tunnel engravings of Dendroctonus. Adult males and females of Dendroctonus sp. were also collected from the tunnels. However, despite the attempts to recover infective forms, all the 60 collected insects were devoid of Deladenus nematode infestation. Nematodes were extracted from the wood cuttings using the Baermann funnel method (Baermann, 1917). Nematode specimens belonging to Deladenus were handpicked from the nematode suspension under a Nikon SMZ 1000 stereomicroscope (Nikon). Specimens were subsequently characterized to species level using morphometric and molecular DNA barcode data.

Fresh nematode specimens were heat-killed, fixed in formaline-glycerine proportion, and processed to pure glycerin according to Seinhorst (1959) as modified by De Grisse (1969). The processed specimens were mounted in dehydrated glycerin on permanent slides and examined under a fluorescence microscope. Morphometric data and photomicrographs were taken using a Zeiss imager Z2 microscope (Carl Zeiss) fitted with Axio-vision, a material science software for research and engineering (Carl Zeiss). Line drawings were made under a drawing tube and redrawn using CorelDRAW® software version 24. Species delineation and diagnosis was done following the diagnostic species compendia presented by Bedding (1974); Chitambar (1991); and Aliramaji et al. (2024).

Genomic DNA was extracted from heat-relaxed morphometrically confirmed single female (2 specimens), and single male specimen of Deladenus using the DNA extraction kit WizPure™ according to Iwahori et al. (2000). Morphological variations in females were considered by extracting DNA from specimens with and without rudimentary post-vulval sac. Three gene fragments – i.e., nearly full-length 18S-rRNA gene, the D2–D3 expansion segment of 28S-rRNA gene, and the partial COI gene – were amplified and sequenced in this study. The 18S-rRNA gene was amplified as two partially overlapping fragments using two primer sets: 988F (5′-CTCAAAGATTAAGCCATGC-3′) and 1912R (5′-TTTACGGTCAGAACTAGGG-3′), 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and 2646R (5′-GCTACCTTGTTACGACTTTT-3′) (Holterman et al., 2006). D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) (Nunn, 1992) primer set was used in the amplification of the D2–D3 expansion segment of 28S-rRNA gene, and the partial COI gene was amplified using the primer set COIF1 (5′-CCTACTATGATTGGTGGTTTTGGTAATTG-3′) and COIR2 (5′-GTAGCAGCAGTAAAATAAGCACG-3′) (Kanzaki and Futai, 2002). Polymerase chain reaction (PCR) was performed with a PCR cycler (T100™, Bio-Rad). The thermal cycles for the primer sets D2A/D3B, 988F/1912R, 1813F/2646R, and COIF1/COIR2 were as described by Mwamula et al. (2023). The PCR products were subsequently purified using QIAquick PCR Purification Kit (Qiagen) and quantified using a quickdrop spectrophotometer (Molecular Devices). The purified products were directly sequenced with the primers specified above at Macrogen Inc. The new sequences were edited and submitted to the GenBank database under the accession numbers PQ814252-PQ814256 (for 18S-rRNA); PQ816571-PQ816575 (for 28S-rRNA); and PQ814257-PQ814261 (for COI gene).

By using the BLAST homology search program, the new sequences (18S-rRNA, 28S-rRNA, and COI gene) were compared with those of related species of Deladenus, and other comparable sequences of related nematode genera published in GenBank (Yu et al., 2017; Heydari et al., 2020; Jalalinasab et al., 2020; Bonab et al., 2023; Kanzaki et al., 2023; Yu et al., 2023; Aliramaji et al., 2024). Multiple alignments for the three genes were built using ClustalX (Thompson et al., 1997). The sequences of Acrobeloides varius (Kim, Kim, & Park, 2017) (MK636580) and Cephalobus cubaensis (Steiner, 1935) (AF202161) were used as the outgroup taxa for the 18S-rRNA gene; Oscheius myriophilus (Poinar, 1986; Sudhaus, 2011) AY602176, and Poikilolaimus oxycercus (de Man, 1895) (DQ059059) were the outgroup taxa for 28S-rRNA gene; and Ektaphelenchus joyceae (Kaisa, Harman & Harman, 1995) (MT712703) and Devibursaphelenchus eproctatus (Sriwati, Kanzaki, Phan & Futai, 2008) (JN122013) were selected as the outgroup taxa for the COI gene. Bayesian inference (BI) of the phylogenies based on sequences of the three genes was performed using MrBayes 3.2.7 (Ronquist et al., 2012), with GTR + I + G model selected for all datasets. Bayesian inference analysis was run with four chains for 1 × 10⁶ generations, and the Markov chains were sampled at intervals of 100 generations. Consensus trees were generated with the 50% majority rule and the generated trees were edited using FigTree v1.4.4 software. Posterior probabilities exceeding 50% are given on appropriate clades. Intraspecific and interspecific sequence variation was analyzed using PAUP* v4.0a169 (Swofford, 2003).

See Table 1.

Deladenus uljinensis n. sp. is characterized by its short and straight body, lateral fields with six to seven lines at mid-body, pharyngeal corpus without a distinct median bulb and lacking a chamber, esophageal-intestinal junction located immediately behind the nerve ring, hemizonid prominent, located 27.0–48.0 μm posterior to nerve ring, excretory pore opening within the contour of hemizonid or just at the base of hemizonid, a large oblong-shaped spermatheca with distinct spheroid sperm, vulva with moderately protuberant lips, and with no lateral vulval flaps, post-uterine sac rudimentary or absent, vulva-anus distance ca. equal to tail length, conoid tail gradually tapering to a broadly rounded terminus, spicules slender and tylenchoid, 18.5–21.5 μm long, gubernaculum thin and short, 5.0–6.0 μm long.

By having a relatively shorter body and the position of excretory pore in relation to hemizonid, Deladenus uljinensis n. sp. is comparable to seven species of the genus, namely D. gilanica (Jalalinasab, Esmaeili, Ye & Heydari, 2020); D. brevis (Heydari, Abolafia & Pedram, 2020); D. pakistanensis (Shahina & Maqbool, 1992); D. oryzae (Bajaj, 2015); D. uteropinusus (Bajaj, 2015); D. aridus (Andrássy, 1957); and D. durus (Cobb, 1922; Thorne, 1941). It differs from D. gilanica by the relatively longer body (504–736 μm vs 314–422 μm), vulva-anus distance ca. equal to tail length vs. less than tail length, tail gradually tapering to a broadly rounded terminus vs. gradually tapering to a pointed tip, long glandular overlap vs. short glandular overlap (57.0–84.0 μm vs. 11–33 μm), longer distance from anterior end to hemizonid/excretory pore (90–111 μm vs. 61–76 μm), a more posterior vulval position (V = 88.6–91.7 vs. 85.4–87.4), and higher c and lower c′ ratios (c = 17.5–24.3 and c′ = 1.7–2.4 vs. c′ = 10.8–14.6 and c′ = 2.4–3.8, respectively); from D. brevis by the relatively longer body (504–736 μm vs. 367–454 μm), longer stylet (8.0–9.5 μm vs. 6–7 μm), hemizonid and excretory pore located posterior to nerve ring vs. hemizonid and excretory pore at the level with nerve ring, vulva lips with no lateral vulval flaps vs. with lateral vulval flaps, lower c′ ratio (1.7–2.4 vs. 2.4–3.2), and longer spicules (18.5–21.5 μm vs. 11.3–14.5 μm); from D. pakistanensis by the stout body (a = 21.1–28.3 vs. 33.0–35.0), its body not abruptly narrowing at posterior of vulva vs. body abruptly or sharply narrowing posterior to vulva, lateral field with 6–7 lines vs. 10–12 lines, excretory pore opening within the contour of hemizonid or at the base of hemizonid vs. excretory pore opening posterior to hemizonid, and vulva lips with no lateral vulval flaps vs. with lateral vulval flaps; from D. oryzae by more anterior vulval position (V= 88.6–91.7 vs. 92.6–93.5), lip region not marked off from rest of body vs. marked off from rest of body, excretory pore opening within the contour of hemizonid or at the base of hemizonid vs. excretory pore opening anterior to hemizonid, lateral fields with 6–7 lines vs. indistinct, pharyngeal corpus without a distinct median bulb and lacking a chamber vs. median pharyngeal bulb present, and with median chamber, vulva-anus distance ca. equal to tail length vs. 1.3 times the tail length, and longer spicules (18.5–21.5 μm vs. 16.0–18.0 μm); from D. uteropinusus by its shorter and stouter body (504–736 μm vs. 760–820 μm, a = 21.1–28.3 vs. 28.0–36.0), lip region not marked off from rest of body vs. slightly marked off from rest of body, more posterior vulval position (V= 88.6–91.7 vs. 87.2–88.5), pharyngeal corpus without a distinct median bulb and lacking a chamber vs. median pharyngeal bulb present, and with median chamber, lateral fields with 6–7 lines vs. indistinct, vulva-anus distance ca equal to tail length vs. 1.4–1.7 times the tail length; tail conoid gradually tapering to a broadly rounded terminus vs. tail cylindrical, with an inverted U-shaped hyaline region; from D. aridus by excretory pore opening within the contour of hemizonid or at the base of hemizonid vs. excretory pore opening posterior to hemizonid; vulva-anus distance ca. equal to tail length vs. almost half the tail length; tail tapering to a broadly rounded terminus vs. acute tip; and from D. durus by its shorter and stouter body (504–736 μm vs. 820–1360 μm, a = 21.1–28.3 vs. 29.0–50.0), more anterior vulval position (V= 88.6–91.7 vs. 91–93), pharyngeal corpus without a distinct median bulb and lacking a chamber vs. median pharyngeal bulb distinct, and with oval median chamber, excretory pore opening within the contour of hemizonid or at the base of hemizonid vs. excretory pore opening posterior to hemizonid, and tail terminus broadly rounded, vs. narrowly rounded or pointed, with cleft, mucro, or with two or more sharp processes (see Chitambar, 1991).

The type population was isolated from the wood of a dead red pine (Pinus densiflora for. erecta) tree stand from the Geumgang pine forest in Uljin, Gyeongsangbuk-do Province. (GPS coordinates: 37° 1′ 12.07″N, 129° 14′ 44.98″E).

Holotype female, 20 female, and 8 male paratypes were deposited in the National Institute of Biological Resources of Korea, and 10 female and 7 male paratypes were deposited in the Nematode Collection of Kyungpook National University (KNU), Republic of Korea.

Deladenus uljinensis n. sp. was recovered from the Geumgang pine forest in Uljin, Gyeongsangbuk-do Province. Thus, the species epithet uljinensis is derived from the locality of its first description, i.e., Uljin.

(Fig. 3).

Figure 3:

Photomicrographs of Deladenus posteroporus (A–J). A: Female anterior region; B: Male anterior region; C: Female pharyngeal and glandular region; D: Excretory pore / hemizonid position; E–G: Variation in female tail region; H: Lateral field; I: Male tail region; J: Vulval region with spermatheca. The two arrows labeled h and e indicate the position of hemizonid and excretory pore, respectively (Scale bars: A–I = 20 μm; J = 10 μm).

See Table 1.

The morphometrics and general morphology of the studied population agree well with the original species description of Yu et al. (2017), especially the U.S. population with the excretory pore located immediately after the hemizonid, except for some variations in a few characters: for instance, a relatively high c ratio in the current population (30.0–49.1 vs. 26.4–37.1). Also, contrary to the original species description, a short post-vulval sac is present in the mycetophagous females in the studied population (see Fig. 3J). However, although noted as absent in the original description of mycetophagous females (see Yu et al., 2017), their line drawings and photomicrographs indicate the presence of post-vulval sac. Thus, these variations are consistent with the paratype data.

Similar to Deladenus uljinensis n. sp., D. posteroporus population was also isolated from the wood of one of the dead red pines (Pinus densiflora for. erecta) from the same locality (Geumgang pine forest in Uljin, Gyeongsangbuk-do Province; GPS coordinates: 37° 1′ 12.07″N, 129° 14′ 44.98″E).

The amplification of the nearly full-length 18S-rRNA for the two species yielded fragments of approximately 1700 bp. No intraspecific sequence variation was recorded in the three newly obtained 18S-rRNA gene sequences (PQ814254-PQ814256) of the new species Deladenus uljinensis n. sp. In the 18S-rRNA gene phylogeny, Deladenus uljinensis n. sp. sequences were grouped in an independent moderately supported subclade in relation to other Deladenus species. The two new 18S-rRNA gene sequences of D. posteroporus (PQ814252, PQ814253) also showed no intraspecific sequence variation and were also identical to D. posteroporus (KY098774) with no sequence variation (0.0%). Sequences of D. posteroporus (PQ814252, PQ814253 and KY098774) clustered with closely homologous sequences of Deladenus proximus (JF304744 and KF908881), Tylenchomorpha sp. (LC147025 and LC147027), and Deladenus siricidicola (AY633447), differing by 9–11 bp (0.6–0.7%), 10–17 bp (0.6–1.0%), and 15 bp (0.9%), respectively.

The D2–D3 region yielded fragments of approximately 750–800 bp. The three D2–D3 sequences of Deladenus uljinensis n. sp. (PQ816573–PQ816575) differed by 1 bp, and clustered with Deladenus gilanica (MF043927) in a separate moderately supported subclade (pp = 87%), differing from MF043927 by 132–133 bp (19.0–19.4%). The two new D2–D3 sequences of D. posteroporus (PQ816571, PQ816572) were identical to the three D. posteroporus sequences available in GenBank database with no sequence variation (100% percent identity). In the LSU phylogeny, the five D. posteroporus sequences were grouped in an independent well-supported subclade with Deladenus sp. (LC801234), Deladenus sp. (MG871254), and D. siricidicola (MW481657), differing by 44–47 bp (5.7–6.1%), 46 bp (6.2–6.9%) and 48–60 bp (6.7–7.8%), respectively.

The amplification of the partial COI gene for the two species yielded fragments of approximately 650 bp. Owing to the limited number of COI gene sequences for the genus in GenBank database, the generated sequences for the two species (Deladenus uljinensis n. sp.: PQ814259–PQ814261 and D. posteroporus: PQ814257, PQ814258) showed relative homology with COI gene sequences of Fergusobia spp. Deladenus proximus and other unidentified species deposited in GenBank database as Allantonematidae spp.; all with percent identities of 80–85% based on the BLAST homology search program. The sequences of Deladenus uljinensis n. sp. (PQ814259–PQ814261) showed a genetic variation of 1 bp., and those of D. posteroporus: (PQ814257, PQ814258) were identical with no recorded sequence variation. Reconstruction of the partial COI gene phylogeny grouped each of the two species into independent clades. Sixty-five 18S-rRNA, sixty-six 28S-rRNA and forty-eight COI gene sequences from member species of Deladenus, and other related genera, including the newly obtained sequences and outgroup taxa constituted the sequence dataset for phylogenetic analyses. Phylogenetic relationships, as inferred from Bayesian analysis of the dataset with GTR + I + G substitution model, are shown in Figs. 4, 5, and 6.

Figure 4:

Bayesian tree inferred under the GTR + I + G model from 18S-rRNA gene sequences of Deladenus spp. and other related species from various genera. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Acrobeloides varius and Cephalobus cubaensis.

Figure 5:

Bayesian tree inferred under the GTR + I + G model from LSU D2–D3 partial sequences of Deladenus spp. and other related species from various genera. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Oscheius myriophilus and Poikilolaimus oxycercus.

Figure 6:

Bayesian tree inferred under the GTR + I + G model from COI gene sequences of Deladenus spp. and other related species from various genera. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Ektaphelenchus joyceae and Devibursaphelenchus eproctatus.