Morphological and molecular characterization of Paractinolaimus uljinensis n. sp. (Nematoda: Actinolaimidae) from Korea, with an updated compendium of the genus

The nematode population was recovered from the bark layer of a dead (wildfire-induced mortality) red pine (Pinus densiflora for. erecta) tree taken from Geumgang pine tree forest in Uljin, Gyeongsangbuk-do Province, Republic of Korea. Nematodes were extracted from the bark cuttings using the Baermann funnel method (Baermann, 1917). Nematode specimens belonging to Paractinolaimus were handpicked from the resultant nematode suspension under a Nikon SMZ 1000 stereomicroscope (Nikon). Specimens were subsequently characterized based on inferences from morphometric and DNA barcode data.

The new sequences (18S-rRNA, 28S-rRNA, and ITS-rRNA gene) were compared with those of related species of Paractinolaimus, including comparable sequences of related species from other genera published in GenBank (Holterman et al., 2006; Nedelchev et al., 2014; Vinciguerra et al., 2016; Kagoshima et al., 2019; Swart et al., 2020; Zhang et al., 2023; Mwamula et al., 2024b) using BLAST homology search program. Multiple alignments for the selected genes were built using ClustalX (Thompson et al., 1997). The sequences of Paravulvus hartingii (de Man, 1880) Heyns, 1968 (AY552976) and Nygolaimus cf. parvus Thorne, 1974 (AY552974) were used as the outgroup taxa for the 18S-rRNA gene; and Mononchus tunbridgensis Bastian, 1865 (AY593063) and Anatonchus tridentatus (de Man, 1876) De Coninck, 1939 (AY593065) were the outgroup taxa for 28S-rRNA gene. Bayesian inference (BI) of the phylogenies based on sequences of the two 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 initiated with a random starting tree and run with four chains for 1 × 106 generations. The Markov chains were sampled at intervals of 100 generations. After discarding burn-in samples, consensus trees were generated with the 50% majority rule. The generated trees were edited using FigTree v1.4.4 software. Posterior probabilities (PP) exceeding 50% are given on appropriate clades. Intraspecific and interspecific sequence distances were analyzed using PAUP* v4.0a169 (Swofford, 2003).

See Table 1.

Paractinolaimus uljinensis n. sp. is characterized by having a medium sized body 2.50 to 2.98 mm long; lip region truncate, angular and offset by a depression; odontostyle 23.5 to 27.0 μm long; basal shield of pharynx present; well-developed, didelphic-amphidelphic female reproductive system; vulval opening wide and longitudinal in profile, positioned slightly anteriorly (V = 42.5–47.7); several advulval papillae; intestine-prerectum junction with an anteriorly directed conical or tongue-like projection; a long female tail (324.0–435.0 μm long, c’ = 10.1–14.2), tapering to a straight middle part, becoming filiform posteriorly, with a finely rounded or pointed tail terminus; a clearly visible copulatory hump; spicules 60.0 to 70.5 μm long; 12 to 15 (mostly 12–14) large contiguous ventromedian supplements and male tail conoid to broadly rounded.

By having advulval papillae, Paractinolaimus uljinensis n. sp. is very close to P. intermedius Altherr, 1968; P. sahandi Pedram, Niknam, Vinciguerra, Ye & Robbins, 2011; P. decraemerae Pedram, Niknam, Vinciguerra, Ye & Robbins, 2010; P. acutus Khan & Park, 1999; and P. macrolaimus (De Man, 1880) Andrássy, 1964. It differs from all, except P. sahandi, by the presence of a higher number of advulval papillae in front and behind the vulva, including the presence of several small papillae without visible innervations in individuals with a few prominent papillae (this configuration is evident in only P. sahandi). Additionally, Paractinolaimus uljinensis n. sp. differs from the closest P. intermedius by its truncate lip region, with angular lips set off from the body by a depression vs lips not angular, rounded, with papillae slightly protruding from their profile, continuous with body or hardly separated by a very slight depression, long female tail (c = 6.9–8.5 vs 13–17; c’ = 10.1–14.2 vs 6–7), a more anteriorly positioned vulva (V = 42.5–47.7 vs 46–54), and a relatively lower number of contiguous ventromedian supplements (12–15) vs relatively higher number of spaced ventromedian supplements (14–19); from P. sahandi by body length (2.5–3.0 mm vs 3.5–4.7 mm in females, and 2.2–2.8 mm vs 3.2–4.4 mm in males), a ratio (40.2–47.9 vs 74.5–88.5), long tail (324.0–435.0 μm vs 198.5–270.0 μm; c = 6.9–8.5 vs 16.2–22.0, c’ = 10.1–14.2 vs 4.5–7.5), a relatively shorter odontostyle (23.5–27.0 vs 27.0–32.5), a more anteriorly positioned vulva (V = 42.5–47.7 vs 47–54), and the presence of a copulatory hump vs no copulatory hump; from P. decraemerae by the angular lips vs rounded, long tail (324.0–435.0 μm vs 200–238 μm ; c = 6.9–8.5 vs 13.3–16.0, c’ = 10.1–14.2 vs 5.5–6.7), males as abundant as females vs very rare, shorter spicules (60.0–70.5 μm vs 76.5 μm), lower number of supplements (12–15 vs 16), and the presence of a copulatory hump vs no copulatory hump; from P. acutus by long body (2.5–3.0 mm vs 2.1–2.4 mm), angular lips offset by a depression vs rounded and continuous with body, longer tail (324.0–435.0 μm vs 285.0–336.0) terminating in a finely rounded or pointed tip vs ending in a distinct acute tip, vulva shape (longitudinally oval, situated in a small depression in P. acutus), and males as abundant as females vs no males.

Regarding comparison with P. macrolaimus, Paractinolaimus uljinensis n. sp. is distinctly different from P. macrolaimus type specimens in almost all attributes: short body (2.5–3.0 mm vs 3.7–4.5 mm), presence of several advulval papillae vs absent, a ratio (40.2–47.9 vs 50), c ratio (6.9–8.5 vs 11.1–11.8), lower number of ventromedian supplements (12–15 vs 16–24), and the presence of a copulatory hump vs absent. However, the new species is comparable to the morphologically divergent African population that was described by Andrássy (1964), and Andrássy (2009) (see Vinciguerra et al. (2013), and Pedram et al. (2011)). Despite the discrepancies in morphological details of this population in relation to the morphology of the type specimens of P. macrolaimus (De Man, 1880), including the subsequent redescriptions by Loof (1961), Paractinolaimus uljinensis n. sp. still differs from this population by its shorter odontostyle (23.5–27.0 vs 27.0–30.0), advulval cuticular configurations (several prominent papillae, including several small papillae without visible innervations in individuals with few prominent papillae), lower number of supplements (12–15 vs 16–19), and the presence of a copulatory hump.

Also, based on morphometric measurements, Paractinolaimus uljinensis n. sp. comes close to P. tuberculatus Choi & Khan, 2000. However, Paractinolaimus uljinensis n. sp. can be qualitatively differentiated from P. tuberculatus by its angular lips (set off by a depression vs rounded and continuous or almost continuous), presence of several advulval papillae vs absent, vulva longitudinal vs transverse slit, presence of a copulatory hump vs absent, ventromedian supplements 12 to 15, contiguous in arrangement vs 15 to 19 (regularly spaced), and the presence of a tubercle-like structure in the lumen of pharynx base in P. tuberculatus, hence its species epithet name.

The nematode population was recovered from the bark of a dead red pine (Pinus densiflora for. erecta) tree stand from Geumgang pine tree forest in Uljin, Gyeongsangbuk-do Province. GPS coordinates: 37°01′50″N, 129°22′83″E).

Holotype female, 25 female, and 24 male paratypes were deposited in the Nematode Collection of Kyungpook National University (KNU), Republic of Korea, and five female and four male paratypes were deposited in the Nematode Collection of Ghent University Museum in Belgium.

Paractinolaimus uljinensis n. sp. was isolated from a red pine tree stand from Geumgang pine tree forest in Uljin, Gyeongsangbuk-do Province. Thus, the species’ epithet uljinensis is derived from the locality of its first description, i.e., Uljin.

The amplification of the nearly full-length 18S-rRNA, the D2-D3 expansion segment of 28S-rRNA gene and ITS1-5.8S-ITS2 of rRNA yielded fragments of approximately 1700 bp, 800 bp, and 1200 bp, respectively. No intraspecific sequence variation was recorded in the three newly obtained 18S-rRNA gene partial sequences (PQ044581–PQ044583). In the 18S-rRNA gene phylogeny, Paractinolaimus uljinensis n. sp. sequences were grouped in a supported subclade with the closely homologous sequences of Paractinolaimus sahandi (GU178030), Trachactinolaimus nanjingensis (ON054242 and ON054246), Paractinolaimus sp. (OQ682610), Enchodelus cf nepalensis (KJ636402), Paractinolaimus macrolaimus (AY284826, AY993978, and KJ636378), Paractinolaimus decraemerae (GU446710), Prodorylaimus sp. (EF207246), Dorylaimus stagnalis (AY284776), and Mesodorylaimus sp. (MG921255), differing by 10 bp (0.8%), 12 to 13 bp (0.8%), 13 bp (0.8%), 14 bp (0.8%), 14 to 16 bp (0.9%), 15 bp (0.9%), 21 bp (1.2%), 21 to 22 bp (1.3%), and 21 to 22 bp (1.3%), respectively.

The three D2-D3 sequences of Paractinolaimus uljinensis n. sp. (PQ045703–PQ045705) were also identical, with no intraspecific sequence variation (0.0%). And, based on the more informative 28S-rRNA gene phylogeny, the sequences of Paractinolaimus uljinensis n. sp. were grouped in a well-supported subclade (PP = 100%) with the sequences of Trachactinolaimus nanjingensis (ON054911 and ON054913), Paractinolaimus sp. (OQ682613), Paractinolaimus decraemerae (GU446711), Paractinolaimus macrolaimus (AY592998 and AY593000), and Paractinolaimus sahandi (GU178031), differing by 44 to 46 bp (5.6–6.0%), 47 bp (6.3%), 45 to 51 bp (6.0–6.3%), 46 to 56 bp (6.1–7.0%), and 53 to 59 bp (7.0–7.3%), respectively. The ITS1-5.8S-ITS2 of rRNA gene sequences of Paractinolaimus uljinensis n. sp. represent the first characterization of the gene for the genus, and therefore, there were no definitive ITS-rRNA gene sequences for Paractinolaimus in GenBank for comparison. However, the three generated sequences (PQ045706-PQ045708) showed relative homology with ITS-rRNA gene sequences of Trachactinolaimus sp. available in GenBank (percentage identity of 89.6%, but with a low query coverage of 43%). 70 18S-rRNA and 69 28S-rRNA gene sequences from member species of Paractinolaimus, 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. 5 and 6.

Figure 5:

Bayesian tree inferred under the GTR + I + G model from 18S-rRNA gene sequences of Dorylaimid species. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Paravulvus hartingii and Nygolaimus cf. parvus.

Figure 6:

Bayesian tree inferred under the GTR + I + G model from LSU D2-D3 partial sequences of Dorylaimid species. Posterior probability values exceeding 50% are given on appropriate clades. The studied population is indicated in bold text. Outgroup taxa: Mononchus tunbridgensis and Anatonchus tridentatus.