Zoosyst. Evol. 101 (2) 2025, 583-596 | DOI 10.3897/zse.101.144271 yee BERLIN Vastnema crassicutaneum gen. et sp. nov. (Nematoda, Dorylaimida, Dorylaimidae), an interesting new taxon from natural areas of Vietnam Tam T. T. Vul*, Joaquin Abolafia?, Anh D. Nguyen’, Thi Mai Linh Le?*, Reyes Pefia-Santiago* Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Graduate University of Science and Technology, Vietnam Academy of Science and Technology. 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Departamento de Biologia Animal, Biologia Vegetal y Ecologia, Universidad de Jaén, Campus "Las Lagunillas" s/n, Edificio B3, 23071- Jaén, Spain FW NM fF Universidad de Jaén, Jaén, Spain https://zoobank. org/B9E64763-486C-429E-AC47-F 78ED360333F Corresponding author: Tam T. T. Vu (vtam7572@gmail.com) Academic editor: A. Schmidt-Rhaesa # Received 12 December 2024 Accepted 24 January 2025 Published 28 February 2025 Abstract A new dorylaimid taxon, Vastnema crassicutaneum gen. et sp. nov., collected in natural habitats of Vietnam, is characterized, including morphological description, morphometrics, SEM observations, and molecular (18S-, 28S-rDNA) analyses. Vastne- ma gen. nov. is distinguished and separated from its relatives by a combination of key traits: large size (body 4.79-6.35 mm long), lip region tapering and continuous with the adjoining body, very short neck (b-ratio 6.3—8.7), presence of a peculiar uterine differentiation, pre-equatorial (V = 38-44) vulva, and 45—58 stomata-like ventromedian supplements. The new species is characterized by its three-layered cuticle, 12.5—15.5 um thick at the anterior region; lip region 26—31 um wide; odontostyle 49-60 um long, or 1.8—2.0 times the lip region diameter; neck 627-888 um long; pharyngeal expansion occupying 46-49% of the total neck length; uterus complex and 571-804 um, or 3.2-4.3 body diameters long; longitudinal vulva; tail short and rounded in both sexes (39-56 um, c = 98-140, c’ = 0.5—0.7); and spicules 138—160 um long. Both morphological and molec- ular data support a close relationship of the new taxon with some Labronematinae representatives but also reveal that internal and external relationships of Dorylaimidae might be more complex than traditionally assumed. Key Words Labronematinae, LSU, morphometrics, phylogeny, SEM, SSU, taxonomy Introduction Alvarez-Ortega et al. (2015, 2016), Nguyen et al. (2016, 2017), Nguyen and Pefia-Santiago (2018, 2020), Hoang Dorylaims, the members of the order Dorylaimida, are probably the most diverse nematode taxon, being present everywhere on the six continents and in all kinds of conti- nental habitats, either freshwater sediments and/or arable and pristine soils (Pefia-Santiago 2021). Traditionally, the study of dorylaimid fauna of Vietnam did not receive too much attention. Actually, Nguyen et al. (2014) compiled previous records of only 25 species and 15 genera. Nev- ertheless, the inventory of species significantly increased throughout the last decade by, among other contributions, et al. (2019), and Vu et al. (2024). Several populations of an interesting dorylaimid form were collected in the course of a nematological survey conducted to explore the nematode fauna of Vietnam. Its study revealed that it was distinguished by a unique combination of features and that it was not comparable to any known genus. Thus, this contribu- tion aims to characterize the new taxon and to discuss its evolutionary relationships. The results are presented in the following. Copyright Vu, T.T.T. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 584 Vu, T.T.T. et al.: Vastnema crassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Materials and methods Sampling, extraction, and processing of nematodes A total of 26 adult specimens, collected in soil sam- ples from three natural areas of Vietnam, were studied. Nematodes were extracted using a modified Baermann funnel technique, heat killed, and fixed in TAF solu- tion (Southey 1986) for morphological observations or in a DESS mixture (Yoder et al. 2006) for molecular analyses. Then, specimens were transferred to anhy- drous glycerol (Seinhorst 1959, 1962), mounted on glass slides for their observation with light microsco- py, and measured and photographed using an Eclipse 801 microscope (Nikon, Tokyo, Japan) equipped with differential interference contrast optics, a drawing tube (camera lucida), and a DS digital camera. Line drawings were made from taken photomicrographs and/or sketches obtained by means of camera lucida. Morphometrics include Demanian indices and other measurements and ratios, some of them presented in a separate table, while others form part of the literal description of species. All measurements were record- ed in um, except body length in mm. After filming and taking pictures, selected specimens were submitted to molecular studies. DNA extraction, polymerase chain reaction (PCR), and sequencing Nematode DNA was extracted from a single indi- vidual as described by Holterman et al. (2006), and DNA extracts were stored at —20 °C until used as PCR templates. The D2, D3 expansion segment of 28S tDNA and 18S was amplified using the forward D2A (5'-ACAAGTACCGTGGGGAAAGTTG-3') and _ re- verse D3B (5'-TCGG AAGGAACCAGCTACTA-3') primers (Subbotin et al. 2006), and the 18S rDNA fragment was amplified using the primers 18S (18F: 5'-TCTAGAGCTAATACATGCAC-3'/18R: 5'-TACG- GAAACCTTGTTACGAC-3’) (Floyd et al. 2005). All PCR reactions contained 12.5 wl of Hot-Start Green PCR Master Mix (2x) (Promega, USA), 1 pl of the for- ward and reverse primer (10 uM each), the 3 unl DNA template, and sterile Milli-Q water to 25 ul of the total volume. All PCR reactions were performed in a Sim- pliAmp thermal cycler (Thermo Fisher Scientific) as follows: an initial denaturation step at 95 °C for 4 min, followed by 40 cycles at 95 °C for 30 s, 54 °C for 30 s, and 72 °C for 60 s with a final incubation for 5 min at 72 °C. Amplicons were visualized under UV illumina- tion after simple safe gel staining and gel electropho- resis. Purified PCR products were sent to Apical Sci- entific Company for sequencing (Selangor, Malaysia). After sequencing, the obtained rDNA sequence frag- ments were deposited in GenBank. zse.pensoft.net Phylogenetic analyses For exploring phylogenetic relationships, analyses were based on 18S and 28S rDNA. The newly ob- tained sequences were manually edited using Chromas 2.6.6 (Technelysium, Queensland, 110 Australia) and aligned with other sequences available in GenBank using the ClustalW alignment tool implemented in MEGA11 (Kumar et al. 2021). Poorly aligned regions at extremes were removed from the alignments using MEGA11. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically se- lected using jModelTest 2.1.10 (Darriba et al. 2012). The phylogenetic tree was generated with the Bayes- ian inference method using MrBayes 3.2.6 (Ronquist et al. 2012). The analysis under the generalized time reversible and invariant sites and gamma distribution (GTR +I1+G) model was initiated with a random start- ing tree and run with the Markov chain Monte Car- lo (Larget and Simon 1999) for 1 x 10° generations. The tree was visualized and saved with FigTree 1.4.4 (Rambaut 2018). Results and discussion Taxonomy and systematics Family: Dorylaimidae de Man, 1876 Subfamily: Labronematinae Pefia-Santiago & Alva- rez-Ortega, 2014 Vastnema Vu, Abolafia & Pefia-Santiago, gen. nov. https://zoobank.org/D80ECAAF-2697-4B75-9521-5EAAD6CB72CC Type and only species. Vastnema crassicutaneum sp. nov. Etymology. The genus name derives from VAST (Vietnam Academy of Science and Technology) and “nema”. Diagnosis. Dorylaimidae. Labronematinae. Large to very large-sized nematodes. Cuticle dorylaimid, very thick, three-layered. Lip region tapering, contin- uous with the adjoining body, with totally fused lips and very small cephalic papillae. Amphid aperture less than one-half of lip region diameter. Odontostyle strong, up to twice the lip region diameter, with wide aperture. Guiding ring, delicate, double. Odontophore rod-like, simple. Pharynx unusually short, entirely muscular, very gradually enlarging, with basal expan- sion occupying less than half of the total neck length. Female genital system di-ovarian with a peculiar uter- ine differentiation, well-developed pars refringens vaginae, and pre-equatorial longitudinal vulva. Tail similar in both sexes, short and rounded. Spicules do- rylaimid, slender. Ventromedian supplements 45—58 in number, very shortly spaced, small, not typical- ly mammiform, with hiatus. Zoosyst. Evol. 101 (2) 2025, 583-596 Morphological separation from its closest genera The general appearance of the new genus (large size, strong odontostyle, double guiding ring, rounded tail in both sexes) resembles the morphological pattern ob- served in some members of Labronematinae Pefia-San- tiago & Alvarez-Ortega, 2014, especially Labronema Thorne, 1939, and Nevadanema Alvarez-Ortega & Pefia-Santiago, 2012. It differs from Labronema, a very heterogeneous genus (needing a good revision, cf An- drassy 2009, 2011) in its larger size (body 4.79-6.35 mm vs. very occasionally exceeding 4.5 mm long), lip region tapering, with rounded anterior margin and continuous with the adjoining body (vs. not tapering, with truncate or even somewhat sunken anterior margin and often offset by deep constriction), very short neck (6 = 6.3-8.7 vs. b-ratio always less than 6.3 and very sporadically more than 5.0), presence (vs. absence) of a peculiar uterine differentiation, pre-equatorial (V = 38-44 vs. equatori- al or post-equatorial vulva, V-ratio never under 44 and very often ca 50 or more), and much more ventromedian supplements (45—58 vs. a maximum of 31) with different shape (very low and resembling stomata of tree leaves vs. typically mammiform). [See also the detailed separation of type species from some atypical members of Labrone- ma.| The new genus Is easily distinguishable from Neva- danema by its much larger general size (vs. body length 2.36—3.40 mm), lip region shape (vs. cap-like and offset by constriction), larger odontostyle aperture (more than one-third vs. less than one-fifth of total length), compara- tively much shorter neck (vs. b = 3.6—4.5), pre-equatorial vulva (vs. V = 49-56), complex (vs. simple) uterus, and male as frequent as female (vs. male absent). Descriptions of species Vastnema crassicutaneum sp. nov. https://zoobank.org/2BDC6FBA-31AA-4079-A389-CE3CC740BFB9 Figs 1-6 Material examined. Sixteen females and eight males from three locations, in good state of preservation, some specimens probably slightly flattened due to their large body, including wide diameter at mid-body. Morphometrics. See Table 1. Type population. (Nature Reserve Nam Xuan Lac, Cho Don district, Bac Can province) 11 females and seven males. Holotype. Female adult, Northern Vietnam, Bac Can province, Cho Don district, Nam Xuan Lac nature reserve, December 2021 (coordinates 22°17'30"N, 105°31'06"E, elevation 800 m), soil from a wild forest. Paratypes. Eleven females and seven males with the same collection details as the holotype. Other material. All in Northern Vietnam. i) Ha Giang province, Bac Me district, Du Gia nature reserve, October 2023 (coordinates 22°43'13"N, 105°11'32"E, elevation 300 m); 11) Ninh Binh province, Cuc Phuong National Park, 585 August 2019 (coordinates 20°21'06"N, 105°35'22"E, ele- vation 430 m), soil from a wild forest. Description. Adult. Moderately slender to slender (a = 25-34) nematodes of large to very large size, 4.79- 6.35 mm long. Body cylindrical, clearly narrowing towards the anterior end, much less towards the posterior extremity, as the tail is short and rounded. Upon fixation, habitus almost straight. Cuticle smooth (but a very fine transverse striation is appreciable with SEM), three-layered, consisting of thin outer and inner layers and much thicker intermediate layer, especially conspicuous at level of anterior region and tail, 12.5—15.5 um thick at anterior region, 9-15 um in midbody, and 14—20 um on tail. Lateral chord 12—29 um wide, occu- pying up to one-sixth (8—16%) of midbody diameter, lacking any differentiation. Body pores very small, the lateral ones arranged in a single row throughout the body length, a few dorsal and ventral pores are present at cervical region. Lip region rounded, occasionally slightly asymmetrical, visibly tapering, less than one-fourth (1 8—24%) of body diameter at neck base, 2.2—2.5 times as wide as high, continuous with the adjoming body or hardly offset by a very shallow de- pression. SEM observations: lips totally amalgamated, oral field wide as inner and outer labial papillae are quite close, perioral area not differentiated, labial papillae button-like, cephalic papillae appearing as very small pores (Fig. 6A—C). Amphidial fovea cup-like, its aperture 10.5—13.5 um wide, occupying less than one-half (33-48%) of lip region diam- eter, appearing as a wide transverse slit visibly occluded at its middle. Cheilostom almost cylindrical, 32-40 um long, with moderately thick walls, but lacking any other spe- cialization. Odontostyle strong, 1.7—2.0 times longer than lip region diameter, 8.3-11.8 times as long as wide, 0.89- 1.13% of body length, its aperture 19-27 um long or less than one-half (36-48%) of the total length. Guiding ring, double, but very delicate and difficult to visualize in some specimens. Odontophore simple, rod-like, 1.3—1.6 times the odontostyle. A mucro 2.5—8.5 x 1.5—3 um Is present in sev- eral specimens at level of odontophore base (Fig. 2A, B, K). Pharynx comparatively very short (6 = 6.3—8.7), strongly muscular, very gradually enlarging into the basal expansion that is 3.46.1 times as long as wide, 1.9-3.0 times longer than body diameter at neck base, and occupies up to one-half (46-49%) of the total neck length; pharyngeal gland nuclei located as follows: DO = 52-56, DN = 53-58, S\N, = 67— 69, SN, = 74-79, S,N = 83-88. Nerve ring located at 228— 308 um or 33-36% of the total neck length from the anterior end. Pharyngo-intestinal junction consisting of a conical to somewhat cylindroid cardia 41-84 x 17-32 um almost en- tirely enveloped by intestinal tissue, and a weak, ring-like structure around the junction of cardia to pharyngeal base. Female. Genital system diovarian, with both branches equally and well-developed, the anterior 823—1250 um long, or 16-24% of body length, the posterior 835—1153 um long, or 15-21% of body length. Ovaries variable in length, 221—704 um the anterior, 185-619 um the posterior, often reaching and surpassing the sphincter level, with oocytes ar- ranged first in several rows and then in a single one. Oviduct variably long too, 157-349 um or 0.8—2.0 body diameters, zse.pensoft.net 586 Vu, T.T.T. et al.: Vastnema crassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Figure 1. Vastnema crassicutaneum gen. et sp. nov. from Vietnam. A. Neck region; B—D. Anterior region, lateral me- dian view; E. Anterior region, lateral surface view; F. Spicule; G. Female, posterior genital branch; H. Pharyngo-intes- tinal junction; I. Male, posterior body region; J. Vagina; K. Female, posterior body region; L. Lateral guiding piece; M. Posterior ovary; N. Female, caudal region; O. Male, caudal region. Scale bars: 100 um (A, G, I, M); 20 um (B, D, F, H, N, O); 10 um (C, E, J, L); 50 um (Ks). zse.pensoft.net Zoosyst. Evol. 101 (2) 2025, 583-596 Figure 2. Vastnema crassicutaneum gen. et sp. nov. from Vietnam (LM). A—G. Anterior region, lateral median view; H. Anterior region, lateral surface view; I, J. Pharyngo-intestinal junction; K. Mucro located at odontophore base. Scale bars: 20 um (A-D, F, G, I, J); 10 um (E, H); 5 um (K). consisting of two sections almost equal in length: distal ca. A weak sphincter separates the oviduct from the uterus. part made of prismatic cells and proximal one an elongated = Uterus 571—804 um or 3.2-4.3 body diameters long, bipar- pars dilatata always with wide lumen inside and containing __ tite, consisting of distal, longer, and slender region with nar- abundant sperm cells, therefore functioning as spermathe- row lumen and a proximal, shorter, and wider region with zse.pensoft.net Vu, T.T.T. et al.: Vastnema crassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Figure 3. Vastnema crassicutaneum gen. et sp. nov. from Vietnam (LM, female genital system); A, B. Posterior branch; C. Vulva, ventral view; D. Oviduct-uterus junction; E. Oviduct; F. Sperm cells inside pars dilatata oviductus; G, H. Uter- us differentiation; I-K. Vagina, lateral view. Scale bars: 100 um (A); 10 um (C, KF, G, E-KK); 20 um (D, H); 50 um (E). zse.pensoft.net Zoosyst. Evol. 101 (2) 2025, 583-596 589 Figure 4. Vastnema crassicutaneum gen. et sp. nov. from Vietnam (LM, neck and posterior region); A. Neck region; B, C. Female, posterior body region; D. Female, rectum and caudal region; E-G. Female, caudal region; H—J. Male, caudal region. Scale bars: 100 um (A); 50 um (B, C); 20 um (D—-J). broad lumen and often containing abundant sperm cells inside; distal region always bearing a very special differen- tiation, resembling the Z-like structure of some dorylaims, with a concentration of many small spindle-shaped ele- ments visibly refringent that adopt a peculiar arrangement (Fig. 3A, B, G, H), all together measuring 13-18 x 22- 26 um. Vagina 54—73 um long, to ca one-third (31-36%) of body diameter: pars proximalis 39-51 x 26—37 um, with divergent walls surrounded by moderately developed cur- cular musculature; pars refringens, consisting of (lateral view) two separated triangular pieces 12—16 x 6-10 um and with a combined width of 26-32 um: pars distalis 5-10 um long. Vulva longitudinal, slightly elliptical, 8-11 um long. Prerectum 2.2—3.3, rectum 0.8—1.3 body diameters long. Anus a weakly arched transverse slit ca 15 um long. Tail short and rounded, inner core 17—22 um long, occupying less than half (37-48%) of tail length, caudal pores two pairs, at the middle of tail, one sublateral, another subdorsal. Male. Prerectum 3.4—5.5, cloaca 1.3—1.5 times longer than body diameter at level of the cloacal aperture. Geni- tal system diorchic with opposite testes. Sperm cells ellip- tical, 8 x 4.5 um. In addition to the pre-cloacal pair, situat- ed at 16—24 um from the cloacal aperture, there 1s a series of 47-58, shortly spaced (7-14, exceptionally reaching 19 um in between), very low, ventromedian supplements, the most posterior of them located at 101—154 um from the adcloacal pair, thus with a perceptible hiatus. As seen with SEM, the ventromedian supplements are not mammiform zse.pensoft.net 590 Vu, T.T.T. et al.: Vastnema crassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Figure 5. Vastnema crassicutaneum gen. et sp. nov. from Vietnam (LM, male traits); A. Posterior body region; B-D. Spicule, arrowheads pointing at the ventral hump; E, I. Lateral guiding piece; F. Ventromedian supplements. Scale bars: 100 um (A); 20 um (B—D, F); 10 um (E, G-D. as so usual in dorylaims but appear as short longitudinal slits that resemble the stomata of plant leaves (Fig. 6K— M). Spicules dorylaimid, relatively slender, 6.9—9.1 times longer than wide, 1.8—2.0 times longer than body diame- ter at level of the cloacal aperture: head 12—25 um long, occupying up to one-sixth (8—16%) of spicule length, 1.2— 1.8 times as long as wide, with its dorsal side visibly lon- ger than the ventral one; median piece 6.5—8.5 um wide, occupying 32-44% of spicule maximum width; posterior tip S—9 um wide; ventral hump and hollow little marked in general, the hump situated at 49-70 um or 33-47% of spicule length; curvature 118—135°. Lateral guiding piec- es 16-24 um long, 4.0-4.8 times wider than long, almost cylindrical but visibly tapering at its posterior third. Tail similar to that of female, maybe slightly more conoid. Other populations. (six females and one male from two locations): Morphologically identical to type mate- rial. Morphometrically very similar too, with totally co- incident or widely overlapping ranges of main measure- ments and ratios, although a few minor differences are zse.pensoft.net also noted; for instance, prerectum length is appreciably longer in Ha Giang specimens: 324-390 vs. 206-321 um in females and 473 vs. 324—390 um in males. Molecular characterization. After sequencing and ed- iting, six (three 18S and three 28S) rDNA sequences were obtained of type material for phylogenetic analyses. The three 28S sequences were 798, 679, and 651 bp long, with 511 bp in common, and a Blast search showed that the longest of them was 96.9% similar to that of Labronema porosum Vu, Elshishka, Nguyen, Le, Mladenov & Peneva, 2024 (acc. PP060468), 96.3% to that of L. bidoupense Vu, Elshishka, Nguyen, Le, Mladenov & Peneva, 2024 (acc. PP060469), 92.0% to that of Labronema vulvapapillatum (Meyl, 1954) Loof & Grootaert, 1981 (acc. AY592997), 91.5% to that of L. vulvapapillatum (acc. AY592996), 91.3% to that of Labronema sp., and 90.8% to that of Ne- vadanema nevadense Alvarez-Ortega & Pefia-Santiago, 2012 (acc. JN242245), all of them rounded-tailed repre- sentatives of Dorylaimidae de Man, 1876, Labronemati- nae Pefia-Santiago & Alvarez-Ortega, 2014. Zoosyst. Evol. 101 (2) 2025, 583-596 Figure 6. Vastnema crassicutaneum gen. et sp. nov. from Vietnam (SEM); A. Lip region, lateral view; B. Lip region, in face view; C. Labial papillae; D. Male, posterior body region, ventral view; E. Lip region, subventral view; F, G. Vulva, ventral view; H. Female, subventral view; I. Anus; J. Male, caudal region; K—M. Detail of ventromedian supplements. Scale bars: 5 um (A, B, E, I); 1 um (C); 100 um (D); 10 um (F); 4 um (G); 20 um (H, J); 3 um (KK); 1 um (L, M). zse.pensoft.net 592 Vu, T.T.T. et al.: Vastnema crassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Table 1. Main morphometrics of Vastnema crassicutaneum gen. et sp. nov. from Vietnam. Measurements in um except L in mm, in the form: average + sd (range). Population Nam Xuan Lac, Bac Can Du Gia, Ha Giang Cuc Phuong, Ninh Binh Holotype Paratypes Paratypes Character n Q 1199 733 42° 3 209 L 6.19 5.51 + 0.47 (5.00-6.35) 5.18 + 0.26 (4.79-5.48) 5.80 + 0.37 (5.26-6.09) 5.66 5.25, 5.63 a 31 29.4 + 2.7 (25-34) 31.1 + 2.4 (27-34) 32.2 + 1.8 (30-34) 33 29, 40 b 7.7 7.1 + 0.7 (6.3-8.4) 7.2 + 0.8 (6.3-8.7) 7.3 + 0.1 (7.2-7.5) 7.6 7.0, 6.3 Cc 129 113 + 11 (98-131) 107 + 17 (94-140) 116 + 22 (101-142) 107 103, 106 V 41 41.8 + 1.6 (39-44) 39.0 + 1.2 (38-41) - ?,44 Cc’ 0.5 0.5 + 0.0 (0.5-0.6) 0.6 + 0.0 (0.5-0.7) 0.5 + 0.1 (0.5-0.6) 0.7 0.5, 0.5 Lip region diameter 31 29.3 + 1.4 (27-31) 28.1 + 1.3 (26-30) 27.0 + 1.2 (26-28) 28 31, 31 Odontostyle length 55 55.2 + 2.7 (51-60) 53.4 + 2.6 (49-57) 55.3 + 2.8 (52-58) 55 51, 70 Odontophore length 80 77.7 + 4.0 (72-84) 75.9 + 3.8 (70-79) 73-0 + 2.4 (70-76) 77 73, 82 Neck length 803 777 + 54 (627-821) 720 + 44 (631-755) 794 + 59 (706-837) 746 746, 888 Pharyngeal expansion length 386 370 + 29 (292-390) 341 + 22 (299-364) 386 + 42 (338-418) 357 357, 449 Body diameter at neck base 167 148 + 10 (133-167) 136 + 12 (122-156) 148 + 4.9 (145-154) 138 142, 137 mid-body 200 188 + 11 (172-210) 168 + 20 (144-194) 180 + 4.3 (175-185) 170 179, 141 anus 96 94.1 + 4.0 (88-102) 79.0 + 3.9 (71-84) 94.3 + 1.5 (93-96) 76 99, 99 Distance vulva — anterior end 2564 2300 + 193 (2035-2585) : 2256 + 91 (2143-2344) - ?, 2491 Prerectum length 321 259 + 39 (206-321) 339 + 57 (265-412) 361 + 30 (324-390) 473 319, 228 Rectum/cloaca length 121 89.9 + 11.8 (79-121) 108 + 7 (96-117) 74 + 5.6 (68-79) 98 97, 90 Tail length 48 48.8 + 3.1 (45-56) 49.0 + 5.1 (39-54) 50.3 + 6.7 (43-56) 53 51, 53 Spicules length - - 149 + 7 (139-160) - 138 - Ventromedian supplements 47-58 - 45 The three 18S sequences were 1698, 1690, and 1689 bp long, with 1684 bp in common, and a Blast search showed that the longest of them was 97.2% simi- lar to that of Aporcelaimellus sp. (acc. JX674034), 97.2% to that of L. ferox Thorne, 1939 (acc. AY552972), 97.2% to that of Aporcelaimellus sp. (acc. PP099686), 97.1% to that of Prodorylaimus sp. (EF207246), and 97.0% to that of Mesodorylaimus sp. (acc. MG921256). Diagnosis. The new species is characterized by its 4.79— 6.35 mm long body, cuticle three-layered and 12.5—15.5 um thick at anterior region, lip region continuous with the adjoining body and 26-31 um wide, odontostyle 49-60 um long or 1.8—2.0 times the lip region diameter, neck 627-888 um long, pharyngeal expansion occupying 46— 49% of the total neck length, female genital system dio- varian, uterus complex and 571—804 um or 3.2-4.3 body diameters long, pre-equatorial (V = 38-44) and longitudi- nal vulva, tail short and rounded in both sexes (39-56 um, c = 98-140, c’=0.5-0.7), spicules 138-160 um long, and 45-58 small ventromedian supplements with hiatus. Separation from some resembling species. The new species resembles a few Labronema species. In having large size, 1t compares to L. magnum Altherr, 1972, a freshwater species known to occur in Nepal, Russia, and Sweden (see taxonomic revision by Pefia-Santiago, 2022), but it differs from this in its much thicker cuticle (vs. for instance, 4-5 um at anterior region), lip region with different shape (vs. anterior region truncate, offset by weak constriction, and one-third as wide as body diame- ter at neck base), comparatively much shorter neck (vs. b = 4.1-6.3), more anterior vulva (vs. V = 44-53), lon- ger spicules (vs. 118-135 um), and more numerous (vs. 21-31) ventromedian supplements with different aspect (vs. typically mammiform). zse.pensoft.net In having continuous lip region, it also resembles several atypical Labronema representatives, including L. hyalinum (Thorne & Swanger, 1936), Thorne, 1939, L. neopacificum Rahman, Jairajpuri, Ahmad & Ahmad, 1987, L. orientale Andrassy, 2011, L. singhalense Andrassy, 2011, L. stech- linense Altherr, 1968, and L. thornei Ferris, 1968, but all of them show smaller general size (body length up to 3.73 mm long, odontostyle up to 48 um long), comparatively longer neck (b-ratio up to 5.2), more posterior vulva (V = 45-55), shorter spicules (up to 120 um long), and much less ventro- median supplements (up to 29) with different shapes. Type material. Female holotype, seven female and three male paratypes, and one male paratype deposited at the De- partment of Nematology, Institute of Ecology and Biolog- ical Resources, VAST, Hanoi, Vietnam. Four (two female and two male) paratypes at the Nematode Collection of the University of Jaén, Spain. Four (two female and two male) paratypes at USDANC, Beltsville, Maryland, USA. Etymology. The species epithet derives from the Latin terms crassus = thick and cutaneum = skin, cuticle, and refers to this peculiar trait of the new taxon. Evolutionary relationships of the new taxon Morphologically, Vastnema crassicutaneum gen. et sp. nov. 1s characterized by a unique combination of very unusual traits within the family Dorylaimidae de Man, 1876, and even the order Dorylaimida. First, its very thick, three-layered cuticle is only observed in some very large dorylaims and should be regarded, with a cladistic approach, as a relevant apomorphy. Second, its compar- atively short pharynx/neck, with b-ratio always exceed- ing 6.0 and very often appreciably more, probably is an Zoosyst. Evol. 101 (2) 2025, 583-596 autapomorphy of the new taxon, especially if its round- ed-tailed condition is considered. Third, the presence of an exclusive uterine specialization, resembling those known as Z-like structures but significantly different from them in its nature, is another recognizable autapomorphic feature too. Fourth, the high number and, in particular, the shape of male ventromedian supplements, appearing as plant leaf stomata rather than button-like or mammiform structures, certainly is (at least) an infrequent apomorphy. Other interesting traits (lip region continuous and with totally fused lips, double guiding ring, pre-equatorial vulva, rounded-tailed sexes) are more usual apomorphic features within dorylaims. As mentioned, the new taxon shares several traits (strong odontostyle, double guiding ring, rounded tail in both sexes) with members of the sub- family Labronematinae, in particular Nevadanema and 100 i Ni2268614"Mononchis funcstas 0.02 100 593 some Labronema representatives, supporting a close re- lationship between them. As derived from the molecular analyses of obtained sequences, whose results are presented in Figs 7 (18S tree), 8 (28S tree), the evolutionary relationships of the new taxon basically agree with those deduced from morphological observations. Thus, and in both trees, Vastnema gen. nov. sequences form part of a maximal- ly (100%) supported clade also including sequences of Labronema representatives. Moreover, the 28S tree, which provides better general resolution, shows that the (Vastnema gen. nov. + Labronema spp.) clade belongs to a much larger (highlighted in yellow, and also maximally supported) clade containing sequences of Dorylaimidae and Actinolaimidae Thorne, 1939 taxa. It is remarkable that this large subclade is divided into two subclades, nf ae GOL hetygnera wyapepatim 109 wofheoOP rS3e45-talanema_| ericum “ob t648967" Falanana baat 100° 169 Baar ipa pronema_| montanum B: neta rGrassicutaneum sp. nm at PQ) is 100 vial “ asin Fu 100 At asin nee cprassicutaneum sp. n. 100 AVSBDQTD The ma ab bronema-b bid oupense onemepofe KiX2o6088 § Neometadorylaimus_ coomansi etador' laimus— ‘coomansi ra m weepiaae Tyenshslamue cyneaai fe Me99197-Tlencholaimus-t do 100. ae etenses: affinis 100 p pesca TMacrolajmus bo jaimus_macrolaimus all ahi eee sof UOC“, Pau Beer SpStaxonchium carmena er y 4809 Pa Him _laetifi [foo ge EF207248 Mesod by pains Eefiipcercus ey etnam hy vietnam ietnam iang, V lint, Bin ac Can 00 1p 1001p colainn S_ major a sa nf a ‘al Wiotora 284831 Leptonchus_ granulosus Y. 4 J636399_Proleptonch cher 94 “oes if gear us weisd ws 61628 Tylendtal =100 i £0. 8 — wa Teno rae Zan janiensis AY284 riexan attenu hoi Bal peices PSconchin pair 38 Synchellaxonchium nan ncheilax couse a4 4 MF32514078y A C ar ve os Pe dira 3 ,bage Ingshanensis elondira pape a_s| 53 100 a Nera i lass ‘bagongshanensis 100 100 ae Payee poten ricus 0 Om NEES ijlamelus virginianus li SSPRES 34 Nigtaxon hium_giennen 9 o5 Hae 1po ja exo lum= enniense. d ie oad Regan tae chum Axon OP cd encholaimus_ helanensis 89 100 89) 00 JX885740 oy ~ B LP Ay 8 ” pporceta g Apors roel mune lee aiiza AB fA dS Aires carat Ros ife eis Ra RNTal SRSeu aus ae fe Fieseley A oreslqimellis-oblusisaudatus ml oe Soret os op hierodorA dest rf HOOHG/S A025 eMiorgsortainus inpiestus tte No gud 77rd f neie dere veces pe: HSESTary RhyssseapusWneutioc Ri: - fe cree Iai cp 400 oP 20 Ry is ed poco dopfarmus.t Fe 00 400 co NS fdorele Rae — ne jane’ Gaeronvars eu riser. bt 100 ‘e Fnkaey pga ecnoceys. bab lon PAHS 100 1Re ra sas enchode Odelus-sardashtensis og 1p0 PY 42953 cho lus macrodorus ay AES EomeS STE a 100 ryla opi 0 ‘100s i ee veer 0G Pun gentus Ss “spe ngens fe ene an ee ae orcelaime! araobtusicaudatus ig } BL — elt SITS Aaridorylaimus_ castaneae - aU ea nya signa tus = Hoo AY 847: ZA eMsso: era cf._nigritulus par 4 68S0! ee ot 00 KJ636386 ae: 10 o ASS RBINE TEER luatides” aquaticus_| 109 00 AY5 Zot? Roaz Agu Bae AY593944_Clavicaudoides_: iS ermeetoette Cc 94, 100 “100 9 86 DQ418791_Romanomermis_culicivorax Figure 7. Bayesian inference tree from the newly sequenced Vastnema crassicutaneum gen. et sp. nov. based on se- quences of the 18S rDNA region. Bayesian posterior probabilities (%) are given for each clade. The scale bar shows the number of substitutions per site. zse.pensoft.net 594 Vu, I.T.T. et al.: Vastnema c rassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Amo AY593016_Allodorylaimus_andrassyi 100 1% AY593015 Allodorylaimusandrassyi bo MKQ07554 Allodorylaimus_sp. 100} 100 97 100 rod Ht! 200 18_Aporce inus_mediterraneus MH184520_Aporcelinus_infundibulicaudatus MK007553_Aporcelinus_floridensis ; ae Aporcelinus_amazonicus 619727 Aporcelinus_sp._elongicaudatus MH619728 Aporcelinus_insularis MKOO: MF 13. anum AY592996_Labronema_vulvapapillatum AY592997/ob-abronema_vulvapapillatum __ 1 060469 _Labronema_bidoupense 100 100-rag- JN c ne 97 106. 100 rr 242245 Nevad EF207241 Prodo AY59299 vo ON133539 Jicus_: GU44671T Paractinolaimus_decraemerae KM067903_ Paractinolaimus_morus. AY592998 Paractinolaimus_macrolaimus 00, om pO EE Ge068 Labronema_porosum AQ Vas! x > la Giang, Vietnam PQ240120_28S Vastnema_crassicutaneum sp. n. Ninh Binh, Vietnam PQ2401217 28S" Vastnema’_crassicutaneum sp. n. Bac Can, Vietnam Z rylaimuS_ sp. 4 Dorylaimus stagnalis estindicus_sp. @d 178031 Paractinolaimus “Sahandi —or— P p QNO5491 — Kiiog92522 :fa 400 t 100 97 100 89 100 054! arhyssocol MH004441. I bt004440_CTrassolabium_Costaricense 0Q92 Ae 87312 Trachactinolaimus_persicus 1_Trachactinolaimus_nanjingensis 549127 Trachactinolaimus_nanjingensis usS_paradoxus ‘ Crassolabium_costaricense 984_Paraxonchium_carmenae 1005 AY593001 Paraxonchium_laetificans Y 593002 Paraxonchium_laetificans AY593004 Dorylaimoides_micoletzkyi AY593003_Dorylaimoides_limnophilus 52 100 100 100 A 84 AY 2930 4 86 95 96 87 100 100 eo 96 100 99 98 100 60 100 100 99 53 100 100 50 100 97 100 400 85 00 O 100 100 89 93 100 100 K JQ778a73_A\ AY593013_Ecumenicus_monohystera iho AY593014 Ecumenicus sp. AY593005 Mesodorylaimus_sp. Y 593006 Mesodorylaimus ‘Sp. AY593011_Oxydirus_nethus AY5930 ( VeOsO10 Sood Sy wel odorylaimus “sylphoides “p00 RY593027. 1 12 Oxydirus_oxycephalus. Iphoides ylencholaimus_mirabilis FF 207243 Tylencholaimugocf._teres . oe pe 2. MG921305_Tylencholaimus_ibericus KU992905_Tylencholaimus_helanensis MN29648%6_Neometadorylaimus _coomansi ; AY593023 ‘Carcharodiscus_banaticus AY593024 Carcharodiscus_Danaticus : EF207238 Discolaimoides_symmetricus MT776559_Discolaimoides_symmetricus MT776558_Discolaimus_major AY593025 Discolaimus_major EF207239 Discolaimus_cf._major AY593026_Discolaimus_major Y750844 Discolaimus_sp. porcella StH ore nam 104H754753 Aporcella_malekimilanii 100 70 KX1517719_Aporcella_charidemiensis 100 KX151720 Aporcella_charidemiensis 00 MW237838 _Aporcella_femina MH727515_Aporcella_talebii MH754747_Aporcella_vitrinus MT079121_Aporcella_dubia MT079124 Aporcella_minor | 10MW243335 Kochinema_farodai : nm KT258985_Belondira_bagongshanensis cain alae ee shanensis 7 oo MF363124 Belondira_coomansi 100, 30 MG921267 Bélondira_sp. _MG921268_Belondira_sp. MG921270_Axonchium_sp. NW@92127T Axonchium_sp. : - po MF325350_Syncheilaxonchium_nairi 400MF 3253517 Syncheilaxonchium_nairi ; co JX885 Axonchoides Smokyensis 100__soo- MG018767 Metaxonchium_toroense MH167347_Metaxonchium_Stenospiculum 100 00 100 00 100 100 100 fo 0 99 OQ099690_ Metaxonchium_magnum Q473054 “Metaxonchium_giennense PP95661G00leaxonchium_olearium J ' Q049740_Persinema_enigmaticum PQ049739_ Persinema_enigmaticum o- AY593054 Enchodelus_macrodorus AY593053 Pungentus_silvestris ; MH346474_ Pungentus_engadinensis MN855359 Pegectus Sufiyanensis : KY881719 | nenonpoldes_signyensis A ; } 400 A oo AY593035_Epidorylaimus_lugdunensis AY593036_Epidorylaimus_lugdunensis AY593037_Eudorylaimus_sp. AY599049_Moorodoryigimus modestus . i px AY593048 Eudorylaimus_minutus #0593047 Eudorylaimus minutus _ toV1235514 Dorydorella_bryophila tipo MH884066_Dorydorella_sp. AN%s593045 Longidorella_sp- ; 100, AY593042_Longidorella_cf. macramphis HM235515_Longidorella_penetrans AY593046_Microdorylaimus_miser KP963965_Heterodorus_brevidentatus . KR184127 Heterodorus_youbertghostai KR84124 Enchodorus_dolichurus veal KP204547_Rhyssocolpus_vinciguerrae 100 100 100 100 100 100 92 100 100 69 00 100 100 00 100 100 100 100 100 100 93 100 : KX691911_Enchodorus_yeatsi JQ827438_Metaporcelaimus_capitatus . KU589226 Sectonema_ciliatum AY593031_Sectonema_barbatoides sodS¥593029_Chrys 78 100 100 100 100 100 100 100 2 EF417153 Roman AY593063_Mononchus_tunbridgensis AY593064 Mononchus truncatus 0.08 JQ927440_Metaporcélaimus_ovogranulosus onema_attenuatum OP221721_Paravulvus_sp. _ OP221726_Parayulvus_hartingii MG921241_Aetholaimus_Totundicauda AY593061_Nygalaimus cf._brachyuris ; 10 eo— —F207237 Clavicaudoides _trophurus EF207236_ Clavicaudoides trophurus EF207234 Clavicaudoides_clavicaudatus ; _OP221727_Solididens_sp. omermis_culicivorax 100 Figure 8. Bayesian inference tree from the newly sequenced Vastnema crassicutaneum gen. et sp. nov. based on se- quences of the 28S rDNA region. Bayesian posterior probabilities (%) are given for each clade. The scale bar shows the number of substitutions per site. one including sequences of Vastnema gen. nov., Labrone- ma, Nevadanema, and Prodorylaimus sp., and another consisting of sequences of Dorylaimus stagnalis Dujar- din, 1845, plus several actinolaims. An integrative approach to the subject, combining mor- phological and molecular information, supports a very close relationship of the new taxon with Labronematinae representatives, but also that internal and external rela- tionships of Dorylaimidae might be more complex than zse.pensoft.net traditionally assumed, a question that will require much further research before its elucidation. Acknowledgements The senior author (RPS) deeply appreciates the invita- tion, support, and reception of the Department of Nem- atology, Institute of Ecology and Biological Resources Zoosyst. Evol. 101 (2) 2025, 583-596 (IEBR), Vietnamese Academy of Sciences and Tech- nology (VAST), Hanoi, Vietnam, to visit its lab and to participate in the project NCXS01.04/23-25. The Spanish authors (JA, RPS) thank the staff (Amparo Martinez-Morales), and this study used the facilities of the equipment of “Centro de Instrumentacion Cientifi- co-Técnica (CICT),” University of Jaén, for assistance to obtain SEM pictures. The work is supported by the Vietnam Academy of Science and Technology (VAST) under the project NCXS01.04/23-25 “Developing the first-class research team on the discovery of diversity and application poten- tial of hymenopterans, myriapods, and soil nematodes in the limestone mountains of northeastern Vietnam.” References Altherr E (1968) Nématodes de la nappe phréatique du réseau fluvial de la Saale (Thuringe) et psammniques du Lac Stechlin (Brandebourg dur nord). Limnologica 6: 247-320. Altherr E (1972) Contribution a la connaissance des Nématodes ri- throstygopsammiques et rithrostygopséphiques de Suede. Re- vue Suisse de Zoologie 79: 881-902. https://doi.org/10.5962/bhl. part.97141 Alvarez-Ortega S, Pefia-Santiago R (2012) Nematodes of the order Dorylaimida from Andalucia Oriental, Spain. Nevadanema nevadense gen. n., sp. n. (Qudsianematidae) from Si- erra Nevada National Park. Nematology 14: 249-264. https://doi. org/10.1163/138855411X589309 Alvarez-Ortega S, Nguyen, TAD, Abolafia J, Vu TTT, Pefia-Santiago R (2015) Three new species of the genus Aporcelaimoides Heyns, 1965 from Vietnam (Nematoda, Dorylaimida, Aporcelaimidae), with an updated taxonomy of the genus. Zookeys 516: 1—26. https:// doi.org/10.3897/zookeys.516.10087 Alvarez-Ortega S, Nguyen TAD, Abolafia J; Vu TTT, Bonkowski M, Pefia-Santiago R (2016) Three new species of Sectonema Thorne, 1930 (Dorylaimida, Aporcelaimidae) from Vietnam. Nematology 18: 517-536. https://doi.org/10.1163/15685411-00002974 Andrassy I (2009) Free-living nematodes of Hungary. III. Pedozoolog- ica Hungarica n° 5. Hungarian Natural History Museum. Budapest, Hungary, 608 pp. Andrassy I (2011) Three new bisexual species of Labronema Thorne, 1939 (Nematoda, Qudsianematidae). Opuscula Zoologica Budapes- tinensis 43: 107—120. https://www.oalib.com/research/2072275 Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. https://doi.org/10.1038/nmeth.2109 Dujardin F (1845) Histoire naturelle des helminthes ou vers intestinaux. Librairie Encyclopédique de Roret, Paris, 654 pp. Ferris VR (1968) Biometric analyses in the genus Labronema (Nemato- da, Dorylaimida) with a description of L. thornei n. sp. Nematologi- ca 14: 276-284. https://doi.org/10.1163/187529268X00480 Floyd RM, Rogers AD, Lambshead PJD, Smith CR (2005) Nematode- specific PCR primers for the 18S small subunit rRNA gene. Molec- ular Ecology Notes 5(3): 611-612. https://doi.org/10.1111/).1471- 8286.2005.01009.x Hoang H, Chu HH, Nguyen TAD, Trinh QP, Abolafia J, Pefia-Santi- ago R (2019) Morphological and molecular characterisation of 595 Chitwoodius coffeae sp. n. (Dorylaimida, Tylencholaimidae) from Vietnam, with a revised taxonomy of the genus. Nematology 21: 509-521. https://doi.org/10.1163/15685411-00003231 Holterman M, Wurff A, Elsen S, Megen H, Bongers T, Holovachov O, Bakker J, Helder J (2006) Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biol- ogy and Evolution 23: 1792-1800. https://doi.org/10.1093/mol- bev/msl044 Kumar S, Stecher G, Tamura K (2021) MEGA 11: Molecular Evolution- ary Genetics Analysis version 11.0. Molecular Biology and Evolu- tion 38: 3022-3027. https://doi.org/10.1093/molbev/msab120 Larget B, Simon DL (1999) Markov Chain Monte Carlo Algorithms for the Bayesian Analysis of Phylogenetic Trees. Molecular Biology and Evolution 16: 750-759. https://doi.org/10.1093/oxfordjournals. molbev.a026160 Loof PAA, Grootaert P (1981) Redescription of Labronema vulvapapil- latum Meyl, (1954) nov. comb. (Dorylaimoidea). Nematologica 27: 139-145. https://doi.org/10.1163/187529281X00197 Man J G de (1876) Onderzoekingen over vrij in de aarde levende Nematoden. Tijdschrift Nederlandsche dierkundige Vereeniging 2: 78-196. Meyl AH (1954) Die bisher in Italien gefundenen freilebenden Erd und Sisswasser-Nematoden. Archivio Zoologico Italiano, Torino 39: 161-264. Nguyen TAD, Pefia-Santiago R (2018) Two new species of the genus Metaxonchium Coomans & Nair, 1975 (Nematoda, Dorylaimida, Belondiridae) from Vietnam. Zootaxa 4415: 150-160. https://doi. org/10.11646/zootaxa.4415.1.7 Nguyen TAD, Pefia-Santiago R (2020) Morphological and molecu- lar characterization of Epidorylaimus procerus sp. n. (Dorylaimi- da, Qudsianematidae) from Vietnam. Journal of Nematology 52: e2020—112 [8 pp]. https://doi.org/10.21307/jofnem-2020-112 Nguyen TAD, Vu TTT, Bonkowski M, Pefia-Santiago R (2014) New data of three rare belondirid species (Nematoda, Dorylaimida, Belondiridae) from Vietnam, with the first record and description of the male of Oxybelondira paraperplexa Ahmad & Jairajpuri, 1979. Biodiversity Data Journal 2: e1156. https://doi.org/10.3897/ BDJ.2.e1156 Nguyen TAD, Abolafia J, Bonkowski M, Pefia-Santiago R (2016) Two new species of the genus Aporcelinus Andrassy, 2009 (Nematoda, Dorylaimida, Aporcelaimidae) from Vietnam. Zootaxa 4103: 550-— 560. https://doi.org/10.11646/zootaxa.4103.6.5 Nguyen TAD, Abolafia J, Dumack K, Bonkowski M, Pefia-Santi- ago R (2017) Two known species of Aporcelinus Andrassy, 2009 (Dorylaimida, Aporcelaimidae) from Vietnam, with the first mo- lecular study of the genus. Nematology 19: 853-868. https://doi. org/10.1163/15685411-00003092 Pefia-Santiago R (2021) Morphology and bionomics of dorylaims (Nematoda: Dorylaimida). Nematology Monographs and Perspec- tives 13 (Series Editors: Cook, R. & Hunt, D.J.). Brill. Leiden, The Netherlands, 278 pp. https://doi.org/10.1163/978900444785 1 Pefia-Santiago R (2022) New synonyms in the genus Labronema Thorne, 1939 (Nematoda, Dorylaimida, Dorylaimidae). Nematolo- gy 24: 953-957. https://doi.org/10.1163/15685411-bjal0175 Pefia-Santiago R, Alvarez-Ortega S (2014) An integrative approach to assess the phylogeny and the systematics of rounded-tailed genera of the subfamily Qudsianematinae (Nematoda, Dorylaimida). Zoo- logica Scripta 43: 418-428. https://doi.org/10.1111/zsc.12058 zse.pensoft.net 596 Vu, T.T.T. et al.: Vastnema crassicutaneum gen. et sp. nov. (Dorylaimida, Dorylaimidae) from Vietnam Rahman MF, Jairajpuri MS, Ahmad I, Ahmad W (1987) Two new species of Labronema Thorne, 1939 (Nematoda, Dorylaimida) from India. Nematologica 32(1986): 367-373. https://doi. org/10.1163/187529286X00264 Rambaut A (2018) Figtree, a graphical viewer of phylogenetic trees. https://github.com/rambaut/figtree/releases/tag/v 1.4.4 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012). MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Seinhorst JW (1959) A rapid method for the transfer of nematodes from fixative to anhydrous glycerine. Nematologica 4: 67-69. https://doi. org/10.1163/187529259X00381 Seinhorst JW (1962) On the killing, fixation and transferring to glycerine of nematodes. Nematologica 8: 29-32. https://doi. org/10.1163/187529262X00981 Southey JF (1986) Laboratory methods for work with plant and soil nematodes. London: Her Majesty’ Stationery Office, 202 pp. zse.pensoft.net Subbotin SA, Sturhan D, Chizhov VN, Vovlas N, Baldwin JG 1949 as inferred from D2 and D3 expansion fragments of the 28S rDNA gen sequences. Nematology 8: 455-74. https://doi. org/10.1163/156854106778493420 Thorne G (1939) A monograph of the nematodes of the superfamily (2006) Phylogenetic analysis of ‘Tylenchida Thorne, Dorylaimoidea. Capita Zoologica 8: 1-261. Thorne G, Swanger HH (1936) A monograph of the nematode genera Dorylaimus Dujardin, Aporcelaimus n. gen., Dorylaimoides n. gen., and Pungentus n. gen. Capita Zoologica 6: 1-223. Vu TTT, Elshishka M, Nguyen AD, Le TML, Mladenov A, Peneva V (2024) Two new species of genus Labronema Thorne, 1939 (Nema- toda, Dorylaimidae) from natural parks of Vietnam with an identifi- cation key to the species with a medium-sized odontostyle. Zookeys 1215: 1-26. https://doi.org/10.3897/zookeys.1215.128183 Yoder M, De Ley I T, King I, Mundo-Ocampo M, Mann J, Blaxter M, Poiras L, De Ley P (2006) DESS: a versatile solution for preserv- ing morphology and extractable DNA of nematodes. Nematology 8: 367-376. https://doi.org/10.1163/156854106778493448