Morphology, development stages, and phylogeny of the Rhabditolaimus ulmi (Nematoda: Diplogastridae), a phoront of the bark beetle Scolytus multistriatus from the elm Ulmus glabra Huds. in Northwest Russia

Nematodes were fixed with hot TAF (2 ml of triethanolamine, 10 ml of formalin, and 90 ml of distilled water) in a water bath (Ryss, 2017a), processed in a glycerol-water series, and mounted in the permanent collection slides using the method described by Ryss (2003, 2017b). Some juveniles fixed in TAF and processed to anhydrous glycerin, were stained with methylene blue stain by adding a 10-µl drop of saturated water solution of methylene blue to a drop of glycerin with the suspended nematode juveniles, on a 75 × 25-mm slide. After 12 hr, the stained suspension was covered with a 20 × 20-mm coverslip previously sealed on its edges with a thin film of glycerin jelly. After quick 60°C heating on a hot plate, the drop spread reaching edges of the coverslip; thus, the suspension was flattened and fixed with a glycerin jelly frame. The stained juveniles were studied and photographed using a Leica compound microscope. Following their examination and identification, a few specimens preserved in glycerin were selected for scanning electron microscopy (SEM) observations. The nematodes were hydrated in distilled water, dehydrated in a graded ethanol and acetone series, critical point-dried, mounted on stubs, coated with gold, and observed with a Zeiss Merlin microscope (Álvarez-Ortega and Peña-Santiago, 2016). The Adobe Photoshop CS2 software was used to adjust the contrast of photos, the ImageJ software for measurements (Collins, 2007), and MS Excel for calculations and statistical processing. Terminology of Fürst von Lieven and Sudhaus (2000) was used to describe the pharynx and stoma structures.

The beetle larvae, pupae, and imagoes were examined for presence and localization of nematode dauers using the stereomicroscope LOMO MBS-10. Surface deworming was used to check the adhesive strength between the nematode and vector. The insect larvae and adults were immersed for 45 min in a 5% solution of acetic acid and 0.02% chlorhexidine and then washed with water on a 200-µm sieve. Beetles and most nematode dauers remained alive and attached to insect mouth parts and cavities, while other nematode stages could not stay attached and were washed out. This test was aimed to identify places in the body of the vector where nematodes could remain viable under aggressive external influences.

DNA was extracted from several pooled nematodes using proteinase K. PCR and sequencing protocols were as described by Tanha Maafi et al. (2003). The forward Rhabditolaimus_D2A-1 – (5′-GGC GAA GCG GAT AGA GTT GAC-3′) and reverse D3B (5′-TCG GAA GGA ACC AGC TAC TA-3′) primers were used for amplification of the D2-D3 expansion segments of 28S rRNA gene. Sequencing was done at Genewiz., Inc (CA, USA). A sequence was deposited in GenBank under the accession number: MW044951.

The D2-D3 expansion segments of 28S rRNA gene sequence of R. ulmi was aligned with sequences of other Rhabditolaimus and related genera (Kanzaki and Giblin-Davis, 2014; Kiontke et al., 2007; Susoy and Herrmann, 2012, Susoy et al., 2015; and others). The alignment was analyzed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) using the GTR + I + G model. BI analysis was as described by Ryss et al. (2021).

Body almost straight, with weak annulation; width of 10 cuticle annuli at mid-body 7.5 µm. Lateral field starts at level of a telostom bulb continuing to the hyaline zone of a tail tip in females and to cloacal opening in males. The field marked by four incisures: its two prominent lateral ridges separated by a hollow where width equals the width of a ridge. On the ventral and dorsal side of the lateral field bordered by 3 to 5 lower ridges, crossed by transverse annulation, the maximum number of these ridges outside the lateral field is five at mid-body; decreasing to three ridges at body extremities. Outside the ridges the cuticle annuli are crossed by longitudinal lines; thus each annulus consists of rectangles where the long sides are parallel to the axis of the body. The prominent dorsal and ventral ridges of the cuticle beginning at the level of combined procorpus and first pharyngeal bulb; the dorsal ridge ends near the hyaline zone of tail tip in female and at the level of the cloaca opening in males; the ventral ridge ends in front of cloacal opening in males and before the anterior lip of anus in females. Cephalic region continuous, low, its sides angular in lateral view and hexagonal radially symmetrical in anterior view; devoid of annulation. With SEM, the cephalic area clearly separated by the first body annulus. Six triangular identical probolae point to the center of the six-lobed oral opening, forming a conical cupola. At the anterior margin of each probola is a tubular inner lip papilla with a central hole. Cephalic papillae absent in female but four tubular cephalic papillae are distinct in male in the middle of two subdorsal and two subventral probolae. Amphids oval, located behind the lateral probolae immediately behind the level of the posterior folds of the oral opening between cephalic lobes; the long amphid axis transversal, it equals the width of the base of probolae, its longitudinal axis is half as wide. Cephalic framework moderate, its basal plate at the probolae bases. Cheilostom cup-shaped, consisting of adradial cuticular plates, devoid of denticles; its front edge is connected to the six-looped oral opening of the body surface cuticle. Tubular base of cheilostom overlaps the anterior margin of triangular flexible gymnostom, encircled by muscular collar where length equals head width. Gymnostom 8-12 times longer than cheilostom, ending in stegostom in the anterior part of a first bulb. Stegostom is anisomorphic with distinct dorsal anterior bulge; the posterior part of stegostom encircled by a cuticular ring with two distinct lateral foramens. The first bulb muscular, its width 3 to 4 times of its length; it consists of two parts weakly separated by interruption of the triangular inner canal lining: its anterior part is a procorpus and its one-third posterior part is a homolog of metacorpus (median bulb of the Rhabditida); the metacorpus part of the muscular bulb includes a pair of unicellular glands with ducts opening into pharyngeal lumen. Isthmus is muscular gradually continuing to the pyriform second (posterior) bulb; canal within isthmus triangular thin walled. Cuticle ring encircled isthmus at the base of median bulb. Nerve ring in the middle of the isthmus, with ganglia anterior and posterior to it. Excretory pore at the posterior edge of the nerve ring. Deirid pore-like, its diameter is half of that of excretory pore, situated on the central hollow of lateral field between the field ridges, 3-5 annuli posterior to excretory pore. Hemizonid three annuli wide immediately anterior to excretory pore. Second bulb without grinder or valve; two pairs of unicellular glands are distinct in the basal part of bulb. Cardium distinct, at 1/3 bulb length, dividing pharynx and mid-intestine, its length twice its diameter; consisting of three rows of three cells. Mid-intestine cellular, wide, its wall consisting of one layer of large granular cells, its inner lumen 1/3 intestine diam. with distinct hyaline layer. Paired phasmids small pore-like with diameter equal to that of deirid, located one anal diameter posterior to anus, in the central hollow between the field ridges.

Body moderately curved to ventral side. Genital system at right side, 2/3 of mid-intestine length. Testis anteriorly reflexed to branch of 1 to 2 body diam. Spermatocytes in multiple rows in reflexed branch, posteriorly arranged in one row of large elongated cells; spermatids as 1 to 3 quartets in the middle zone of gonad; followed by the tubular vas deferens, filled with secretory granules and small spherical sperm; the gonad opening into cloaca sac ventral, at 20% of spicule length, while the mid-intestine opens via small valve into cloaca dorsally between heads of spicules; three large rectal glands at sides of valve. Spicules paired and equal, very long, slightly curved, needle-like, heading small, needle-lug-shaped. Gubernaculum Y-shaped. Bursa alae enveloping tail tip, starting anteriorly at the level of last third of spicules. Hyaline tail tip narrowly conical, surrounded by bursa. Cloacal opening is a transverse slit anteriorly arch-shaped. Unpaired papilla-like papilla P1 two cuticle rings anteriorly to cloaca opening. It is transversely elongated, twin, with two connected tubular pores.

It is reasonable to divide the rest of the male tail papillae into three groups according to their positions in structure rows (i) outer paired papillae on the fold of bursal alae (7 pairs of papillae: OP1…OP7); (ii) inner paired papillae on the ventral tail surface (two pairs, their numeration continues that of unpaired papilla P1, abdominal: P2...P3); a pair of paired pore-like papillae, presumably homologous to phasmids of the female, locating on the tail surface at the inner base of lateral wings at the level of upper edge of triad OP4, OP5, OP6. The papilla-like papillae located on folds of bursal alae form bursal ribs. A pair of OP1 one anal diameter anteriorly to the cloacal opening mark start points of the bursal alae. Paired OP2 at the level of half the distance from OP1 to the cloacal opening. Paired OP3 at the level of mid- tail. Pairs OP4, OP5 and OP6 form a group that are close to the posterior edge of the bursa. Paired OP7 at the dorsal side of the bursal alae at a distance of two cuticle annuli from the bursal terminus, immediately behind the grouping of OP4, OP5 and OP6. The paired OP7 devoid of ribs; they form the posterior horns of the shovel-shaped end of the bursa, without ribs. Inner (abdominal) tail papillae. P2 at OP2 level at the base of the bursal alae; P3 anteriorly to the OP3 locating from the latter at the distance equal to that of OP3-OP4, i.e. three cuticular rings.

Genital system paired, anterior branch at right side and posterior to left side of intestine. Ovaries reflexed (antidromous) with multiple rows of oocytes in both terminal endings. The mature oocyte enlarged at the loop of ovary flexure. Spermatheca filled with large cytoplasmic sperm, oval, its length twice of its diam. Spermatheca is the dorsally branching blind appendix of uterus that joins with ovary via narrow duct. Mature large oocyte passes the oviduct to uterus; and thus pushes the spermatheca; the latter injects sperm into uterus where the oocyte insemination occurs. Uterus paired, oval, anteriorly joined with oviduct ventrally and spermatheca dorsally; posteriorly the uterus connected with small oval uteral chamber bordered laterally with two pairs of dense thick-walled cells; the uteral chamber separated with two sphincters from both uteri: anteriorly from anterior uterus and posteriorly from posterior uterus. The chamber opens into cuticle vagina, perpendicular to ventral body surface; vagina opens into transversal vulva surrounded by valve, pyriform in lateral view. The valve surrounded with massive vulval muscles. The vulval hole is rounded, surrounded by dense cuticle folds that form a wrinkled oval-shaped vulval zone, the length of this zone is one and a half times more than its width. On the outside, the wrinkled zone is surrounded by a smooth round cuticle area, the width of the smooth zone equals to the width of the wrinkled zone. Outside of the smooth zone, the cuticle is annulated with a rectangular pattern typical to the rest of body surface. In front of the vulva at the edge of the smooth and annulated zones, two pore-like papillae are located at the level of front edge of the wrinkle zone. Their arrangement may be asymmetrical – anterior vulval papilla may be situated by the width of one cuticle ring at front of the other one.

Rectum 1.2 to 1.5 anal body diam., consisting of capillary posterior part and broad pyriform anterior part with wide lumen, bordered by three large rectal glands. Tail conical, 4 to 5 times its diam., gradually tapering to hyaline narrowly conical tail tip with hyaline zone of 1/6 of tail length. The anterior lip of female anus rounded, arch-shaped with marked cuticular border. Posterior anal lip is bulged, smooth in front and posteriorly annulated; annuli are crossed by longitudinal incisures breaking the cuticle rings into longitudinal elongated rectangles, just behind the posterior anal lip seven rows of rectangles form a V-shaped figure, posteriorly at a distance of one anal body width from anus the number of rows decreases to five and they are parallel.

Egg is large, its length 58 to 65 µm; 2.5 times of its diam. Eggs are laid by a female at different stages of cleavage: from single cell stage up to the second stage juvenile after the first molt inside eggshell; in latter case it is the ovoviviparity. Eggs are usually laid in chains of 2 to 5 units. Eggshell is smooth but after a juvenile breaks the shell its surface is marked with longitudinal slit.

Propagative females laid eggs at different stages: from a blastula to an egg containing a completely developed J1 or J2; the latter case is ovoviviparity. The J1 juvenile inside the eggshell had a distinct pharynx with a small medial bulb without a valve, a posterior bulb and cardia. The stoma is indistinguishable. The second stage juvenile J2 inside the eggshell had an external shed cuticle and a massive long bulbous pharynx corpus (first bulb) with a triangular cuticular lumen, which is similar to that structure in adult females of the propagative generation. The genital primordium of at least six cells is visible. At this stage, the J2 juvenile was hatching from the eggshell, breaking through a longitudinal slit by its head end. The J2 juvenile within egg rolled up in three curves. After leaving the egg the J2 straightened, its body length elongated.

Except for genital structures, the J2’s general morphology is similar to that of the adult female. Small elongated genital primordium in the middle of the mid-intestine part of the body. Primordium consists of eight cells: four small somatic cells in the central part, each of two primordium extremities with one large germinal cell and one small apical cell.

Except for genital structures, the J3 male’s general morphology is similar to that of the adult male. Small drop-like genital primordium in the middle of the mid-intestine part of the body. Four large germinal cells with an apical cell in posterior pole; in anterior part of primordium a small hook-like part with somatic nuclei reflexed posteriorly. This part in later stages will grow to cloaca primordium. Cloaca primordium present, encircling rectum, with its ventral side narrow and elongated anteriorly and dorsal side widely expanded.

Except for genital structures, the J3 female’s general morphology is similar to that of the adult female. Vulval primordium absent. Genital primordium is a small Z-shaped to horseshoe shaped structure with central somatic zone and two flexed branches, each of them with two large germinal cells and small apical cell, situated in the middle of mid-intestine section of the body.

Except for genital structures, the J4 male’s general morphology is similar to that of the adult male. Genital primordium horseshoe-like, in the middle of the mid-intestine part of the body, its length 5 to 7 times of its diam. Main part of primordium with 10 to 15 large germinal cells and with a small apical cell on posterior pole. Anteriorly primordium has a hook-like reflexed part with somatic nuclei which grows during this stage in the direction of cloaca primordium, finally joining with the latter. Cloaca primordium encircling the expanded rectum canal; its ventral side narrow and elongated anteriorly and dorsal side widely expanded. Primordium has very dense structure filled with numerous somatic nuclei.

General morphology is the same as in adult female with smaller sizes of organs. The only difference is the genital structures. Genital primordium 4 to 5 body diam. long; it consists of two equal reflexed branches with central part with vulval primordium attached to the ventral body wall in the middle of the mid-intestine section of the body. Anterior branch at right side and posterior branch at left side of the mid-intestine. Vulval primordium 1/5 body diam., massive, containing 30 to 40 somatic nuclei, with the central transverse lens-like invagination under cuticle. Genital primordium attached to dorsal surface of vulval primordium. Reflexed part of each branch of genital primordium consists of ca 10 large germinal cells and the apical cell, almost reaching the vulval primordium invagination. Non-reflexed part of genital primordium tubular, consisting of somatic cells.

The molting individuals (between stages JN and JN + 1) are characterized by the presence of shed cuticle and the genital primordia with structures which are intermediate between those typical for the non-molting specimens of JN and JN + 1. Two preadult molting phases are described below.

Body in shed molting cuticle, separated from body at extremities; it is remarkable that the shed elongated conical tail cuticle envelops the rounded male tail with bursa. All structures as in adult male, including caudal bursa and its papillae, except for the weak and very transparent spicules in cloaca canals.

Body in shed molting cuticle, separated from body at extremities. All structures as in adult female, except genital tube. Vulval-vaginal primordium is a transparent ventral wide invagination under cuticle which dorsal bottom is shaped as provisory uteral chamber. Two branches of genital primordium equal in size, fused with vulval-vaginal primordium attaching to the provisory uteral chamber. Each branch is divided into sections of future uterus, spermatheca, oviduct and the reflexed provisory ovary of 15 to 20 germinal cells reaching the vaginal primordium from dorsal side. Anterior genital primordium branch at right side and posterior at left side of mid-intestine. Tail conical as in female, anus and rectum well developed, three rectal glands visible.

All nematodes described above were initially collected from the bark and wood and then multiplied in agar cultures with the fungus Botryotinia fuckeliana. Additionally, transmissive dauer juveniles (DJ3) were collected from the undersides of the elytra of Scolytus multistriatus.

(Fig. 9 and Table 1).

Body straight. Annulation weak, four incisures in lateral field. Deirid indistinct. Cephalic region continuous, high, conical, probolae absent, lips amalgamated; with labilal disc. Cephalic framework weak hyaline-like. Stoma slit-like, thick-walled, short, 5 to 8 its widths. Pharynx narrow. Procorpus and metacorpus joined in elongated pyriform corpus, but with distinct border between long cylindrical procorpus and compact ellipsoid metacorpus, the latter with distinct inner triangular canal and devoid of cuticle valve. Isthmus narrow, with capillary canal, posterior bulb elongated ovoid; with two paired gland ducts opening into the canal. Cardium of ¼ bulb length, collar-like, separating pharynx from mid-intestine. Nerve ring at mid-isthmus; excretory pore at its posterior border; hemizonid 3 annuli wide just anterior to excretory pore. Genital primordium ventral, at ½ distance of mid-intestine; in female juveniles U-shaped with terminal germinal zones each with two large cells and central somatic part with ca 10 small nuclei; in male juveniles primordium drop-like with anterior hook-like somatic part and the germinal part of 4 large cells directed posteriorly. Rectum 1.5 times anal body diam., posterior part thickened, anus distinct. Tail conical, straight, 9 to 10 times anal body diam; terminal hyaline zone equal to rectum length. Phasmid small, at anterior third of tail. Male dauer juvenile differs from female dauer juvenile in presence of cloaca primordium around the thickened posterior half of rectum; its diam. equals to ¾ of anal body diam. According to 300 to 360 µm body size and genital primordium with 4 germinal cells the dauer juveniles correspond to the J3 juveniles of the propagative generation and therefore identified as the DJ3 stage of transmissive generation. After 3 to 6 hr in water or on PA media the DJ3 molted to DJ4 and then to adult nematodes with well-developed stoma and pharynx as described above for males and females.

Cephalic region and stoma hexagonal; female with six lip sensilla and two amphids, male with additional four cephalic sensilla. Didelphic females and males with peloderan bursa; papilla formula: three preanal pairs, one unpaired precloacal; seven postanal pairs (five subventral papilla-like, one subdorsal papilla-like; one ventral pore-like glandular). Corpus massive, 3 to 4 diam. long; stoma 0.8 times corpus length. Lateral field 2 ridges and hollow (4 incisures), additional 3 to 5 ridges from each side of lateral filed crossed by cuticle annuli. Commensals of bark beetles Scolytus spp.

Rhabditolaimus ulmi similar to R. zamithi, R. robiniae and R. walkeri in combination of didelphic female genital system and peloderan bursa. R. ulmi differs by having longer spicules: 34 to 53 µm vs 25 to 28 µm in R. zamithi, 30 to 35 µm in R. robiniae, and 24 to 29 (27) µm in R. walkeri. R. ulmi has the highest corpus ratio (length to diam.): 3 to 4 vs 3 or less in R. zamithi, R. robiniae, and R. walkeri. Value c′ 5.7 to 7.4 (6.6) is the highest value for females in R. ulmi vs 4.9 to 5.0 in R. robiniae and 3.4 to 4.9 (4.4) in R. walkeri. Additionally, the male caudal papillae of R. ulmi pattern (3 + 1 + 7) is unique in the presence of unpaired pre-cloacal papilla and the subventral pore-like papillae pair which are possible phasmids homologs. R. zamithi, R. robiniae and R. walkeri have the pattern of 3 preanal + 6 postanal papilla pairs.

Key to juvenile stages and adults of Rhabditolaimus ulmi:

- Copulatory structures functional, body length equal to or longer than 550 μm. ...............2

Park of St Petersburg State Forest Technical University (59.991923°N, 30.342697°E), St Petersburg, Russia.

Cultures on B. fuckeliana-PA medium were started from individuals isolated from the bark and soft wood (1 cm deep) obtained from a dying elm, Ulmus glabra (Ulmaceae) showing symptoms of Dutch elm disease, i.e., wilting, dark-colored ring in cross-section of wilted branches, trunk with galleries of larvae, pupae and imagoes of S. multistriatus (Curculionidae: Scolytinae). Additional cultures on B. fuckeliana-PA medium were started from dauers collected under elytra of imagoes of S. multistriatus.

Collection slides were deposited at the State Collection of the Zoological Institute RAS (20 slides) and the University of California, Davis Nematode Collection, CA, USA (10 slides).

Rhabditolaimus Fuchs, 1914.

(Generic synonymy according to Susoy and Herrmann, 2012).

Diagnosis (emended): Diplogastridae. Lateral field of two ridges separated by hollow (four incisures); outside the field the additional less prominent ridges crossed annules. Cephalic region and stoma hexagonal; female with six lip sensilla and two amphids, male with additional four cephalic sensilla. Cheilostom with inner cuticular plates, without denticles; its outer wall overlaps anterior end of gymnostom. Gymnostom flexible, very long, 10 times of its width and longer, ratio: gymnostom to corpus 0.4 to 1.3; a ratio stoma (cheilostom and gymnostom) to pharynx 0.2 to 0.4. Stegostom in the anterior part of corpus, asymmetric with anterior dorsal bulge and with posterior inclined subventral plates without denticles; it is encircled posteriorly with cuticular ring with two lateral foramens. Corpus strong cylindrical muscular bulb, 2 to 5 widths long, combining procorpus and metacorpus which borders are marked by interruption of the lumen triangular lining. Excretory-secretory pore at second bulb which devoid of grinder valve, or the pore shifted to base of pharynx. Hemizonid just anterior to excretory pore. Deirid a small pore at nerve ring or shifted to the base of pharynx. Female genital system didelphic with central vulva, or monoprodelphic with vulva shifted posteriorly (V = 55 or more). In females two small pore-like phasmids in the anterior part of the long conical tail. Male with well-developed peloderan or leptoderan narrow bursa, in type-species and bursa enveloping tail but with a mucronate 2 to 10 µm spike outside the bursal flap. Generally three preanal and six postanal caudal papilla pairs (sometimes 2 preanal and 5, 7 or 8 postanal pairs); the postanal papilla located at bursa alae and ventrally, a group of closely situated 3 papilla pairs (ribs) at the posterior margins of alae; one of postanal papilla is subdorsal. In some species unpaired pre-cloacal papilla detected and one of the paired postanal papilla may be pore-like: they are the ‘glandular papillae’ which may be homologs of phasmids. Male spicule narrow, widely varied in size from 17 to 600 µm. Gubernaculum present, with complicated species-specific shape. Saprophages and phoronts of bark-beetles with the specific dauer juveniles of transmissive generation. Some species associated with soil and decaying matter.

Here is used the list of species names accepted as valid in Susoy and Herrmann (2012) with their synonyms.

R. anoplophorae (Kanzaki and Futai, 2004) Susoy and Herrmann (2012)

R. carolinensis (Massey, 1967) Susoy and Herrmann (2012)

R. curzii (Goodey, 1935) Susoy and Herrmann (2012)

R. dendrophilus (Kinn, 1984) Susoy and Herrmann (2012)

R. erectus (Massey, 1960) Susoy and Herrmann (2012)

R. goodeyi (Rühm, 1959) Susoy and Herrmann (2012)

R. inevectus (Poinar, Jackson, Bell and Wahid, 2003) Susoy and Herrmann (2012)

R. kishtwarensis (Hussain, Tahseen, Khan and Jairajpuri, 2004) Susoy and Herrmann (2012)

R. macrolaimus (Schneider, 1866) Susoy and Herrmann (2012)

R. nacogdochensis (Massey, 1974) Susoy and Herrmann (2012)

R. neolongistoma (Hussain, Tahseen, Khan and Jairajpuri, 2004) Susoy and Herrmann (2012)

R. pellucidus (Cobb, 1920) Susoy and Herrmann (2012)

R. picei (Fuchs, 1931)

R. pini (Fuchs, 1931)

R. platypi (Kanzaki, Kobayashi, Nozaki and Futai, 2006) Susoy and Herrmann (2012)

R. rifflei (Massey and Hinds, 1970) Susoy and Herrmann (2012)

R. robiniae (Harman, Winter and Harman, 2000) Susoy and Herrmann (2012)

R. ulmi (Goodey, 1930) Susoy and Herrmann (2012)

= Cylindrogaster ulmi (Goodey, 1930)

= Rhabditolaimus schuurmansi (Fuchs, 1933)

= Goodeyus ulmi scolytus (Rühm, 1956)

= Myctolaimus ulmi (Goodey, 1930) Sudhaus and Fürst von Lieven (2003)

R. vitautasi (Korenchenko, 1975) Susoy and Herrmann (2012)

R. walkeri (Hunt, 1980) Susoy and Herrmann (2012)

R. zamithi (Lordello in Andrássy, 1984) Susoy and Herrmann (2012)

Tabular key to species of Rhabditolaimus is given in Table 4. The data were obtained from the original species descriptions and drawings. Missing values are calculated from the data in original taxonomic species descriptions and drawings, or in detailed re-descriptions.

Sequence of D2-D3 of 28S rRNA gene from R. ulmi was obtained in the present study. The D2-D3 of 28S rRNA gene alignment (786 bp) included 47 sequences of diplogastrids, including R. ulmi and two sequences of outgroup taxa. Phylogenetic analysis resulted in the Bayesian consensus tree given in Figure 10. Representatives of the genus Rhabditolaimus (= Myctolaimus) formed a highly supported clade (PP = 100%). Rhabditolaimus ulmi sequence from Russia (GenBank no. MW044951, CD3323a) was identical to that deposited by Kiontke et al. (2007).

Figure 10: