Ashrafi, Samad, Mugniery, Didier, Heese, Evelyn YJ van, Aelst, Adriaan C. van, Helder, Johannes & Karssen, Gerrit, 2012, Description of Meloidoderita salina sp. n. (Nematoda, Sphaeronematidae) from a micro-tidal salt marsh at Mont-Saint-Michel Bay in France, ZooKeys 249, pp. 1-26: 3-14
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Females, males and second-stage juveniles: See Table 2. Embryonated eggs (n= 44): Length: 102.5 ± 5.0 (94.4-112) µm; diam.: 41.7 ± 1.9 (38.4-46.4) µm; length/width ratio: 2.5 ± 0.2 (2.1-2.9). Cystoids (n=18): Length: 224 ± 34.5 (176-336) µm; Width: 187.5 ± 33.1 (145.6-280) µm; length/width ratio: 1.2 ± 0.1 (1.0-1.7).
Female. Body swollen with a small posterior protuberance, pearly white to light brown, oval to pear-shaped. Neck region distinct, irregular shaped, usually twisted, 49 to 82 µm in length (Figs 2, 8). Body cuticle thick, without annulation. Head continuous with body, without annules. Cephalic framework weakly developed, lip region flattened. Stylet well developed, with posteriorly sloping oval-shaped knobs; stylet cone longer than shaft, slightly curved dorsally, shaft cylindrical (Fig. 2C). Dorsal gland orifice (DGO) close to basal knobs; vestibule extension visible. Secretory-excretory (S-E) pore well developed with clear cuticular lobes, located posterior to the neck, about 35 (20-56)% from anterior end of body; S-E duct markedly sclerotized, running posteriorly. Pharyngeal lumen from stylet to valve of metacorpus prominent. Metacorpus usually oval-shaped, situated at the posterior part of neck region, with distinct sclerotized valve apparatus, distance from middle of metacorpus to anterior end about 58 ± 10 µm long. Posterior gland bulb extending into anterior portion of swollen body cavity. Reproductive system extending towards pharyngeal region, monodelphic, spermatheca not observed; vulva with noticeable protruding lips, positioned usually at the posterior extremity of the body, rarely subterminal. Vulval lips forming thickened and muscular area around vulval slit (vulval area). Anus faint, opening pore-like, diffi cult to observe by LM, located at the base of dorsal vulval lip, apparently not functional (Figs 5E, 8C). Uterus swollen, prominent, bordered by a thick hyaline wall, becoming enlarged and filled with eggs, transforming into a cystoid within the female cuticle.
Male.Body slender, vermiform, tapering at both ends but more posteriorly, usually slightly curved ventrally at tail region. Cuticle marked by fine annulations, about 0.9 µm wide. Young males usually still enveloped in the last cuticle of second-stage juveniles (Fig. 4D). Lateral field beginning with 2 weak lines, roughly between head end and S-E pore level, and continuing with four weak lines behind S-E pore level. Head continuous with body, rounded-conoid, without annules and separated lips, distinct but weak cephalic framework present; amphidial apertures slit-like, angled, adjacent to oral opening surrounded by a small elevated oral disc (Fig. 7B). Pharyngeal region degenerated except for the posterior bulb, no stylet observed. S-E pore well developed, adjacent to hemizonid. S-E duct strongly sclerotized anteriorly (Fig. 4E). Deirids small, located just above S-E pore level (Fig. 7C). Monorchic, outstretched, testis well developed, with small vas deferens about6 µm long. Spicules paired, equal, not fused, arcuate, with rounded manubrium. Gubernaculum slightly curved. Cloacal tube about 2 µm long. Bursa-like structure visible by SEM (Fig. 7E). Phasmids small, posterior to cloacal opening. Tail conical, tapering to rounded terminus, marked with one or rarely two mucrones; if two are present, ventral mucro usually smaller; terminal mucro positioned ventrally, length 0.6‒3.2 µm (Fig. 1 K–N).
Second-stage juvenile. Body slender, vermiform, tapering at both ends but more so posteriorly, slightly ventrally curved at tail region; cuticle with fine annulations, annules about 1 µm wide. Lateral field with two visible outer lines in some specimens; in SEM, lateral field starts with three lines about 30 µm from head at neck region, four lines at 20%, and five lines at 33% of body length. Head continuous with body, rounded-conoid with slightly elevated concave oral disc, with distinct but relatively weak cephalic framework, without annules; two open slit-like amphidial apertures adjacent to slightly elevated concave oral disc surrounding the oral aperture, as visible by SEM (Fig. 6A). Lips not visible as distinct structures. Stylet well developed; cone tapering towards fine point; shaft straight; knobs rounded, prominent, sloping slightly posteriorly, set off from shaft (Fig. 1D). DGO close to stylet base. Metacorpus slightly elongated, with weak valves. S-E pore posterior and adjacent to hemizonid, located at isthmus level; hemizonid 2-3 annules long (Fig. 3D). Isthmus slender, distinct. Pharyngeal glands slightly overlapping intestine ventrolaterally. Deirids small, located just above S-E pore level. Genital primordium located posteriorly at 68‒77% of body length. Anus small, weakly developed, obscure by LM, pore-like (Fig. 6E). Phasmids small, difficult to observe by LM, located at about 19 µm from tail tip. Tail conical, slightly curved ventrally, tapering to finely pointed terminus, with finger-like projection. Hyaline tail part clearly delimitated anteriorly (Fig. 3 G–I).
Cystoid. Irregularly spherical to oval, filled with embryonated and non-embryonated eggs. Colour ranging from light in young cystoids to brown in older cystoid bodies. Body wall thickness 5.3 ± 1.2 (3.2-8.3) µm, containing bead-like outgrowths, displaying a specific sub-cuticular hexagonal beaded pattern (Figs 5, 8).
Egg mass.Females and cystoids usually completely surrounded by a gelatinous matrix (egg-mass) measuring about 316 ± 71.0 µm in length and 275 ± 54.0 µm in diameter (Fig. 5F).
Eggs. Oblong, translucent, egg shell without any visible markings, enveloped in a gelatinous matrix or within a cystoid.
Type host and locality.
Collected from rhizosphere and roots of the salt marsh halophytic shrub Atriplex portulacoides L. (= Halimione portulacoides (L.) Aell.), the most abundant species in ungrazed European salt marshes ( Bouchard et al. 1998), growing in cohesive muddy soil of the macro-tidal salt marshes of 'Le Vivier-sur-Mer’ at 48°36'32"N latitude and 1°47'00"W longitude at Mont-Saint-Michel bay, France.
Holotype female (slide WT 3591) and paratypes (second-stage juveniles, females, cystoids and males) (slides WT 3592-WT 3595) deposited in the Wageningen Nematode Collection (WaNeCo), Wageningen, The Netherlands. Additional second-stage juvenile, female, cystoid and male paratypes deposited at each of the following collections: Biology Department, Gent University, Gent, Belgium; Central Science Laboratory (CSL), Sand Hutton, York, UK.
The specific epithet refers to salty soil (saline environment) and is derived from the Latin word sal or salis meaning “salt”.
Diagnosis and relationships.
Meloidoderita salina sp. n. is characterized by sedentary mature females having a small swollen body with a clear posterior protuberance, stylet 19.9 (19-22) µm long, stylet cone slightly curved dorsally and longer than shaft, with posteriorly sloping knobs, neck region irregular in shape and twisted, well developed S-E pore, prominent uterus bordered by a thick hyaline wall and filled with eggs. Meloidoderita salina sp. n. is further distinguished by the cystoid having a unique sub-cuticular hexagonal beaded pattern.
Male without stylet, pharyngeal region degenerated, S-E duct prominent, spicules 18.4 (15.3-21.1) µm long, deirids just above S-E pore level, small phasmids posteriorly to cloaca opening and situated at 5.9 (3.2-7.7) µm from tail end, conical tail ending in a rounded terminus with one (rarely two) ventrally positioned mucro.
Second-stage juvenile body is 470 (419-496) µm long, with a 16.4 (14.7-17.3) µm long developed stylet, prominent rounded knobs set off from the shaft, hemizonid anterior and adjacent to S-E pore, tail 38.7 (33.9-44.2) µm long tapering to a finely pointed terminus with a finger-like projection.
On the basis of morphology, the female of Meloidoderita salina sp. n. resembles other species of the genus ( Meloidoderita kirjanovae , Meloidoderita safrica and Meloidoderita polygoni ) in the shape of the neck region (twisted, irregular and variable in size), the shape of the vulva (protruded), and the shape of the uterus (prominent, with large cells and a thick wall). Males of the four species are similar in lack of a stylet, degenerated pharyngeal region, the shape of the spicules (arcuate), the shape of the cloacal opening (ventrally protruded), and the shape of the tail (slightly curved ventrally, ending in a terminal mucro). Second-stage juveniles have a continuous head region, weakly sclerotized cephalic framework, similar shape of the tail (conically tapering to a pointed terminus, often with a finger-like terminal mucro), obscure anus, and position of hemizonid (anterior and adjacent to S-E pore).
Meloidoderita salina sp. n. differs from the previously described species by a smaller female body, a longer J2 body, the male with a longer body length and (except Meloidoderita kirjanovae described by Poghossian (1975)) by the present of a bursa-like structure, and by having a smaller cystoid body with a unique body cuticle surface pattern (displaying a hexagonal beaded pattern vs a spine-like structure in Meloidoderita kirjanovae , Meloidoderita polygoni and Meloidoderita safrica ). It also differs from them in known hosts and the saline habitat.
The new speciesdiffers in other characters from Meloidoderita kirjanovae by females having a longer stylet length and a much shorter distance from anus to vulval slit. Male differs from those characterized by Golden and Handoo (1984), and Vovlas et al. (2006) by having longer spicules length (15.4-21.1 vs 13.4-16.1, and 13-15 µm, respectively), and by a lateral field with 2-4 vs 3 incisures, and 4 incisures in Meloidoderita kirjanovae as redescribed by Kirjanova and Poghossian (1973). The second-stage juvenile of Meloidoderita salina sp. n. differs from Meloidoderita kirjanovae characterized by Golden and Handoo (1984), Siddiqi (1985) and Vovlas et al. (2006) in having a longer stylet (14.7-17.3 vs 12.9-14, 12-14, and 12-15 µm, respectively), lateral field (with 3-5 vs 3 incisures), a shorter hyaline tail with 6.4-9.6 µm long vs 8.1-13.3 µm long in those reported by Golden and Handoo (1984), 9-14 µm long in Siddiqi (1985), and 14-15 µm long in those of Meloidoderita kirjanovae re-described by Kirjanova and Poghossian (1973). Second-stage juveniles also differ from those reported by Golden and Handoo (1984) and Vovlas et al. (2006) by a shorter tail (33.9-44.1 vs 38-51, and 41-50 µm, respectively).
Meloidoderita salina sp. n. differs from Meloidoderita safrica by the female having DGO closer to base of stylet (2.5-4.0 vs 8.1-22.1µm), shorter distance from vulval slit to anus (13.4-23.0 vs 22.4-24.3 µm), by the male having a shorter testis (62-137 vs 190-319 µm), and by the J2 having a longer distance from anterior end to base of pharynx (111-144 vs 51.8-75.4 µm).
It differs from Meloidoderita polygoni females having a longer stylet (19.0-22.0 vs 15.0-17.4 µm), shorter distance from vulval slit to the anus (13.4-23.0 vs 32.0-86 µm), and a shorter vulval slit (16.0-22.5 vs 22.0-34.0 µm), and by the male without stylet vs visible anterior stylet part, a shorter tail (27.5-41.6 vs 32.0-56).
The new species is morphologically close related to the genus Sphaeronema , particularly to Sphaeronema alni Turkina & Chizhov, 1986. According to their observed phylogenetic relationships, they form together a highly supported clade. The absence of a cystoid stage in Sphaeronema is the most import differences compared to Meloidoderita .Additionally Meloidoderita salina sp. n. differs from Sphaeronema alni by females having a head region continuous with body vs head cap set off from neck and the lip region lacking annulations vs 2 annuli. The second-stage juveniles has a tail conically tapering to a pointed terminus, often with a finger-like projection, whereas in Sphaeronema alni the tail tapers gradually to a finely rounded terminus.
Molecular characterization and phylogenetic position of Meloidoderita salina sp. n.
The nearly complete rDNA sequence length of SSU rDNA obtained for Meloidoderita salina sp. n. (GenBank FJ969126 and FJ969127) both spanned1728 bp. A local alignment (1883 aligned position) included 39 nearly full length SSU rDNA sequences from related taxa and representatives of the genus Ecphyadophora were selected as outgroup. The SSU rDNA sequence analysis and the gene tree represented by the Bayesian and RAxML trees (Fig. 9) revealed a robust sister relationship between the new species and Sphaeronema alni within the Criconematina , and the two combined were positioned at the basal part of the local tree. The phylogenetic position of the suborder Criconematina has been analyzed several times ( Subbotin 2005, Vovlas et al. 2006, Holterman et al. 2009, van Megen et al. 2009, Palomares-Ruis et al. 2010). However, for conclusive statements on the positioning of this genus among the Criconematina , more rDNA sequence from representatives of the genus Meloidoderita are required. Further phylogenetic analyses using SSU rDNA and more taxon sampling are needed to infer intra-generic relationships and the position of Meloidoderita salina sp. n. within the Criconematina .
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