Morphological and molecular characterization of Labronema montanum sp. n. (Dorylaimida, Dorylaimidae) from Spain

Nematodes of medium size are stout to moderately slender (a = 18–26), with length 1.56–2.08 mm. The body is cylindrical, tapering toward both ends, but more so toward the anterior end, as the tail is short and rounded. Upon fixation, habitus visibly curved ventrad, more regularly in females, to an open C shape, and more perceptibly at posterior body region in males, to a J shape. Cuticle is three layered, especially obvious at caudal region, 3.5 to 6.5 µm thick at anterior region, 5.0 to 8.5 µm in mid-body, and 10.5 to 13.5 µm on female tail and 9 to 12 µm on male tail; outer layer is thin, with a very fine transverse striation (only appreciable in SEM images, Fig. 4A,B) and there is a constant thickness throughout the body; intermediate layer is much thicker than the outer one, particularly at the caudal region; inner layer is much thinner than the intermediate layer, especially distinct at the caudal region. The lateral chord is 10 to 22 µm broad, occupying 12 to 26% of mid-body diameter. Several ventral and dorsal pores are present at the cervical region. The lip region is offset by a deep constriction, 2.8 to 3.7 times broader than high and less than one-third (23–31%) of body diameter at the neck base. SEM observations are as follows: lips are mostly amalgamated, the lateral ones are barely differentiated from their adjacent subdorsal and subventral lips; papillae are button like with a pore at their center; labial papillae are surrounded by a circular striation that does not exist around the cephalic papillae; oral field is relatively wide, as the inner labial papillae are situated far from the oral aperture, bearing coarse radial incisures reaching the outer margin of the lip region, perioral area is differentiated in distinct but weakly protruding liplets. Amphid with funnel-shaped fovea is present; its aperture is 8 to 11 µm, occupying two-fifths to one-half (41–50%) of the lip region diameter. Cheilostom is nearly cylindrical, with thick walls, but it is lacking any other differentiation. Odontostyle is typical of the genus, strong, 5.3 to 7.1 times as long as broad, somewhat longer (1.1–1.3 times) than the lip region diameter, and 1.15 to 1.86% of total body length; aperture is 7 to 11 µm long, occupying one-third to two-fifths (33–42%) of the odontostyle length. Odontophore is rod-like; its length is 1.4 to 1.8 times the odontostyle. Pharynx is conspicuously muscular, with its slender portion enlarging very gradually and basal expansion being 5.1 to 7.9 times as long as wide, 2.5 to 3.6 times longer than body diameter at neck base, and occupying ca one-half (47–53%) of the neck length; pharyngeal gland nuclei are located as follows: DO = 46 to 55, DN = 49 to 58, S₁N₁ = 61 to 68, S₁N₂ = 69 to 78, S₂N = 82 to 87, DN nucleolus 5 µm in diameter, S₁N₁ 2 µm and very close to its outlet, S₁N₂ 4 µm and at 3 µm from its outlet, and S₂N 4.5 µm and at 9 µm from its outlet. The nerve ring is situated at 142 to 185 µm or 30 to 38% of the total neck length from the anterior end. Pharyngo-intestinal junction is well developed; it consists of conoid to conical, 25–52 × 16–26 µm, cardia, enveloped by the intestinal wall and a distinct ring-like structure protruding in three lobes that in some specimens show gland-like bodies (see remarks). Intestine is present with no relevant differentiation. The tail is short, convex conoid to rounded, often showing some kind of indentations, mainly on its dorsal side, probably as result of fixation process affecting the very thick cuticle at this region.

Prerectum is 3.2 to 7.4 and cloaca is 1.5 to 1.9 times longer than the body diameter at cloacal aperture. Genital system is diorchic, with opposed testes, and spindle-shaped sperm cells. In addition to the ad-cloacal pair, located at 8 to 11 µm from the cloacal aperture, there is a series of 20 to 25 contiguous, 5.5 to 11 µm apart, ventromedian supplements, ending in front of the anterior end of spicules, at 59 to 86 µm from the adcloacal pair; therefore, a distinct hiatus exists. Spicules are 4.6 to 5.8 times as long as wide, 1.4 to 1.9 times longer than body diameter at the cloacal aperture: head is 1.8 to 3.2 times longer than wide, occupying 31 to 36% of the total spicule length, and with slender walls, the dorsal one is longer than ventral and it is slightly curved; median piece is narrow, occupying hardly one-third (19–36%) of the maximum width of spicule, and reaching the posterior end, which is 4.5 to 6 µm broad; curvature is 130 to 140°; ventral hump and hollow are poorly demarcated but appreciable; the former is situated at 36 to 58% of the spicule length from its anterior end. Lateral guiding piece is curved ventrad, 19 to 25 µm long, 5.4 to 6.3 times longer than broad, and with somewhat bifid terminus.

Three D2-D3 28S-rRNA sequences were obtained, one with 769, 772 and 783 pb long. These three fragments agree in 729 pb long, all of them were 100% identical. Their analysis has facilitated a study of the evolutionary relationships of the new species. The results are presented in Figure 5.

Figure 5:

The new species is characterized by its 1.56 to 2.08 mm long body, with lip region being offset by constriction and 19 to 24 µm broad, and with low perioral liplets, odontostyle 21 to 29 µm long at its ventral side, with aperture occupying 35 to 42% of its length, neck 417 to 551 µm long, pharyngeal expansion 205 to 272 µm long or occupying 47 to 53% of the total neck length, the presence of three cardiac lobes at the pharyngo-intestinal junction, didelphic–amphidelphic female genital system, a long and tripartite uterus, a longitudinal vulva (V = 57–60), short and rounded caudal region (19-35 µm, c = 56–86, c’ = 0.5–0.8), spicules 65–76 µm long, and 20–25 nearly contiguous ventromedian supplements with hiatus.

In its general morphology and morphometry, L. montanum sp. n. much resembles three Mediterranean species recently re-described by Peña-Santiago and Vinciguerra (2019), namely, L. angeloi Vinciguerra and Clausi, 1994, L. carusoi Vinciguerra & Orselli, 1998, and L. duhouxi (Altherr, 1963) Álvarez-Ortega & Peña-Santiago, 2013 (=L. pulchrum Vinciguerra and Zullini, 1980), from which it can be distinguished by small but significant differences. Thus, it differs from L. angeloi in its much thicker body cuticle (for instance, 10.5–13.5 vs 4–8.5 µm on female tail), broader lip region (19–23 vs 15–20 µm), longer neck (417–551 vs 386–402 µm), and pharyngeal expansion (205–272 vs 150–191 µm), the presence (vs absence) of three large lobes at pharyngo-intestinal junction, and a higher number of ventromedian supplements (20–25 vs 15–17). It differs from L. carusoi in its much thicker body cuticle (for instance, 10.5–13.5 vs 4.5–5.5 µm on female tail), longer neck (417–551 vs 342–382 µm), and pharyngeal expansion (205–272 vs 162–175 µm), the presence (vs absence) of three large lobes at pharyngo-intestinal junction, and longer spicules (65–76 vs 52–57 µm) with different morphology (large vs very small head). Also, it differs from L. duhouxi in its larger general size (body 1.56–2.08 vs 1.1–1.42 mm long, neck 417–551 vs 324–371 µm long, pharyngeal expansion 205–272 vs 142 µm), much longer spicules (65–76 vs 45–47 µm), and a higher number of ventromedian supplements (20–25 vs 15–16).

Evolutionary relationships, as derived from the analysis of D2-D3 28S-rRNA gene sequences, are presented in molecular tree of Figure 5. The new species forms part of a highly supported clade, predominantly constituted by representatives of the family Dorylaimidae, namely Crassolabium, Labronema, Mesodorylaimus, and Nevadanema sequences, and also by sequences of the genera Ecumenicus (Qudsianematidae) and Opisthodorylaimus (Thornenematidae). These results totally match recent previous findings (Varela-Benavides and Peña-Santiago, 2018), and confirm the present difficulties to elucidate the phylogeny of Dorylaimina. The internal relationships of the clade are not satisfactorily resolved, as the sequences corresponding to members of the subfamily Labronematinae (Crassolabium, Labronema, and Nevadanema) appear separated in two subclades, with both of them even including sequences of different species of the same genus, Labronema.

The new species was collected from a grassy area with stony soil at 1,800 elevation in Pandera mountain, Jaén province, southern Iberian Peninsula, Spain.

Female holotype, 14 female paratypes and 7 male paratypes were deposited in the nematode collection of the University of Jaén, Spain. Two female paratypes and two male paratypes were deposited in the USDANC, Beltsville, Maryland, USA.

The specific epithet is a Latin term that means belonging to the mountain, and it refers to type habitat where the new species was collected from.

As mentioned above, the new species is similar to the three originally described in Italy, and together, they probably form a quite homogeneous group within the genus Labronema (cf. Peña-Santiago and Vinciguerra, 2019). They have been mainly reported in Mediterranean countries (Albania, Italy, Spain), and also in other neighboring territories (Romania and Switzerland).

The existence of three large lobes at the pharyngo-intestinal junction is a remarkable feature of the new species, already mentioned (as cardiac glands), in L. pulchrum (cf. Peña-Santiago and Vinciguerra, 2019). The present observations suggest that these lobes/glands are not comparable to those recognizable in nygolaims (suborder Nygolaimina). Actually, a more or less developed ring-like structure is easily perceptible in many Dorylaimina species, including Labronema representatives (for instance, L. ferox Thorne, 1930; see Peña-Santiago, 2019), which, in particular taxa, shows additional differentiation resulting in a large dorsal lobe, as observed in members of the genus Aporcelinus Andrássy, 2009, or three large lobes that apparently display a tri-radial symmetry.