The genus
A nematological survey conducted in November 2017 to explore the dorylaimid diversity in a mountain area of the southern Iberian Peninsula yielded a
Nematodes were collected from a grassy area with stony soil at 1,800 elevation in Pandera mountain, Jaén province, southern Iberian Peninsula, Spain. They were extracted using Flegg’s (1967) sieving method and a modified Baermann’s (1917) funnel technique; they were killed with heat, fixed into 4% formalin, transferred to pure glycerine following Siddiqi’s (1964) method, and mounted on permanent glass slides.
Observations, measurements, line illustrations, and LM photomicrographs were made using a Nikon Eclipse 80i (Nikon, Tokio, Japan) microscope with differential interference contrast (DIC) optics, a drawing tube (
Specimens preserved in glycerine were selected for observation under SEM according to Abolafia (2015). They were hydrated in distilled water, dehydrated in a graded ethanol–acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss®, Oberkochen, Germany).
Nematode DNA was extracted from single fresh individuals using the proteinase K protocol and PCR assays, as described in the study of Castillo et al. (2003). The specimen was cut in small pieces using a sterilized needle on a clean slide, with 18 ml of AE buffer (10 mM Tris-Cl + 0.5 mM EDTA; pH 9.0), it was transferred to a microtube and 2 μl proteinase K (700 μg/ml−1) (Roche®, Basel, Switzerland) was added, and it was stored at −80°C for 15 min (for several days). The microtubes were incubated at 65°C for 1 hr, followed by 95°C for 15 min. The microtube was centrifuged at 13,000 r.p.m. or 15,900× g for 3 min, and 2 μl of the supernatant extracted DNA was transferred to a microtube containing 2.5 μl 10× PCR reaction buffer 5 μl Q-solution 5×, 0.5 μl dNTPs mixture (10 mM each), 1 μl of each primer (10 mM), 0.2 μl Taq DNA Polymerase (Qiagen®, Venlo, the Netherlands), and ddH2O with a final volume of 25 μl. The primers used for amplification of the D2-D3 region of 28S rRNA gene were the D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and the D3B (5′-TCGGAAGGAACCAGCTACTA-3′) primers (De Ley et al., 1999). PCR cycle conditions were as follows: one cycle of 94°C for 3 min, followed by 35 cycles of 94°C for 1 min + annealing temperature of 55°C for 45 s + 72°C for 2 min, and finally one cycle of 72°C for 10 min. After DNA amplification, 5 μl of product was loaded on a 1% agarose gel in 0.5% Tris-acetate-EDTA (40 mM Tris, 20 mM glacial acetic acid and 2 mM EDTA; pH = 8) to verify the amplification using an electrophoresis system (Labnet Gel XL Ultra V-2, Progen Scientific®, London, UK). The bands were stained with RedSafe (20,000×), which was previously added to the agarose gel solution. The sequencing reactions were performed at Sistemas Genómicos (Valencia, Spain). The sequences obtained (with 769, 772 and 783 pb) were submitted to the GenBank database under accession numbers MK894244, MK894245 and MK894246.
For phylogenetic relationships, analyses were based on 28S rDNA. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with another D2–D3 expansion segments of 28S rRNA gene sequences available in GenBank, using MUSCLE alignment tool implemented in the MEGA7 (Kumar et al., 2016). The ambiguously aligned parts and divergent regions were known using the online version of Gblocks 0.91b (Castresana, 2000) (
In total, 17 females and 11 males, from one location, were examined.
See Table 1.
Morphometrics of
Holotype | Paratypes | Paratypes | |
---|---|---|---|
Character | ♀ | 17♀♀ | 11♂♂ |
L | 1.74 | 1.83 ± 0.14 (1.56–2.07) | 1.87 ± 0.12 (1.68–2.08) |
a | 17.8 | 21.3 ± 2.0 (17.8–24.4) | 22.7 ± 1.8 (20.3–25.9) |
b | 3.7 | 3.8 ± 0.3 (3.3–4.3) | 3.7 ± 0.2 (3.3–4.1) |
c | 62 | 68.1 ± 9.4 (56–86) | 72.2 ± 5.5 (65–84) |
V | 58 | 58.4 ± 1.2 (56.5–60.2) | – |
c’ | 0.6 | 0.6 ± 0.1 (0.5–0.8) | 0.6 ± 0.0 (0.6–0.7) |
Lip region diameter | 23 | 22.0 ± 1.1 (19–23) | 22.2 ± 1.3 (19–24) |
Odontostyle length ventral side | 27 | 25.0 ± 1.9 (21–28) | 26.0 ± 2.0 (22–29) |
Odontostyle length dorsal side | 28 | 26.5 ± 2.1 (23–30) | 27.3 ± 2.0 (23–30) |
Odontophore length | 43 | 42.0 ± 1.9 (37–44) | 42.7 ± 2.2(38–45) |
Neck length | 474 | 476 ± 27 (417–514) | 500 ± 31 (450–551) |
Pharyngeal expansion length | 238 | 242 ± 19 (205–272) | 247 ± 16 (212–264) |
Body diam. at neck base | 92 | 80.6 ± 8.3 (65–98) | 78.9 ± 6.1 (72–91) |
mid-body | 98 | 85.9 ± 7.6 (72–99) | 82.8 ± 8.9 (71–101) |
anus/cloaca | 44 | 43.9 ± 3.3 (39–51) | 42.3 ± 3.7 (36–47) |
Distance vulva – anterior end | 1015 | 1068 ± 88 (889–1232) | – |
Prerectum length | 93 | 125 ± 23 (84–160) | 202 ± 34 (150–268) |
Rectum/cloaca length | 61 | 60.0 ± 4.1 (52–68) | 71.6 ± 5.6 (61–80) |
Tail length | 28 | 27.4 ± 4.5 (19–35) | 26.1 ± 2.9 (20–30) |
Spicules length | – | – | 71.3 ± 3.6 (65–76) |
Ventromedian supplements | – | – | (20–25) |
Nematodes of medium size are stout to moderately slender (
Genital system is amphidelphic, with both branches well and equally developed; the anterior is 207 to 399 µm or occupying 13 to 22% of the total body length; the posterior is 226 to 380 µm or 13 to 23% of the total body length. Ovaries are reflexed, variably sized; the anterior is 57 to 183 µm long, whereas the posterior is 72 to 158 µm long, usually reaching the oviduct–uterus junction, with oocytes first in two or more rows and then in a single row. Oviduct is 78 to 180 µm long or 1.0–2.3 times longer than the body diameter, consisting of a distal, slender section made of prismatic cells and a moderately developed proximal
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.
The Bayesian tree inferred from known and the newly sequenced
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 (
In its general morphology and morphometry,
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
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
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