The genus
In spite of the nearly cosmopolitan distribution of the genus, none of its species was hitherto recorded in Vietnam. However, a nematological survey conducted to study nematode fauna associated with natural habitats in the country located an interesting
Soil samples were collected in Cao Bang Province, a natural area of Northern Vietnam, and temporarily stored in plastic bags for transport to the laboratory. Nematode extraction was done following the methods of Baermann (1917) and Flegg (1967). Specimens were relaxed and killed with heat, fixed in 4% formaldehyde, processed to anhydrous glycerin according to Siddiqi’s (1964) technique, and mounted on permanent glass slides for handling and observation with light microscopy.
Specimens were measured, drawn, and identified with a Nikon Eclipse 80i light microscope equipped with differential interference contrast (DIC) optics and a drawing tube (
DNA was extracted from single individuals using the proteinase K and Worm Lysis Buffer protocol (William et al., 1992). Each nematode was transferred to a 0.5-ml Eppendorf tube containing 18 µl of Worm Lysis Buffer (WLB) (50 mM KCL, 10 mM Tris, pH 8.3, 2.5 mM MgCl2, 0.45% NP 40, and 0.45% Tween 20) and 2 µl of proteinase K (600 µg ml−1) (Thermo Scientific). The tubes were incubated at 65°C (1 hr) and then at 95°C (15 min). PCR was performed in a 30-µl final volume containing 24.9 µl of sterile water, 0.6 µl of each PCR primer, 0.6 µl of dNTP, 0.3 µl of Taq-polymerase, 3 µl of Buffer 10 x Thermo Scientific Green, and 1 µl of DNA-extracted solution. The PCR amplification profile consisted of 4 min at 94°C, 35 cycles of 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C, followed by a final step of 10 min at 72°C. The primers used for amplification were D2A (5´-ACAAGTACCGTGAGGGAAAGTTA-3´) and D3B (5´-TCCTCGGAAGGAACCAGCTACTA-3´) for amplification of the D2-D3 region of 28S (Subbotin et al., 2006).
PCR products were purified with the GeneJET PCR Purification Kit (#K0701, Thermo Scientific, USA), following the manufacturer’s manual. The sequencing reaction was performed with 15 ng of purified template, 4 µL of BigDye Terminator v3.1 Ready Reaction Mix, 2 µL of 5X Sequencing Buffer, and 3.2 pmol of forward/reverse primers for a total of 10-µL volume. The mixture was heated for 10 sec at 96°C, then 5 sec at 55°C, repeated for 32 cycles followed by 4 min at 60°C. The sequencing was performed on 3500 x L Genetic Analyzers (Applied Biosystems, Foster City, California) at the National Key Laboratory of Gene Technology (IBT – VAST, Hanoi). The sequences obtained were submitted to the GenBank database under accession numbers MT612084 and MT612088.
The obtained sequences were aligned with 35 other D2-D3 expansion segments of 28S rDNA gene sequences available in GenBank, using SeaView with the muscle algorithm followed by manual refining (V4.5.3, Gouy et al., 2010). Outgroup taxa were chosen according to previously published data (Holterman et al., 2008; Álvarez-Ortega et al., 2013). The sequence dataset was analyzed with Bayesian inference (BI) and maximum likelihood (ML) using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). We chose the commonly used model GTR + I + G that then was used to calculate phylogenetic trees in PhyML 3.1 with 100 replicates and MrBayes with a burn-in of 25% and the final split frequencies of less than 0.01 (settings: mcmc ngen = 1,000,000; sample freq = 500; print freq = 500; diagn freq = 5,000; Ronquist and Huelsenbeck, 2003; Altekar et al., 2004). Both trees where then combined into one reconstructed phylogeny in Adobe Illustrator© (Creative Suite 6).
In total, there were 15 females from one location in excellent state of preservation.
Morphometricssee Table 1.
Morphometrics of
Holotype | Paratypes | |
---|---|---|
♀ | 15♀♀ | |
Character | ||
|
2.37 | 2.28 ± 0.09 (2.16-2.46) |
|
30.8 | 31.4 ± 1.2 (28.8-32.9) |
|
5.5 | 5.2 ± 0.2 (4.9-5.6) |
|
13.0 | 13.3 ± 0.7 (12.1-14.4) |
|
41.5 | 41.2 ± 0.8 (39.8-42.6) |
|
5.4 | 4.9 ± 0.4 (4.4-5.5) |
Lip-region diameter | 17 | 15.9 ± 0.6 (15-17) |
Odontostyle length | 32 | 33.4 ± 1.2 (32-35) |
Odontophore length | 32 | 30.2 ± 1.5 (28-33) |
Neck length | 432 | 435 ± 18 (415-461) |
Pharyngeal expansion length | 204 | 212 ± 13 (194-238) |
Body diam. at neck base | 61 | 65.0 ± 4.0 (61-73) |
Mid-body | 77 | 72.6 ± 3.4 (68-79) |
Anus/cloaca | 36 | 35.2 ± 1.3 (32-36) |
Distance vulva – anterior end | 982 | 938 ± 43 (885-1012) |
Prerectum length | 91 | 91.8 ± 18.4 (70-123) |
Rectum length | 42 | 42.5 ± 4.6 (35-49) |
Tail length | 182 | 171 ± 9.2 (157-186) |
There were moderately slender (
The lip region was truncate, offset by a distinct constriction, 1.9 to 2.3 times broader than high and one-fifth to one-fourth (21-26%) of body diameter at the neck base; the lips were amalgamated, with visibly protruding labial and cephalic papillae. Amphid fovea appeared to be cup-like, its aperture measured 4 to 5.5 µm, occupying up to one-third (28-33%) of lip-region diameter. Cheilostom was a truncate cone, without any particular differentiation. Odontostyle was relatively long and slender, 10.7 to 11.5 times longer than wide, length about twice (1.9-2.1 times) the diameter of the lip-region diameter, and about 1.38 to 1.51% of the total body length; the aperture measured 6.5 to 8.0-µm long, occupying up to one-fourth (20-25%) of odontostyle length. Odontophore was rod-like, nearly equal (0.8-1.0 times) in length to odontostyle. The pharynx was entirely and conspicuously muscular, with its slender portion enlarging gradually, and the basal expansion being 5.4 to 6.5 times as long as wide, 3.0 to 3.6 times longer than body diameter at the neck base, and occupying
Unknown.
The new species is distinguished from other
Evolutionary relationships, as derived from the analysis of D2-D3 28S-rRNA gene sequences, are presented in a molecular tree (Fig. 3). The new species forms part of a highly supported (99.9%) clade with
Vietnam, Cao Bang Province, Cao Bang Natural Reserve (GPS coordinates: 22° 34′07″ N, 105° 52′34″ E), in a tropical evergreen forest soil with
Female holotype and nine female paratypes were deposited with nematode collection of the University of Jaén, Spain. Five female paratypes were deposited with the nematode collection of the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.
The specific epithet is the Latin term