Morphological and molecular characterization of Epidorylaimus procerus sp. n. (Dorylaimida: Qudsianematidae) from Vietnam

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 (camera lucida). Morphometrics included Demanian ratios and other measurements. Microphotographs were taken with the same microscope provided with a Nikon Digital Sight DS-U1 camera. Raw photographs were edited using Adobe^® Photoshop^® CS.

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.

The new species is distinguished from other Epidorylaimus spp. by a combination of the following character states and morphometrics: body 2.16 to 2.46-mm long, lip region offset by constriction, width 15 to 17-µm broad, odontostyle 32 to 35-µm long with aperture occupying 20 to 25% of its length, neck 415 to 461-µm long, pharyngeal expansion 194 to 238-µm long (47-52% of the total neck length); the female genital system was described as follows: didelphic-amphidelphic, uterus simple and 76 to 130-µm long (1.0-1.5 body diameters), and vulva transverse (V = 40-43); the caudal region was conically elongated (157-186 µm, c = 12.1-14.4, c′ = 4.4-5.5) with blister-like bodies and hyaline portion occupying one-fourth its length.

Epidorylaimus procerus sp. n. resembles E. mellenbachensis (Alther, 1974) Andrássy, 1986 and E. rivalis Gagarin, 1991., and can be easily distinguished from other species of the genus (see key and table compendium by Ahmad et al., 2016) in having odontostyle longer than 25 µm. It differs from the German freshwater species E. mellenbachensis in its larger general size (body length 2.16-2.46 vs 1.70-2.00), less slender body (a = 29-33 vs 42-50), longer odontostyle (32-35 vs 28-29 µm; 1.9-2.1 vs 1.25 times lip-region diameter), much more anterior vulva (V = 39-42 vs V = 50-55), and relatively longer tail (c = 12-14 vs c = 19-38; c′ = 4.4-5.5 vs c′ = 3-4). From the Russian species E. rivalis, it differs in being longer (2.16-2.46 vs 1.46-2.29 mm, n = 15), the lip region is less differentiated (weak vs deep constriction), and it has a longer odontostyle (32-35 vs 28-30 µm). Males of E. rivalis are as abundant as females, whereas they are unknown in E. procerus n. sp. and thus likely to be rare or absent.

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 E. lugdunensis de Man, 1880, the type species of the genus, supporting monophyly of Epidorylaimus. Both species appear closely related (100% support) to Crassolabium circuliferum Loof, 1961, a member of the family Dorylaimidae, and form part of a highly supported larger clade that includes members of the families Nordiidae and Qudsianematidae.

Figure 3:

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 Dipterocarpus sp. and Cinnamomum sp. as dominant plant species.

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 procerus = slim or svelte, and refers to the slender figure of these nematodes.