Two atypical new species of the genus Sectonema Thorne , 1930 ( Nematoda , Dorylaimida , Aporcelaimidae ) from Vietnam

Two new species of the genus Sectonema from natural habitats of northern Vietnam are studied. This paper includes their descriptions, measurements, line illustrations, and light microscope (LM) and scanning electron microscope (SEM) pictures. Sectonema tropicum sp. nov. is characterized by a 2.56– 3.24 mm long body, 19–21 μm broad lip region, odontostyle 20–21 μm long at its ventral side, 730– 834 μm long neck, pharyngeal expansion occupying 52–59% of total neck length, uterus a simple tube-like structure 150–242 μm long or 1.2–2.5 times the body diameter, pars refringens vaginae present, V = 48–52, short (31–40 μm, c = 70–91, c’ = 0.5–0.6) and rounded tail, 91–97 μm long spicules, and only one weakly developed ventromedian supplement. Sectonema vietnamense sp. nov. is characterized by its slender (a = 33–49) and 2.71–4.25 mm long body, 14–16 μm broad lip region, odontostyle 8–9 μm long at its ventral side, 716–918 μm long neck, pharyngeal expansion occupying 63–67% of total neck length, uterus simple and 209–242 μm long or 2.5–2.9 times the corresponding body diameter, pars refringens vaginae absent, V = 54, short (34–39 μm, c = 70–115, c’ = 0.6–0.8) and rounded tail, 59–75 μm long spicules, and three or four irregularly spaced ventromedian supplements bearing hiatus. Both species are also characterized by their nearly continuous lip region, an atypical feature in this genus. Molecular analysis of S. tropicum sp. nov. confi rms that Sectonema is a natural (monophyletic) taxon, very close to Metaporcelaimus.


Introduction
This contribution presents partial results of two nematological projects that overlap in their objectives.On the one hand, it is the second in a series of papers devoted to studying the nematode diversity of aporcelaims (family Aporcelaimidae Heyns, 1965) from natural areas of Vietnam, the first one (Álvarez-Ortega et al. 2015) dealing with the representatives of the genus Aporcelaimoides Heyns, 1965.On the other hand, it is part of a general revision of the taxonomy and systematics of this dorylaimid group, in which the genera Aporcelaimellus Heyns, 1965, Metaporcelaimus Lordello, 1965and Sectonema Thorne, 1930 have already received some attention (see for instance, and respectively, Álvarez-Ortega & Peña-Santiago 2013; Álvarez-Ortega et al. 2013a;Peña-Santiago & Álvarez-Ortega 2014a).
Interesting material belonging to the genus Sectonema was collected by the first author during several nematological surveys conducted in natural areas of Vietnam over the last years.The specimens are characterized by a nearly continuous lip region, indeed a rather atypical feature in Sectonema.Its detailed study revealed it to belong to two unknown forms, which are herein described.

Nematodes
Nematodes were collected from pristine areas in Northern Vietnam, extracted from soil samples using the methods by Baermann (1917) and Flegg (1967), relaxed and killed by heat, fixed in 4% formaldehyde, and processed to anhydrous glycerine following Siddiqi's (1964) technique.Finally, the specimens were mounted on permanent glass slides to allow handling and observation under LM.

Light microscopy
Specimens were measured using a light Olympus BH-2 microscope equipped with differential interference contrast (DIC).Morphometrics included de Man's indices and the usual measurements.The location of the pharyngeal gland nuclei is expressed according to Loof & Coomans (1970) and spicule terminology follows Peña-Santiago et al. (2014).Some of the best preserved specimens were photographed with a Nikon Eclipse 80i microscope and a Nikon DS digital camera.Raw photographs were edited using Adobe® Photoshop® CS version 8.0.1.Drawings were made using a camera lucida.

Scanning electron microscopy
After examination and identification, a few of the preserved specimens were selected for observation under SEM following the protocol by Abolafia & Peña-Santiago (2005).The nematodes were hydrated in distilled water, dehydrated in a graded ethanol and acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope.
The PCR products were purified using Exo-SAP PCR cleanup containing 7 µl of PCR product, 0.15 µl Exonuclease I (Exo), 0.9 µl Shrimps Phosphatase Alkali (SAP) and 1.95 µl of sterile water.They were incubated at 37°C for 20 min and then heated up to 85°C for 15 min; then the purified solution was diluted ten times.
The sequencing reaction was performed with 1 µl of DNA purified template, 0.25 µl BigDye v3.1, 2.25 µl 5x BigDye sequencing buffer, 0.25 µl of one primer.The mixture was heated up for 10 s at 96°C, 5s at 55°C repeated for 32 cycles followed by 4 min at 60°C.The sequencing was performed at the Cologne Center for Genomics (CCG).The sequences obtained were submitted to the GenBank database under accession numbers KT868957 and KT868958.

Phylogenetic analyses
The newly obtained sequences were aligned with other forty two D2-D3 expansion segments of 28S rRNA gene sequences available in GenBank using ClustalX 1.83 (Thompson et al. 1997).Outgroup taxa were chosen according to the results of previously published data (Holterman et al. 2008;Álvarez-Ortega et al. 2013b).Sequence alignments were manually edited using GenDoc 2.6.002(Nicholas et al. 1997).The sequence dataset was analysed with Bayesian inference (BI) and Maximum Likelihood (ML) using MrBayes 3.1.2(Huelsenbeck & Ronquist 2001;Ronquist & Huelsenbeck 2003) and MEGA 6 (Tamura et al. 2013), respectively.The best fit model of DNA evolution for BI was obtained using the program MrModeltest 2.2 (Nylander 2002) with the Akaike Information Criterion in conjunction with PAUP* 4b10 (Swofford 2003).BI analysis under the GTR + G + I model was initiated with a random starting tree and run with the four Metropolis-coupled Markov chain Monte Carlo (MCMC) for 10 6 generations.ML analysis was implemented under the best-fitting evolutionary model (GTR + I + G), obtained using the program MEGA 6, and 1000 bootstrap replications.The topologies were used to generate a 50% majority rule consensus tree.Posterior probabilities (PP) are given on appropriate clades.The trees were visualised with the program FigTree v1.4.0 and drawn with Adobe Acrobat XI Pro 11.0.1.

a
= body length / greatest body diameter b = body length / distance from anterior end to pharyngo-intestinal junction c = body length / tail length c' = tail length / tail diameter at anus or cloaca DN = distance from body anterior end to the nucleus of pharyngeal dorsal gland expressed as percentage (%) of total neck length DO = distance from body anterior end to the outlet of pharyngeal dorsal gland expressed as percentage (%) of total neck length L = overall body length n = number of specimens on which measurements are based S 1 N 1 = distance from body anterior end to the anterior nucleus of first pair of ventro-sublateral pharyngeal glands expressed as percentage (%) of total neck length S 1 N 2 = distance from body anterior end to the posterior nucleus of the first pair of ventro-sublateral pharyngeal glands expressed as percentage (%) of total neck length S 2 N = distance from body anterior end to the nuclei of the second pair of ventro-sublateral pharyngeal glands expressed as percentage (%) of total neck length V = distance from body anterior end to vulva expressed as percentage (%) of the body length.

Etymology
The specific epithet refers to the tropical area where the new species was collected.

Type material examined
Holotype VIETNAM: ♀, in acceptable state of preservation, Northern Vietnam, Cao Bang Province, Pia Oac Natural Reserve, 22º 36'28'' N, 105º 52'15'' E, tropical evergreen forest soil associated with Machilus sp. and Dimocarpus sp., deposited in the nematode collection of the University of Jaén, Spain.

Paratypes
VIETNAM: 4 ♀♀, 3 ♂♂, in acceptable state of preservation, same data as holotype; 1 ♀, 1 ♂, in acceptable state of preservation, same locality, deposited in the nematode collection of the Institute of Ecology and Biological Resources, Hanoi, Vietnam; 1 ♂, same locality, used for SEM.

Description Adult
Moderately slender to slender nematodes of medium to big size, 2.56-3.24mm long.Body cylindrical, distinctly tapering towards the anterior end, less so towards the posterior end as the caudal region is short and rounded.Habitus curved ventrad after fixation, especially in posterior body region, C-, G-or spiral-shaped.Cuticle 3.0-4.5 μm thick at anterior region, 5-6 μm in mid-body and 8-10 μm on tail; consisting of three layers, especially distinguishable at caudal region: thinner outer layer bearing very fine transverse striation across the entire body, thicker intermediate layer with radial striation, and thin inner layer.Lateral chords 13-21 μm wide at mid-body, occupying about one-sixth (13-18%) of midbody diameter.Two ventral and two dorsal body pores often present at level of odontostyle-odontophore. Lip region hardly offset from the adjacent body by weak, but perceptible depression, 2.8-3.0 times wider than high and up to one-fourth (17-25%) of body diameter at neck base; lips (under SEM) mostly amalgamated, but their perioral part distinctly separated by the existence of six radial, interlabial, deep incisures delimiting six perceptible liplets; button-like labial papillae, the inner ones located at the margin of the oral field and surrounded by two or three concentric annuli, whereas the outer papillae, located  a little behind the inner papillae, are surrounded by only one annulus; cephalic papillae pore-rather than button-like, also surrounded by only one ring-like annulus; oral aperture a dorso-ventral, slightly hexagonal orifice, the lip region hence showing a bi-radial symmetry.Amphid fovea cup-shaped, its opening occupying 9-10 μm or less than one-half (43-49%) of lip region diameter.Cheilostom nearly cylindrical, without any differentiation.Stomatal protruding structure apparently a reduced odontostyle 6.6-7.1 times longer than wide (see: Remarks), its ventral side 1.0-1.1 times longer than lip region diameter and 0.65-0.83% of body length.Guiding ring simple, plicate, at 0.8-0.9lip region diameters from the anterior end.Odontophore linear, rod-like, 1.7-1.9times the odontostyle length, somewhat irregular at its base and with (in lateral view) the ventral side slightly longer than the dorsal one (Fig. 2A).Anterior region of pharynx enlarging very gradually; basal expansion 7.1-10.7 times longer than wide, 3.6-5.6times as long as body diameter, and occupying 52-59% of total neck length; gland nuclei often obscure, located as follows: DN = 60-62 (n = 4); S 1 N 1 = 75 (n = 2); S 1 N 2 = obscure; S 2 N = obscure.Nerve ring located at 182-216 μm from anterior end or 25-26% of total neck length.Cardia rounded conoid, 13-17 × 13-17 μm; a weak ring-like structure is present surrounding its junction to pharyngeal base.

Male
Genital system diorchic, with opposite testes.In addition to the ad-cloacal pair, situated at 18-21 μm from cloacal aperture, one weakly developed ventromedian supplement, lying out the range of retracted spicules and located at 80-131 μm from the ad-cloacal pair.Spicules distinctly robust and massive, especially in its posterior half, 3.6-4.2times its maximum width, 1.4-1.6 times the body diameter at level of the cloacal aperture: dorsal contour regularly convex, ventral contour slightly concave, with distinct hump and hollow; curvature 121-124°; head occupying 11-16% of spicule total length, its dorsal side visibly curved at its anterior end and longer than the ventral one, which is shorter and almost straight; median piece 5.4-6.2 times longer than wide, occupying 51-59% of spicule maximum width, reaching the posterior tip; posterior end 7-8 μm wide.Lateral guiding pieces 23-29 μm long, 5.3-7.1 times longer than wide.Prerectum 3.2-4.6,cloaca 0.9-1.3times the corresponding body width long.Cloacal aperture, as seen under SEM, a curved anteriad, transverse slit.Tail similar to that of female; caudal pores two pairs, one lateral, another subdorsal.
Moreover, in having a lip region nearly continuous with the adjacent body, the new species resembles S. mucrodens Siddiqi, 1984 andS. truxum Siddiqi, 1984, but it can be easily distinguished from these by the more anterior position of the stomatal protruding structure (its anterior tip in front of the level of guiding ring, just behind the base of lip region vs its anterior tip distinctly behind the level of guiding ring, far from the lip region base) and pars refringens vaginae well (vs faintly) developed.Moreover, it differs from S. mucrodens by its shorter (730-834 vs 1086-1157 μm) neck, shorter (occupying 52-59 vs 69% of total neck length) pharyngeal expansion, shorter (32-39 μm, c' = 0.5-0.6 vs 44-50 μm, c' = 0.7) female tail, and male present (vs absent).It differs from S. truxum in its narrower (19-21 vs 24 μm broad) lip region, shorter (730-834 vs 837-1070 μm) neck, more anterior (V = 48-52 vs V = 54-56) vulva, and male present (vs absent).

Molecular characterisation
Two sequences of the D2-D3 28S rRNA gene were obtained from one female and one male.Both sequences are very similar (99%) and differ by two nucleotides only.The evolutionary relationships of the new species with representatives of the order Dorylaimida are presented in Figs 5-6.The two S. tropicum sp.nov.sequences are clustered together with other available sequences of Sectonema representatives and form a highly supported clade together with members of the genus Metaporcelaimus, thus confirming previous results (Álvarez-Ortega et al. 2013a, b).

Remarks
The nature of the stomatal protruding structure in the new species is difficult to interpret and deserves further explanation.The specimens examined bear a reduced odontostyle (comparable to that described in the type species S. ventrale Thone, 1930; see recent description by Peña-Santiago & Álvarez-Ortega 2014a) rather than a mural tooth as the base of its dorsal side is visibly sclerotized and seems to join the dorsal stomatal wall (vs dorsal side not scletorized at the base, somewhat sigmoid and not joining the dorsal stomatal wall).Nonetheless, some doubt persists in this matter.

Etymology
The specific epithet refers to the geographical origin of the species.

Type material examined
Holotype VIETNAM: ♀, in good state of preservation, Northern Vietnam, Cuc Phuong National Park, pristine tropical forest, deposited in the nematode collection of the University of Jaén, Spain.

Paratypes
VIETNAM: 4 ♂♂, 3 juvs, in good state of preservation, same data as holotype; 1 ♂, in good state of preservation, same locality, deposited in the nematode collection of the Institute of Ecology and Biological Resources, Hanoi, Vietnam; 1♂, 1 juv., same locality, used for SEM.

Other material examined
VIETNAM: 2 ♂♂, in acceptable state of preservation, Northern Vietnam, Thái Bình province, intensively cultured field, deposited in the nematode collection of the University of Jaén, Spain.

Adult
Very slender nematodes of big size, 3.13-4.25mm long.Body cylindrical, distinctly tapering towards the anterior end, less so towards the posterior one as the caudal region is rounded.Habitus regularly curved ventrad after fixation, to a more or less open C, occasionally more curved at posterior body region.Cuticle 2.0-2.5 μm thick at anterior region, 3.0-4.5 μm in mid-body and 8-10 μm on tail; consisting of three layers especially distinguishable at caudal region: thinner outer layer bearing very fine transverse striation through the entire body, thicker intermediate layer with radial striation, and thin inner layer.Lateral chords 6-12 μm wide at mid-body, occupying up to one-sixth (8-15%) of mid-body diameter.Two ventral and two dorsal body pores often present at level of stoma, and four distinct lateral pores 6-20 μm apart visible behind the amphid fovea.Lip region nearly continuous (a shallow depression is, however, often perceptible), weakly angular, visibly narrower than adjacent body, 2.1-2.3 times wider than high and about one-fifth (18-23%) of body diameter at neck base; it appears (under SEM) marked by radial (oral field) or longitudinal (remaining lip region) incisures, with amalgamated lips and protruding, pore-like labial and cephalic papillae, all of them surrounded by a ring-like annulus, and the inner labial papillae visibly larger than the outer ones; oral field perceptibly hexagonal, oral aperture a dorso-ventral slit, the lip region hence showing a bi-radial symmetry.Cupshaped amphid fovea, its opening at level of cephalic depression and occupying 10-11 μm or 68-72% of lip region diameter.Nearly cylindrical cheilostom, without differentiation.Odontostyle comparatively short and occupying the whole stomatal lumen, its ventral side 0.5-0.6 times longer than lip region diameter and 0.20-0.29% of body length.Guiding ring simple, somewhat plicate, at 0.5 times the lip region diameter from anterior end.Odontophore linear, rod-like, 3.7-4.2times the odontostyle length.Tripartite pharynx, consisting of an anteriorly thickened section behind the odontophore base, a perceptible narrower intermediate section with the nerve ring surrounding it, which enlarges very gradually into the basal expansion 9.9-15.0times longer than wide, 6.7-7.3 times longer than body diameter and occupying 63-67% of total neck length; gland nuclei mostly obscure, only S 2 N are visible, at 77-78% (n = 3) of total neck length.Nerve ring located at 147-178 μm from anterior end or 19-22% of total neck length.Cardia rounded conoid, 15-19 × 12-17 μm; a ring-like structure is present surrounding its junction to pharyngeal base.

Female
Genital system didelphic-amphidelphic, with almost equally and well developed branches, the anterior 473 μm long or 13% of body length and the posterior 387 μm long or 10% of body length, each branch bearing one uterine egg.Moderately sized ovaries, not surpassing the sphincter level, the anterior 191 μm, the posterior 141 μm long; oocytes arranged first in two or more rows, then in a single row.Oviduct 117, 142 μm long or 1.4, 1.7 times the corresponding body diameter, and consisting of a slender part made of prismatic cells and a well developed pars dilatata with wide lumen and sperm cells inside.Oviduct-uterus junction marked by a sphincter.Uterus a simple, tube-like structure 209, 242 μm long or 2.5, 2.9 times the corresponding body diameter, containing abundant sperm cells and one uterine egg.Vagina extending inwards 36 μm or about three-sevenths (44%) of body diameter: pars proximalis 23 × 28 μm, somewhat sigmoid walls and surrounded by weak musculature; pars refringens absent; pars

Fig. 5 .
Fig. 5. Bayesian 50% majority rule consensus trees as inferred from D2-D3 expansion segments of 28S rRNA gene sequence alignments under the GTR + I + G model.Posterior probabilities are given for appropriate clades.Newly obtained sequences are indicated by bold letters.

Fig. 6 .
Fig. 6.Maximum Likelihood tree as inferred from D2-D3 expansion segments of 28S rRNA gene sequence alignments under the GTR + I + G model.Bootstrap values are given for appropriate clades.Newly obtained sequences are indicated by bold letters.

Table 1 .
Morphometrics of Sectonema tropicum sp.nov.Measurements in μm (except L, in mm), and in the form: mean ± standard deviation (range).