Three new species of the genus Aporcelaimoides Heyns, 1965 from Vietnam (Nematoda, Dorylaimida, Aporcelaimidae), with an updated taxonomy of the genus

Abstract Three new species of Aporcelaimoides from natural habitats in Vietnam are studied, described and illustrated, including line drawings, LM and/or SEM pictures. Aporcelaimoides brevistylum sp. n. is characterized by its body 1.95–2.90 mm long, lip region offset by deep constriction and 17–18 µm broad, ventral side of mural odontostyle 11–14 µm long with aperture occupying 62–71% of its length, neck 663–767 µm long, pharyngeal expansion occupying 58–66% of total neck length, uterus a simple tube 85–182 µm long, pars refringens vaginae absent, V = 55–63, tail short and rounded (34–46 µm, c = 49–76, c’ = 0.6–0.8), spicules 67–86 µm long, and one ventromedian supplement out the range of spicules. Aporcelaimoides minor sp. n. is distinguished in having body 2.09–2.61 mm long, lip region offset by deep constriction and 19–20 µm broad, mural odontostyle 14–16 µm long at its ventral side with aperture occupying 73–84% of its length, neck 579–649 µm long, pharyngeal expansion occupying 57–66% of total neck length, uterus a simple tube 44–69 µm long, pars refringens vaginae well developed, V = 48–56, female tail very short, rounded conoid or truncate (14–26 µm, c = 90–146, c’ = 0.3–0.6), and male unknown. Aporcelaimoides silvaticum sp. n. is characterized by its body 2.09–2.60 mm long, lip region offset by depression and 17–18 µm broad, mural odontostyle 11–12 µm long at its ventral side with aperture occupying 60–66% of its length, neck 597–720 µm long, pharyngeal expansion occupying 58–64% of total neck length, uterus a simple tube 128–243 µm long, pars refringens vaginae well developed, V = 58–60, tail short and rounded (27–37 µm, c = 67–94, c’ = 0.6–0.7), spicules 64–75 µm long, and two or three widely spaced ventromedian supplements bearing hiatus. The genus Aporcelaimoides is restored, its diagnosis emended, and three species of Sectonema, namely Sectonema amazonicum, Sectonema haguei and Sectonema moderatum, transferred to it. An updated list of its species, a key to their identification and a tabular compendium with the most important morphometric features are also presented.


Introduction
The genus Aporcelaimoides is an interesting aporcelaimoid taxon, created by Heyns (1965) to accommodate two new species, namely A. probulbum (type species) and A. californicum. It was originally characterized among other features by its "Spear dorylaimoid, with a large dorsal aperture, but the basal part of the spear much narrower than the lumen of the pharynx, and situated ventrally in the pharynx". Later, Siddiqi (1995) regarded it as a junior synonym of Sectonema Thorne, 1930, an action that, several years later, was followed by Andrássy (2009). However, this taxonomical decision deserves further analyses since important morphological differences exist between both genera, being especially important those affecting the nature of the stomatal protrusible structure.
The study of dorylaimid fauna from Vietnam has received poor attention. Several authors (Vu et al. 2010;Nguyen et al. 2011;Gusakov 2012 and2013a,b;Nguyen et al. 2014) discovered some new species and reported for the first time information about other known species. It is suggested that Vietnamese dorylaimid fauna might be highly diverse. This is the first contribution in a series devoted to study the aporcelaimid fauna of this Asian country.
During a general nematological survey conducted during the last five years to study the diversity of the Vietnamese nematode fauna, several specimens of the genus Aporcelaimoides were collected from natural areas in Vietnam. Their detailed examination revealed they belonged to three unknown forms, which are herein described. Besides, the study of this nematode material has confirmed relevant data to reconsider the identity of the genus Aporcelaimoides.

Nematodes
Nematodes were collected from several natural areas in Vietnam, extracted from soil samples using the methods of Baermann (1917) and Flegg (1967) somewhat modified, 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
Nematodes were measured using a light microscope. Morphometrics included de Man's indices and most of the usual measurements. The location of the pharyngeal gland nuclei is expressed according to Loof and 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. Drawings were made using a camera lucida.

Scanning electron microscopy
After their examination and identification, a few specimens preserved in glycerin were recycled to their observation under SEM following the protocol by Abolafia and 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.
Measurements. See Table 1. Description. Adult. Moderately slender to slender nematodes of medium size, 1.95-2.90 mm long. Body cylindrical, distinctly tapering towards the anterior end, less so towards the posterior one because the caudal region is rounded. Habitus regularly (often strongly) curved ventrad after fixation, usually spiral-shaped. Cuticle threelayered, especially distinguishable at caudal region, where it consists of thinner outer layer bearing very fine transverse striation through the entire body, thicker intermediate layer with radial striation and thin inner layer; thickness 3-5 µm at anterior region, 4-7 µm in mid-body and 9.0-12.5 µm on tail. Lateral chord 8-20 µm wide at mid-body, occupying one-eighth to less than one-fifth (12-18%) of mid-body diameter. Three ventral and three dorsal body pores are usually present at level of mural odontostyle-odontophore, their corresponding ducts appearing especially thickened beneath intermediate cuticle layer. Lip region offset by deep constriction, 2.7-3.3 times as wide as high and one-fifth to two-sevenths (18-30%) of body diameter at neck base; lips (under SEM) amalgamated; labial papillae button-like, very perceptible and protruding under LM, surrounded by a ring-like annulus (occasionally two annuli), the inner ones at the margin of oral field; cephalic papillae larger than the labial ones, with an oval transverse slit; oral aperture a dorso-ventral, slightly hexagonal orifice, the lip region hence showing a biradial symmetry. Amphid fovea cup-shaped, its opening occupying 9-11 µm or one-half to two-thirds (52-64%) of lip region diameter. Cheilostom nearly cylindrical, lacking any differentiation. Mural odontostyle attached subventrally and comparatively short, 4.1-5.4 times as long as wide, 0.6-0.8 times as long as lip region diameter, and 0.43-0.61% of body length; aperture 8-9 µm long or up to five-sevenths (62-71%) its length. Guiding ring simple, somewhat plicate, at 0.6-0.8 lip region diameters from anterior end. Odontophore linear, rodlike, 3.4-4.2 times the mural odontostyle length. Anterior region of pharynx enlarging very gradually; basal expansion 9.5-12.6 times as long as wide, 4.6-7.2 times as long as body diameter, and occupying 58-66% of total neck length; gland nuclei obscure in most specimens examined, DN = 50 (n=1) and S 2 N = 84 (n=1). Nerve ring located at 154-185 µm from anterior end or 21-26% of total neck length. Cardia rounded conoid, 10-14 × 14-18 µm; a ring-like structure is present surrounding its junction to pharyngeal base. Tail short and rounded; inner core with irregular shape at tail end. Caudal pores two pairs, one lateral, another sub-lateral.
Male. Genital system diorchic, with opposite testes. In addition to the ad-cloacal pair, situated at 15-20 µm from cloacal aperture, there is only one ventromedian supplement located out the range of spicules, at 48, 58 (n=2) µm from ad-cloacal pair. Spicules distinctly robust and massive, especially in its posterior half, 3.4-4.6 times its maximum width, 1.2-1.7 times the body diameter at level of the cloacal aperture: dorsal contour regularly convex, ventral contour very weakly concave, with shallow or weak hump and hollow; curvature 126-142°; head occupying 7-21% of spicule total length, its dorsal contour conspicuously curved at its anterior end and longer than the ventral one, which is short and straight; median piece 7.2-10.9 times as long as wide, occupying 35-50% of spicule maximum width, reaching the posterior tip; posterior end 5-9 µm wide. Lateral guiding pieces 13-17 µm long, 3.5-5.1 times as long as wide. Prerectum 2.9-4.4, cloaca 1.1-1.3 the corresponding body widths long.
Other locality and habitat. Vietnam, Lam Dong Province, Bidoup-Nui Ba National Park, from soil of a pristine forest, collected in June 2013.
Type material. Female holotype and seven female and nine male paratypes deposited in the nematode collection of the University of Jaén, Spain. One female and one male paratypes deposited in the nematode collection of the Institute of Ecology and Biological Resources, Vietnam.
Etymology. The specific epithet is a compound Latin term referring to the short mural odontostyle that characterizes this species.
Remarks. The two populations examined are very similar in their morphological features and morphometrics, but some minor differences have been also noted, which are herein regarded as intraspecific variation. Thus, the population from Dak Lak province shows a shorter mural odontostyle (ventral side 11-13 vs 13-14 µm, dorsal side 13-14 vs 14-17 µm, in females) and comparatively longer neck (b = 3.3-3.4 vs b = 3.5-3.7).
Measurements. See Table 2. Description. Female. Moderately slender to slender nematodes of medium size, 2.09-2.61 mm long. Body cylindrical, distinctly tapering towards the anterior end, less so towards the posterior one as the caudal region is very short and rounded to truncate. Habitus regularly curved ventrad after fixation, often spiral-shaped. Cuticle three-layered, especially distinguishable at caudal region: thin outer layer bearing fine transverse striation through the entire body, a much thicker intermediate layer with radial striation, and a thin inner layer; thickness 3.0-4.5 µm at anterior region, 4.5-6.5 µm in mid-body and 6.5-9.5 µm on tail. Lateral chord 7-13 µm wide at mid-body, occupying one-tenth to less than one-sixth (9-15%) of mid-body diameter. Two ventral and two dorsal body pores are usually present at level of mural odontostyle-odontophore, their corresponding ducts appearing especially thickened beneath intermediate cuticle layer. Lip region offset by deep constriction, 2.8-3.3 times as wide as high and one-fifth to less than one-third (21-30%) of body diameter at neck base; lips mostly amalgamated, somewhat angular; papillae perceptible, somewhat protruding. Amphid fovea cup-shaped, its opening occupying 8-10 µm or up to one-half (44-50%) of lip region diameter. Cheilostom nearly cylindrical, lacking any differentiation. Mural odontostyle attached subventrally, 6.7-7.7 times as long as wide, 0.7-0.9 times as long as lip region diameter, and 0.54-0.72% of body length; aperture 11-13 µm long or up to six-sevenths (73-84%) its length. Guiding ring simple, somewhat plicate, at 0.6-0.8 lip region diameters from anterior end. Odontophore linear, rod-like, irregular at its base, in lateral view with the ventral side longer that the dorsal one (figure 7A, C), and 2.0-2.4 times the mural odontostyle length. Anterior region of pharynx enlarging very gradually; basal expansion 8-12 times as long as wide, 4.1-5.3 times as long as body diameter and occupying 57-66% of total neck length; gland nuclei obscure in most specimens examined, DN = 54 (n=1) and S 2 N = 92 (n=1). Nerve ring located at 158-177 µm from anterior end or 24-31% of total neck length. Cardia rounded conoid, 11-19 × 14-18 µm; a ring-like structure is present surrounding its junction to pharyngeal base. Genital system didelphic-amphidelphic, with both branches almost equally and well developed, the anterior 174-207 µm long or 7-9% of body length and the posterior 168-220 µm long or 7-9% of body length. Ovaries variably sized, the anterior 93-191 µm, the posterior 84-175 µm long; oocytes arranged first in two or more rows, then in a single row. Oviduct 73-103 µm long or 0.9-1.1 times the corresponding body diameter, and consisting of a slender part with prismatic cells and a weakly developed pars dilatata. Oviduct-uterus junction marked by a sphincter. Uterus a short, simple, tube-like structure 44-69 µm long or 0.5-0.9 times the corresponding body diameter, lacking sperm cells inside. Vagina extending inwards 38-51 µm or two-fifths to one-half (43-53%) of body diameter: pars proximalis 25-35 × 15-20 µm, with somewhat sigmoid walls and surrounded by weak musculature; pars refringens with two small, triangular to drop-shaped pieces measuring 8-10 × 6-8 µm and with a combined width of 14-19 µm; and pars distalis short, 3.0-5.5 µm long. Vulva a transverse slit. Prerectum 1.4-2.1, rectum 0.8-1.1 anal body diameters long. Tail very short and rounded to truncate. Caudal pores two pairs, one sublateral, another sub-dorsal.
Besides, in having short mural odontostyle (11-14 µm at its ventral side) the new species resembles A. brevistylum sp. n. and A. californicum, but it differs from these in its well developed pars refringens vaginae (vs absent). Moreover, it differs from A. brevistylum sp. n. in its shorter neck (579-649 vs 663-767 µm), wider lip region (19-20    Type material. Female holotype and one female paratype deposited in the nematode collection of the University of Jaen, Spain. One female paratype deposited in the nematode collection of the Institute of Ecology and Biological Resources, Vietnam.
Etymology. The specific epithet means 'small' and refers to the comparatively small general size of the new species.
Remarks. In spite of it was collected from three localities, the material examined is very similar in its main morphological features and morphometrics. Nevertheless, some differences have been also observed, especially affecting the female tail shape as some specimens show a short and rounded-conoid caudal region whereas it becomes extremely short and truncate in other individuals. Material examined. Two females and four males, in variable state of preservation.
Measurements. See Table 2. Description. Adult. Moderately slender to slender nematodes of medium size, 2.09-2.60 mm long. Body cylindrical, distinctly tapering towards the anterior end, less so towards the posterior one because the caudal region is rounded conoid. Habitus regularly curved ventrad after fixation, to a more or less open C, occasionally more curved at posterior body region, spiral-shaped in only one male specimen. Cuticle three-layered, especially visible distinct at caudal region, consisting of thin outer layer bearing fine transverse striation through the entire body, a much thicker intermediate layer with radial striation, and a thin inner layer; thickness 2.5-4.0 µm at anterior region, 5-6 µm in mid-body and 8-10 µm on tail. Lateral chord 7-12 µm wide at mid-body, occupying one-tenth to one-eighth (10-12%) of mid-body diameter. Three ventral and three dorsal body pores are usually present at level of odontostyle-odontophore, their corresponding ducts appearing especially thickened beneath inner cuticle layer. Lip region visibly narrower than adjacent body, offset by depression, 2.2-2.6 times as wide as high and one-fifth to one-fourth (19-27%) of body diameter at neck base; lips (under SEM) amalgamated; papillae button-like, the inner labial ones rather close the margin of oral field and surrounded by one or two ring-like annuli, whereas the outer labial ones are surrouned by only one annulus and the cephalic ones lack a such differentiation; oral aperture a dorsoventral, nearly hexagonal orifice, the lip region hence showing a biradial symmetry. Amphid fovea cup-shaped, its opening at level of cephalic depression and occupying 12-13 µm or up to three-fourths (72-75%) of lip region diameter. Cheilostom nearly cylindrical, lacking any differentiation. Mural odontostyle attached subventrally and comparatively short, 4.5-4.9 times as long as wide, 0.6-0.7 times as long as lip region diameter, and 0.44-0.54% of body length; aperture 7-8 µm long or up to two-thirds (60-66%) its length. Guiding ring simple, somewhat plicate, at 0.5-0.7 lip region diameters from anterior end. Odontophore linear, rod-like, 3.8, 4.1 (3.5, 3.7 in males, n=2) times the odontostyle length. Anterior region of pharynx enlarging very gradually; basal expansion 8.3-11.0 times as long as wide, 5.0-5.8 times as long as body diameter, and occupying 58-64% of total neck length; gland nuclei obscure in all the specimens examined. Nerve ring located at 139-169 µm from anterior end or 23-26% of total neck length. Cardia rounded conoid, 10-16 × 12-18 µm; a ring-like structure is present surrounding its junction to pharyngeal base. Tail short, rounded to rounded conoid, its inner core bearing a finger-like projection at tail end. Caudal pores two pairs, one lateral, another sub-lateral.
Female. Genital system didelphic-amphidelphic, with both branches almost equally and well developed, the anterior 246, 387 µm long or 10, 15% of body length and the posterior 301 µm long or 12% (n=1) of body length. Ovaries moderately sized, usually not surpassing the sphincter level, the anterior 82, 119 µm, the posterior 66, 103 µm long; oocytes arranged first in two or more rows, then in a single row. Oviduct 99-107 µm long or 1.2, 1.3 times the corresponding body diameter, and consisting of a slender part with prismatic cells and a moderately developed pars dilatata with visible lumen but no sperm cell. Oviduct-uterus junction marked by a sphincter. Uterus a short, simple, tube-like structure 128-243 µm long or 2.1, 2.8 times the corresponding body diameter, one female containing abundant sperm cells inside. Vagina extending inwards 31, 32 µm or about three-eighths (36%, n=1) of body diameter: pars proximalis 23, 24 × 25, 26 µm, with somewhat sigmoid walls and surrounded by weak musculature; pars refringens with two small, triangular to drop-shaped pieces measuring 5 × 4, 5 µm and with a combined width of 9, 10 µm; and pars distalis 1.0, 1.5 µm long. Vulva a post-equatorial transverse slit. Prerectum 2.3, 2.5, rectum 0.8, 1.2 anal body diameters long.
Male. Genital system diorchic, with opposite testes. In addition to the ad-cloacal pair, situated at 16-19 µm from cloacal aperture, there is a series of two or three widely spaced (22-42 µm apart) ventromedian supplements, the posteriormost of which lying out the range of spicules, but very close to the spicules end, being situated at 42-63 µm from ad-cloacal pair. Spicules relatively robust, 3.5-4.6 times its maximum width, 1.3-1.6 times the body diameter at level of the cloacal aperture: dorsal contour regularly convex, ventral contour bearing weak hump and hollow; curvature 140-143º; head occupying 8-10% of spicule total length, with both contours nearly straight, and its dorsal side longer than the ventral one; median piece 6.5-8.3 times as long as wide, occupying 45-54% of spicule maximum width, reaching the posterior tip; posterior end 5-6 µm wide. Lateral guiding pieces 19-23 µm long, 6.4-7.8 times as long as wide. Prerectum 3.0-3.3, cloaca 1.2-1.4 times the corresponding body width long.
Type locality and habitat. Northern Vietnam, Cuc Phuong National Park, where the new species was collected from soil of a pristine tropical forest in 2009. Type material. Female holotype and one female and three male paratypes, deposited in the nematode collection of the University of Jaen, Spain. One male paratype deposited in the nematode collection of the Institute of Ecology and Biological Resources, Vietnam.
Etymology. The specific epithet is a Latin term meaning 'from the jungle', and refers to the habitat where the species dwells.

On the taxonomy of Aporcelaimoides
As mentioned in the introductory section, the identity of Aporcelaimoides has been matter of some controversy. In his original description of this genus, Heyns (1965) highlighted the differences between it and Sectonema, mainly based on the nature of the stomatal protrusible structure, "a dorylaimid spear which is set ventrally … similar in position to the mural tooth of Sectonema", putting hence emphasis on the existence of a large dorsal aperture in the protrusible structure of Aporcelaimoides and the absence of a such aperture in the mural tooth of Sectonema. Heyns (op. cit.) also noted that "the basal part of the spear (is) much narrower than the lumen of the pharynx (stoma)" [text between brackets incorporated by the authors]. Subsequent contributions by Andrássy (1976) and Jairajpuri and Ahmad (1992) assumed Heyns' point of view. Siddiqi (1995), however, stated (p. 99) that "…Since there is a great variation in the mural tooth of Sectonema (see Siddiqi 1984), there is no justification for holding Aporcelaimoides as a valid genus". And, later, Andrássy (2009) followed Siddiqi's opinion.
Very recently, Peña-Santiago and Álvarez-Ortega (2014a) redescribed S. ventrale Thorne, 1930, the type species of Sectonema, and conclude that (p. 1103) "the protrusible structure of Sectonema, as observed in its type species, is not a typical mural tooth as seen in nygolaims, but a reduced odontostyle with its base occupying most (if not whole) the stomatal lumen". It means that mural odontostyle of Aporcelaimoides significantly differs from the reduced odontostyle of Sectonema. Thus, Siddiqi's (1995) action might be not well supported as there are morphological arguments to separate both genera and to restore Aporcelaimoides as valid genus. Unfortunately, there is no molecular information of the latter, which would be especially useful to confirm the morphological data.
A revised diagnosis of Aporcelaimoides as well an updated list of its species, three of them transferred from Sectonema, and a key to their identification are given in the following. Besides, a compendium of their main morphometrics is presented in Table 3.

Diagnosis (emended)
Aporcelaimidae. Slender to very slender nematodes (a = 23-75) of medium to large size, 1.95-5.66 mm long. Cuticle three-layered, especially obvious at caudal region, with the intermediate layer more refringent and thicker than the outer and the inner ones. Oral aperture a dorso-ventral, nearly hexagonal slit. Lip region offset by a more or less distinct constriction, but by depression in A. silvaticum sp. n. Mural odontostyle attached subventrally, comparatively short and with wide aperture, often occupying more than one-half its length. Guiding ring simple and plicate. Odontophore rod-like. Pharynx enlarging gradually, with basal expansion occupying three-fifths to two-thirds of total neck length. Female genital system didelphic-amphidelphic; pars refringens vaginae present or absent; and vulva a transverse slit. Tail similar in both sexes, short, rounded conoid, rounded or truncate. Spicules dorylaimoid, well developed. Ventromedian supplements in low number (0-4), widely separated, always with pre-cloacal space (hiatus).

Relationships
As mentioned, Aporcelaimoides is morphologically very similar to Sectonema, from which it differs in the nature of the stomatal protrusible structure. It can be easily distinguished from the typical species of Sectonema, for instance S. ventrale -the type species of the genus, recently re-described by Peña-Santiago and Álvarez-Ortega (2014a) -in having a mural odontostyle attached to the ventral side of stoma (vs a reduced axial odontostyle), much narrower than (vs occupying the whole) stomatal lumen, with (in lateral view) its dorsal and ventral sides parallel and distinctly perceptible (vs dorsal side nearly lost) and a perceptible dorsal aperture often occupying more than half of its total length (vs nearly the total odontostyle length). Besides, Aporcelaimoides compares to other atypical species of Sectonema, for instance S. demani Altherr, 1965 (see recent description by Peña-Santiago and Álvarez-Ortega 2014b) and S. septentrionale Peña-Santiago & Álvarez-Ortega, 2015, which are characterized by having a mural tooth (a protrusible structure lacking a distinct aperture, resembling that found in nygolaims) with asymmetrical sides as (in lateral view) the dorsal side is visibly sigmoid and distinctly longer than the ventral one.