Five new and five known species of the genus Tylencholaimus de Man, 1876 (Nematoda: Dorylaimida: Tylencholaimoidea) from Western Ghats of India

Five new and five known species of the genus Tylencholaimus de Man, 1876 are described from the Western Ghats of India. Tylencholaimus macroamphidius sp. nov. has a 0.61–0.85 mm long body, angular lips, large amphid, 8.0–9.0 μm long odontostyle, odontophore with asymmetrical basal knobs, pharyngeal bulb expanding gradually, female genital system mono-prodelphic, and a rounded to conoid tail. Tylencholaimus shamimi sp. nov. has a 0.57–0.71 mm long body, 6.0–7.0 μm long odontostyle, odontophore with basal thickening, pharyngeal bulb expanding gradually, female genital system amphidelphic and a convex-conoid tail. Tylencholaimus southindicus sp. nov. has a 0.44–0.55 mm long body, lip region with distinct labial disc, 4.5–5.5 μm long odontostyle, odontophore with minute basal knobs, pharyngeal expansion abrupt, female genital system mono-prodelphic, and a roundedconoid to rounded tail. Tylencholaimus striatus sp. nov. has a 0.30–0.34 mm long body with distinctly striated cuticle, 5.0–5.5 μm long odontostyle, odontophore with minute basal knobs, pharyngeal bulb expanding abruptly, female genital system mono-prodelphic, and a conoid tail with bluntly rounded terminus. Tylencholaimus tamiliensis sp. nov. has 0.51–0.58 mm long body, 5.5–6.0 μm long odontostyle, odontophore with minute basal knobs, pharyngeal bulb expanding gradually, female genital system mono-prodelphic and tail rounded to conoid with sunken terminus. Five known species of the genus Tylencholaimus viz. T. mirabilis, T. teres, T. micronanus, T. ibericus and T. cosmos also recorded from the region and redescribed/illustrated.


Introduction
The Western Ghats in India is recognized as a biodiversity hotspot (Myers et al. 2000) and is one of the world's eight "Hottest biodiversity hotspots". It represents some of the best tropical evergreen forests with highly mosaic topography. Its climate varies with altitudinal gradation and equatorial distance. Although, the Western Ghats covers about 6% of the total Indian land area, it retains very rich floral and faunal diversity (CEPF 2007). Several species of soil-inhabiting nematodes have been described from this biodiversity hotspot (Ferris et al. 1979;Ahmad & Jairajpuri 1982, 1983, 1984, 1986, 1988, 2002Ahmad 1993;Dhanam & Jairajpuri 1999;Tabinda et al. 2013), however, the species diversity of the genus Tylencholaimus de Man, 1876 which is one of the most speciose genus representing the superfamily Tylencholaimoidea Filipjev, 1934 of the order Dorylaimida Pearse, 1942, has not been extensively studied in this region. The nematodes belonging to this genus mostly occur in undisturbed forest soils and have worldwide distribution. Several authors (Jairajpuri 1965;Loof & Jairajpuri 1968;Ali & Chisty 1972;Ahmad & Jairajpuri 1979;Khan & Laha 1982;Rahman et al. 1987;Khan et al. 1989;Dhanachand 1994;Khan & Ahmad 1994;Dhanam & Jairajpuri 1999;Mohilal & Dhanachand 2003;Mushtaq et al. 2007 andAhad &Ahmad 2016) added species to this genus from India, but currently there is not much information from the Western Ghats. Only three species viz., Tylencholaimus cosmos Peña-Santiago, 2008, T. ibericus Peña-Santiago & Coomans, 1994and T. vulvulatus Rahman et al., 1987 have been recorded so far (Dhanam & Jairajpuri 1999) from this region.
During the present study, several populations representing the genus Tylencholaimus were collected from different localities of this region. After examination, they were found to represent five new and five known species, which are described and illustrated in this paper.

Material and methods
Soil samples were collected from different localities of the Western Ghats of India. The nematodes were extracted from soil samples following Cobb's (1918) sieving and decantation and modified Baermann's funnel techniques. The extracted nematodes were fixed in hot triethanolamine-glycerol fixative, dehydrated by the slow evaporation method (Seinhorst 1959), and mounted in anhydrous glycerine. Permanent mounts were prepared using the paraffin wax ring method (de Maeseneer & d'Herde 1963). The measurements were taken using an ocular micrometre, and position of pharyngeal gland nuclei and their orifices were calculated according to Loof & Coomans (1970). Line drawings were prepared using a drawing tube, and photographs were taken with a Nikon DS digital Camera attached with Nikon Eclipse 80i microscope. Raw photographs were edited using Adobe ® Photoshop ® .
Type and others specimens are deposited in the nematode collection of the Department of Zoology, Aligarh Muslim University, India (AMU/ZD/NC), as well as in the nematode collection of the Zoological Survey of India, Kolkata, India.
List of abbreviations a = body length / greatest body diameter b = body length / neck length c = body length / tail length c ' = tail length / body diameter at anus or cloaca DN = position of dorsal pharyngeal gland nucleus from anterior end × 100 / total neck length DO = orifice of the dorsal pharyngeal gland nucleus from anterior end × 100 / total neck length DO-DN = distance of dorsal gland nucleus from the orifice of dorsal gland (expressed as percentage of total neck length) G1 = length of anterior genital branch × 100 / body length G2 = length of posterior genital branch × 100 / body length L = total body length n = number of specimens European Journal of Taxonomy 774: 58-105 (2021) S1N1 = nucleus of the first gland of the first subventral pair of pharyngeal glands × 100 / total neck length S1N2 = nucleus of the second gland of the first subventral pair of pharyngeal glands × 100 / total neck length S2N = nuclei of the second subventral pair of pharyngeal glands × 100 / total neck length S2O = orifice of the second subventral pair of pharyngeal glands × 100 / total neck length V = distance of vulva from anterior end × 100 / body length

Description Female
Small sized nematodes, slightly curved ventrad upon fixation; body cylindrical, tapering gradually towards both extremities but more so towards the anterior end. Cuticle with two distinct layers, 1.0-1.5 µm thick at anterior region, 2.0-2.5 μm at midbody and 3.0-4.0 μm on tail. Outer cuticle thin, finely striated; inner layer thick, its outline somewhat irregular, with distinct radial refractive elements. Lateral chords occupying about 15-25% of midbody diameter. Body pores distinct, lateral pores: one at odontostyle-odontophore region, 2-5 in neck region, 5-8 at pharyngeal base to vulva and 5-10 in post-vulval region; dorsal body pores: one at odontostyle-odontophore region, 1-3 in neck region, 3 from pharyngeal base to vulva and 1-2 in post-vulval region; ventral body pores: 1-3 in neck region, 1-3 from pharyngeal base to vulva and 3 in post-vulval region. Lip region cap-like, offset by deep constriction, 1.8-2.2 times as wide as high or about ⅓ of the body diameter at neck base. Lips rounded, amalgamated, inner part slightly elevated. Labial and cephalic papillae distinct but not interfering with the labial contour. Amphids cup-shaped, their aperture occupying about ⅓ to ½ of lip region diameter.   (Bütschli, 1873). All measurements are in μm and in the form: mean ± s.d. (range) for females, and measurements for each male (n = 2) separated by a comma.  Stoma a truncate cone. Odontostyle 0.7-0.9 times the lip region diameter long, its aperture about ¼ to ⅓ of its length. Odontophore rod-like, with distinct basal swelling, 1.2-1.6 times the odontostyle length. Guiding ring simple, refractive, at 0.6-0.8 times the lip region diameter from anterior end. Pharynx consisting of a slender, weakly muscular anterior part, expanding abruptly into a cylindrical basal bulb, with thick-walled lumen, separated by a constriction, occupying about 41-46% of total neck length. Pharyngeal gland nuclei and their orifices are located as follows: DO = 59-62, DN = 62-65, DO-DN = 1.7-3.7, S1N1 = 72-76, S1N2 = 76-80, S2N = 89-91, S2O = 90-93. Nerve ring at 34-42% of neck length from anterior end. Cardia rounded to conoid, about ⅓ to 2/5 of the corresponding body diameter long.

Male
General morphology similar to that of female, except for posterior region being more ventrally curved. Genital system diorchic, testes opposed, sperm cell spindle-shaped. In addition to adcloacal pair at 7.0-8.0 µm from cloacal aperture, there are four ventromedian supplements, located outside the range of spicules, first ventromedian supplement at 37-38 μm from adcloacal pair, second at 7.0-8.0 μm from first, third at 22-23 μm from second and fourth at 10-11 μm from third ventromedian supplement. Spicules typically dorylaimoid, curved ventrad, relatively robust, 4.0-4.4 times as long as wide and 1.2-1.3 times as long as body diameter at level of cloacal aperture, dorsal contour regularly convex, ventral contour bearing a moderately developed hump and hollow, curvature 123-125º, head occupying about 22% of total spicules length, median pieces 9.5-10.2 times as long as wide, occupying about 30% of the spicules maximum width, reaching the spicules tip, posterior end 3.0-3.5 μm wide. Lateral guiding pieces distinct, rod-like, 3.4-4.0 times as long as wide or ¼ of the spicules length. Prerectum 6.0-7.0 and rectum 1.2-1.5 times cloacal body diameter long. Tail short, convex-conoid, with bluntly rounded terminus, 1.4 cloacal body diameter in length, with a pair of caudal pores on each side.

Remarks
Bütschli (1873) described Tylenchus mirabilis from Germany for which de Man (1876) proposed the genus Tylencholaimus with T. mirabilis as its type species. The type specimens were not preserved and the identity of the species remained uncertain. De Man (1880, 1884) collected specimens from soil near Apeldoorn, The Netherland in July, 1879 and described them as Tylencholaimus mirabilis. Tarjan (1953) described Discomyctus brevicaudatus from Rhode Island, United States, which he later (Tarjan 1956) transferred to Tylecholaimus. Coomans (1962) redescribed T. brevicaudatus from Belgium and considered it closely similar to T. mirabilis (Bütschli, 1873). Loof & Jairajpuri (1968) in their revision of the genus Tylencholaimus, redescribed this species based on seven females from the type locality and one male from Switzerland, and designated neotype as well as topotype from these specimens. They also agreed with de Man's (1876) opinion that T. mirabilis is the type species of the genus Tylencholaimus. Vinciguerra (1986) described this species from Italy, while Peña-Santiago & Coomans (1994b), in their revision of the genus Tylencholaimus, made a detailed study of the type population and of another population collected from Spain. The morphometrics of the present specimens collected from India conform well with the type population, except in having slightly smaller spicules (27 vs 31 μm). The present specimens also conform well with populations described earlier by Tarjan (1953), Loof (1961) and Coomans (1962), except for the presence of males (vs absent). The present specimens also conform with Spanish and Italian populations described by Peña-Santiago & Coomans (1994b) and Vinciguerra (1986). This is the first report of T. mirabilis from India. Thorne, 1939 Fig. 3, Table 2 Tylencholaimus teres Thorne, 1939: 58-59.

Male
General morphology similar to that of female, except for posterior region being more ventrally curved. Genital system diorchic, testes opposed, sperm cell spindle-shaped. In addition to the adcloacal pair at 6.0 µm from cloacal aperture, there are three ventromedian supplements, located outside the range of spicules, first one at 12 μm from adcloacal pair, second at 16 μm from first and third one at 18 μm from second ventromedian supplement. Spicules typically dorylaimoid, curved ventrad, relatively slender, 5.7 times as long as wide and 1.4 times cloacal body diameter long, dorsal contour regularly convex, ventral contour bearing a moderately developed hump and hollow, curvature 140°, head occupying about 25% of total spicules length, median piece 10.6 times as long as wide, occupying about 42% of the spicules maximum width, reaching the spicules tip, posterior end 2.5 μm wide. Lateral guiding piece distinct, rod-like, about 5.0 times as long as wide or about 1/5 of the spicules length. Prerectum 5.7 and rectum 1.5 times the cloacal body diameter long. Tail short, rounded to conoid, 0.93 times the cloacal body diameter long, with a pair of caudal pores on each side.

Remarks
Dhanam & Jairajpuri (1999) described Amphitylencholaimus cosmos from Malnad tracts of Karnataka, India. Peña-Santiago (2008) did not accept the generic status of Amphitylencholaimus and transferred A. cosmos to Tylencholaimus. Ahad & Ahmad (2016) reported this species from Kaziranga National Park, Assam, India. Dhanam & Jairajpuri (1999) characterized this species by having the anterior slender part of pharynx expanding gradually to form the cylindroid basal part but in their fig. 1C, the anterior part of pharynx appears slender, expanding abruptly to form the basal expanded part. In the present specimens, the anterior part of pharynx is also slender, expanding abruptly to form the basal expanded part. The morphometrics of the present populations conform well with the type population except in having a shorter body length (0.55-0.76 vs 0.7-0.9 mm), slightly lower b value (3.1-4.2 vs 3.9-5.0) and shorter odontophore length (8.5-10 vs 12-14 μm). The present populations also conform well with the Assam population except in having a slightly higher lip region (3.0-3.5 vs 2.0-3.0 μm) and a longer rectum (17-22 vs 13-14 μm).

Etymology
The new species is named Tylencholaimus macroamphidius sp. nov. because of its characteristically large amphids.

Type habitat and locality
Soil samples collected from around the roots of shrubs (unidentified) from Naduvattum, Nilgiri Hills, Tamil Nadu State.

Female
Small sized nematodes, slightly curved ventrad upon fixation; body cylindrical, tapering gradually towards both extremities but more so towards the anterior end. Cuticle with two distinct layers, 1.5-2.0 μm thick at midbody and 2 μm on tail. Outer cuticle thin, finely striated; inner layer thick, its outline somewhat irregular, with distinct radial refractive elements, more distinct in caudal region. Lateral chords occupying about 25-29% of midbody diameter. Lateral, dorsal and ventral body pores indistinct. Lip region cap-like, offset by deep constriction, 2.0-2.5 times as wide as high or about ½ to 2/5 of the body diameter at neck base. Lips moderately separate, angular, inner part slightly elevated and protruding. Labial and cephalic papillae distinct but not interfering with the labial contour. Amphids large, stirrup-shaped, their aperture occupying about ½ to 3/5 of lip region diameter. Stoma a truncate cone. Odontostyle 0.8-1.0 times the lip region diameter long, its aperture about ⅓ of the odontostyle length. Odontophore 1.0-1.1 times of the odontostyle length with asymmetrical basal knobs, the subventral knobs always larger than dorsal one. Guiding ring simple, refractive, at 0.6-0.9 times the lip region diameter from anterior end. Pharynx consisting of a slender, slightly muscular anterior part, expanding gradually into a cylindroid basal bulb, with thick-walled lumen, occupying about 38-43% of total neck length. Pharyngeal gland nuclei and their orifices are located as follows: DO = 61-64, DN = 63-67, DO-DN = 1.5-3.1, S1N1 = 73-76, S1N2 = 77-81, S2N = 88-90, S2O = 90-91. Nerve ring at 33-39% of neck length from anterior region. Cardia rounded to conoid, about ⅓ to 3/5 of the corresponding body diameter long.

Male
General morphology similar to that of female, except for the posterior body region being comparatively more ventrally curved. Genital system diorchic, testes opposed, sperm cell spindle-shaped. In addition to adcloacal pair at 3.0-5.0 μm from cloacal aperture, there are three ventromedian supplements, located outside the range of spicules, first one at 16-20 μm from adcloacal pair, second at 8.0-10 μm from first, and third at 8.0-14 μm from second ventromedian supplement. Spicules typically dorylaimoid, curved ventrad, relatively robust, 4.9-5.8 times as long as wide, 1.0-1.4 times as long as cloacal body diameter, dorsal contour regularly convex, ventral contour bearing a moderately developed hump and hollow, curvature 120-123º, head occupying about 14-17% of total spicules length, median pieces 11.3-12.5 times as long as wide, occupying about 37% of the spicules maximum width, reaching the spicules tip, posterior end 2.0-2.5 μm wide. Lateral guiding pieces slender, about 4.0-6.0 times as long as wide or about ⅓ of total spicules length. Prerectum 4.2-5.0 and rectum 1.0-1.2 cloacal body diameter long. Tail short, conoid, 1.1-1.2 times cloacal body diameter long with a pair of subdorsal caudal pores.

Etymology
The new species is named after Prof. Mohammad Shamim Jairajpuri in recognition of his contribution to nematode taxonomy.

Type habitat and locality
Soil sample collected from around the roots of grasses (unidentified) from Kudyathoor, Idukki district, Kerala State.

Other habitat and locality
Soil sample collected from around the roots of grasses and shrubs (unidentified) from Yellapur, Uttara Kannada district, Karnataka State.

Male
General morphology similar to that of female, except for posterior region being more ventrally curved. Genital system diorchic, testes opposed, sperm cell spindle-shaped, 8.0-9.0 μm long. In addition to adcloacal pair, situated at 5.0-6.0 µm from cloacal aperture, there are two to three spaced ventromedian supplements, located outside the range of spicules; first one at 32-36 μm from adcloacal pair, second at 23-35 μm from first and the third (n = 1) at 20 μm from second ventromedian supplement. Spicules typically dorylaimoid, curved ventrad, relatively slender, 4.6-4.8 times as long as wide and 1.4 times as long as cloacal body diameter, dorsal contour regularly convex, ventral contour bearing a moderately developed hump and hollow, curvature 128-131º, head occupying about 10-13% of total spicules length, median pieces 10.5-14.3 times as long as wide, occupying about 30-40% of the spicules maximum width, reaching the spicules tip, posterior end 1.5-2.0 μm wide. Lateral guiding pieces distinct, rod-like, 4.3-4.6 times as long as wide or about ⅓ of the spicules length. Prerectum 3.9-5.5 and rectum 1.5-1.6 cloacal body diameter long. Tail short, convex-conoid, with rounded terminus, 0.9-1.0 times cloacal body diameter long, with a pair of caudal pores on each side and a distinct terminal pore.

Etymology
The new species is named Tylencholaimus southindicus sp. nov. because of its distribution in south India.

Type habitat and locality
Soil samples collected from around the roots of shrubs (unidentified) from Manikandanchal, Ernakulam district, Kerala State.

Other habitat and locality
Soil samples collected from around the roots of shrubs (unidentified) and forest trees (unidentified) from Bhagamandala, Kodagu district, Karnataka State.

Male
General morphology similar to that of female, except for posterior region being more ventrally curved. Genital system diorchic, testes opposed, sperm cell spindle-shaped. In addition to adcloacal pair at 4.0-5.0 µm from cloacal aperture, there are two ventromedian supplements, located outside the range of spicules, first one at 22-31 μm from adcloacal pair, second at 16-31 μm from first ventromedian supplement. Spicules typically dorylaimoid, curved ventrad, 4.3-5.1 times as long as wide and 1.3-1.5 times as long as body diameter at level of cloacal aperture, dorsal contour regularly convex, ventral contour bearing a moderately developed hump and hollow, curvature 125-130º, head occupying about 10-11% of total spicules length, median pieces 7.5-11.3 times as long as wide, occupying about 37-57% of the spicules maximum width, reaching the spicules tip, posterior end 1.5-2.0 μm wide. Lateral guiding pieces distinct, rod-like, about 3.3-5.0 times as long as wide or about ¼ to ⅓ of the spicules length. Prerectum 5.1-7.9 and rectum 1.5-1.7 times cloacal body diameter long. Tail short, rounded to conoid, 0.7-1.0 times cloacal body diameter long; a pair of subdorsal and a prominent terminal caudal pore present.

Etymology
The new species is named Tylencholaimus striatus sp. nov. because of its distinctly striated cuticle.

Type habitat and locality
Soil samples collected from around the roots of shrubs and forest trees (unidentified) from Bhagamandala, Karnataka State.

Description Female
Small sized nematodes, slightly curved ventrad to open C-shaped upon fixation; body cylindrical, tapering gradually towards both extremities but more so towards the anterior end. Cuticle with two distinct layers, 1.0-1.5 μm thick at midbody and 2.0-2.5 μm on tail. Outer cuticle strongly striated; inner layer thick, loose, irregular outline with distinct radial refractive elements. Lateral chords occupying about 26-36% of midbody diameter. Lateral dorsal and ventral body pores indistinct. Lip region caplike, offset by slight constriction, 1.7-2.0 times as wide as high or about ⅓ to 2/5 of the body diameter at neck base. Lips rounded, amalgamated, inner part slightly elevated. Labial and cephalic papillae distinct but not interfering with the labial contour. Amphids cup-shaped, their aperture occupying about ⅓ to 2/5 of lip region diameter. Stoma a truncate cone. Odontostyle 0.8 times the lip region diameter long, its aperture about ⅓ of the odontostyle length. Odontophore rod-like, as long as odontostyle, with minute basal knobs. Guiding ring simple, refractive, at 0.7-0.9 times the lip region diameter from anterior end. Pharynx consisting of a slender, slightly muscular anterior part, expanding abruptly into a cylindrical basal bulb, with thick-walled lumen, occupying about 36-41% of total neck length. Pharyngeal gland nuclei and their orifices are located as follows: , DO-DN = 2.2-3.1, S1N1 = 74-77, S1N2 = 78-82, S2N = 89-91, S2O = 91-92. Nerve ring at 39-44% of neck length from anterior end. Cardia rounded conoid, about ⅓ of the corresponding body diameter long.

Male
Not found.

Etymology
The new species is named Tylencholaimus tamiliensis sp. nov. because of its type locality Tamil Nadu.

Type habitat and locality
Soil samples collected from around the roots of shrubs (unidentified) from forest near Naduvattum, Nilgiris district, Tamil Nadu State.

Female
Moderately sized nematodes, slightly curved ventrad upon fixation; body cylindrical, tapering gradually towards both extremities but more so towards the anterior end. Cuticle with two distinct layers, 1.5-2.0 μm thick at midbody and 2.5-3.0 μm on tail. Outer cuticle with fine transverse striations; inner layer thick, its outline loose irregular, with distinct radial refractive elements. Lateral chords occupying about 22-31% of midbody diameter. Lateral body pores indistinct, dorsal body pores one at odontostyle-odontophore region, 1-2 in neck region, one between pharyngeal base to vulva and one at post-vulval region; ventral body pores: 1-2 in neck region, and 2 in post-vulval region. Lip region cap-like, offset by deep constriction, 2.0-2.3 times as wide as high or about ⅓ of the body diameter at neck base. Lips rounded, amalgamated, inner part slightly elevated. Labial and cephalic papillae distinct but not interfering with the labial contour. Amphids small, cup-shaped, their aperture occupying about ⅓ of lip region diameter. Stoma a truncate cone. Odontostyle 0.7-0.9 times the lip region diameter long, its aperture about ¼ to 1/5 of the odontostyle length. Odontophore rod-like, with minute basal knobs, about 1.0-1.1 times the odontostyle length. Guiding ring simple, refractive, at 0.6-0.8 times the lip region diameter from anterior end. Pharynx consisting of a slightly muscular anterior part, expanding gradually into a cylindrical basal bulb, with thick-walled lumen, occupying about 40-43% of total neck length. Pharyngeal gland nuclei and their orifices are located as follows: DO = 58-62, DN = 61-64, DO-DN = 2.1-3.5, S1N1 = 74-77, S1N2 = 77-82, S2N = 87-89, S2O = 89-92. Nerve ring located at 37-41% of neck length from anterior region. Cardia rounded conoid, about ⅓ to 2/5 of the corresponding body diameter long.

Male
Not found.

Discussion
The genus Tylencholaimus is one of the most widely distributed and speciose taxa under the superfamily Tylencholaimoidea of the order Dorylaimida. Its representatives generally occur in undisturbed natural soils, predominantly recorded from temperate Northern Hemisphere i.e., North America and Eurasia (Golhasan et al. 2019). A large number of species have been reported from India, representing about 40% of its total species. The Western Ghats in India constitutes a range of tropical rainforests with a very high degree of species richness and endemism. Only three species, representing the genus Tylencholaimus have so far been recorded from this region by Dhanam & Jairajpuri (1999). In the course of the present study, several populations of this genus were recorded from soil samples collected from different habitats and localities. The presently reported populations represented five known species (T. mirabilis, T. teres, T. micronanus, T. ibericus and T. cosmos) and five new species. With the addition of the presently described new species, the total number of valid species under this genus is raised to 62 (Ahad & Ahmad 2016;Wu et al. 2018;Golhasan et al. 2019), 27 of them are recorded from India, and 11 species are recorded from the Western Ghats. Tylencholaimus mirabilis, the type species is reported here for the first time from India. Two known species T. micronanus and T. teres are reported here for the first time from the Western Ghats and a male specimen was also described for T. ibericus. As a result of this survey, the Western Ghats comprises 11 species of the genus Tylencholaimus, which constitutes about 40% of the genus diversity in India and 17% of the World's Tylencholaimus diversity.