Characterization of Iberian species of the genus Pungentus Thorne & Swanger , 1936 ( Nematoda , Dorylaimida , Nordiidae )

Several populations of four known species of the genus Pungentus (P. clavatus, P. engadinensis, P. marietani and P. silvestris), collected in the wild and in cultivated soils from the Iberian Peninsula, are studied. Detailed redescriptions and morphometrics are presented for each species. Illustrations are provided, including line drawings, light microscopy pictures of the four species as well as scanning electron microscopy observations of P. engadinensis. The Iberian populations are compared to type and other known populations, and new data are given that provide a better characterization of these taxa. Pungentus engadinensis is the most widely distributed species in the Iberian Peninsula.


Introduction
The genus Pungentus Thorne & Swanger, 1936 is an interesting dorylaimid nematode genus.Its species are common inhabitants of natural forest areas of the Holarctic range.So far, five species of Pungentus have been recorded for the Iberian fauna (see compendium in Peña-Santiago et al. 2003).A sixth species, namely P. intertextus (Thorne & Swanger, 1936) was recently transferred to Stenodorylaimus Álvarez-Ortega & Peña-Santiago, 2011.Nevertheless, no morphological or taxonomical study of these Iberian taxa was ever carried out and their identity was never confirmed.General nematological surveys, mainly conducted in the southern Iberian Peninsula during the last three decades to explore the dorylaimid fauna of this region, yielded many populations of Pungentus.

Material and Methods
Nematodes were collected mainly in natural areas of the southern Iberian Peninsula during several nematological surveys conducted in the last three decades.They were extracted from soil samples using the methods by Baermann (1917) and Flegg (1967), relaxed and killed by heat, fixed in 4% formaldehyde, processed to anhydrous glycerine following Siddiqi's (1964) technique, and mounted on permanent glass slides to allow handling and observation under light microscopy (LM).Morphometrics (Table 1) included de Man's indices and most of the usual measurements; the location of pharyngeal gland nuclei (DN, S 1 N and S 2 N) is provided after the scheme proposed by Loof & Coomans (1970).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 attached to the light microscope.After their examination and identification, a few specimens of Pungentus engadinensis, preserved in glycerine, were processed 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 JEOL JSM-5800 microscope.

Abbreviations 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 orifice of dorsal gland 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

Diagnosis
This species is distinguished by the length of its body (1.37-1.91 mm), the lip region being offset by a constriction and 13-15 μm wide, an odontostyle being 30-35 μm long and 2.3-2.5 times lip region width or 1.7-2.1% of total body length, neck length of 352-437 μm, a pharyngeal expansion 190-221 μm long or 50% of total neck length, a monodelphic-opisthodelphic female genital system, with the anterior genital branch reduced to a very short prevulval sac up to 16 μm long or up to 0.4 times the body diameter, a uterus being a simple tube 101-129 μm long or 2.3-2.8 times the body diameter, V = 39-47, and the female tail being rounded and somewhat clavate (22-31 μm, c = 41-75, c' = 0.7-1.0).

Material examined
Nine females from two locations, in various states of preservation.

Description
Female Slender to very slender nematodes of medium size, 1.37-1.83mm long.Habitus after fixation ventrally curved, especially in posterior body region, G-shaped.Body cylindrical, tapering towards the anterior end.Cuticle two-layered, 2.0-3.0 μm thick in anterior region, 2.0-3.0 μm at mid-body and 4.0-6.0μm on dorsal side of tail; its outer layer with very fine transverse striation and thinner than inner one.Lateral chord 9-11 µm wide or occupying 21-28% of mid-body diameter, very granular but lacking any differentiation.Ventral body pores perceptible along the entire body.Lip region offset by constriction, 2.2-2.4 times as wide as high and ca.one-third (30-34%) of body diameter at neck base.Lips moderately angular and separate; labial and cephalic papillae distinct, but not especially protruding above cephalic contour.Amphid fovea cup-shaped, opening at level of cephalic constriction, occupying 6.5-8.0 μm or one-half to three-fifths of lip region diameter.Cheilostome almost cylindrical, its walls distinctly thick and sclerotised at the anterior half; perioral refractive dots very conspicuous.Odontostyle 14-18 times as long as wide, 2.3-2.5 times longer than lip region diameter or 1.7-2.1% of total body length; aperture 4.0-5.5 μm long or 12-17% of its length.Odontophore rod-like, almost equal (0.9-1.1 times) to odontostyle.Guiding ring double, located at 20-22 μm or 1.5-1.6 times the lip region diameter from anterior end.Pharynx enlarging very gradually; basal expansion 9.5-12.1 times as long as wide and 5.3-5.4 times the corresponding body diameter, occupying one-half (50%) of total neck length.Pharyngeal gland nuclei located as follows: DO = 52-53, DN = 54-55, S 1 N 1 = 73, S 1 N 2 = 74, S 2 N = 83-88.Nerve ring at 144-156 μm from anterior end or 36-38% of total neck length.Cardia rounded conoid, 10-11 x 10-11 μm.Genital system monodelphic-opisthodelphic; anterior branch reduced to a short to vestigial, uterine, prevulval sac up to 16 μm long or up to 0.4 body diameters; posterior branch well developed, 180-305 μm long.Ovary 44-218 μm long, often reaching and surpassing the sphincter level; oocytes first in two or more rows, then in one row.Oviduct 77-124 μm long, or 1.9-2.4times the body diameter, consisting of a slender portion with prismatic cells and a moderately developed pars dilatata.Sphincter distinct between oviduct and uterus.Uterus a simple tube-like structure, 101-129 µm long or 2.3-2.8 times the body diameter.Sperm cells absent within the genital tract.Uterine eggs 96-108 x 36-40 μm.A distinct dorsal cell mass or coelomocyte (see Fig. 2D) is present at level of oviduct or uterus in two of the specimens examined.Vagina extending inwards 20-25 μm or about one-half (43-54%) of body diameter; pars proximalis almost as long as wide, 13-18 x 12-16 μm, with convergent walls and enveloped by weak circular musculature; pars refringens with two weakly sclerotized pieces; pars distalis 5.5-7.0 μm long.Vulva a pre-equatorial, short transverse slit, which is preceded by a depression of body surface.Prerectum 2.5-2.6 anal body diameters long.Rectum 30-31 μm long, almost equal (0.9-1.0 times) to anal body diameter.Tail short and rounded, more or less clavate.Caudal pores two pairs, one subdorsal, another sublateral, at the middle of tail.

Male
Unknown.

Remarks
Although new data are provided and the ranges of many morphometric features are extended, the present description agrees very well with the original one.In addition to the type locality in India, P. clavatus has been reported from Italy (Vinciguerra & Giannetto 1987), Poland (Winiszewska-Slipinska 1987a) and Spain (Hernández et al. 1988).Winiszewska-Slipinska (op.cit.) also mentioned its occurrence in Canada.No significant interpopulational variation has been reported in this species.The most relevant and characteristic feature of the species is the clavate shape of its caudal region.Pungentus clavatus might be a widely distributed species in the Holarctic.(Altherr, 1950) Figs 1D-H, 3-4

Material examined
More than one hundred females and one male from more than 20 locations, in various states of conservation.

Description
General features of adults Slender nematodes of small to medium size, 0.77-1.21mm long.Habitus after fixation curved ventrad, C-shaped in females, G-shaped in male.Body cylindrical, tapering towards the anterior end.Cuticle three-layered, especially distinguishable at caudal region, 1.5-2.5 μm thick in anterior region and at mid-body and 2.5-4.0 μm on dorsal side of tail; outer layer with very fine transverse striation.Lateral chord 5.5-6.5 µm wide or occupying 19-24 % of mid-body diameter, very granular but lacking any differentiation.Ventral body pores perceptible along the entire body.Lip region offset by constriction, 2.4-3.2 times as wide as high and ca.one-third (31-38%) of body diameter at neck base.Under SEM, lips mostly amalgamated and somewhat angular, lacking any distinguishable striation; labial and cephalic papillae very distinct and visibly protruding, the inner labial papillae situated at the top of low but perceptible lobes; perioral area with (apparently) four small elevations (liplets) surrounding the short dorsoventral, slit-like oral opening.Amphid fovea cup-shaped, opening at level of cephalic constriction, occupying 5.0-5.5 μm or about three-fifths of lip region diameter.Cheilostome almost cylindrical, but a little wider at its posterior half and with its walls distinctly thicker at the anterior half; circum-oral refractive dots very conspicuous.Odontostyle 11-14 times as long as wide, 1.8-1.9times longer than lip region diameter or 1.5-2.2% of total body length; aperture 2.5-3.0 μm long or less than one-sixth its length.Odontophore rod-like, but with small thickenings at its base, nearly as long (0.9-1.1 times) as odontostyle.Guiding ring double, located at 10-11 μm or 1.1-1.3times the lip region diameter from anterior end.Pharynx enlarging very gradually; basal expansion 5.5-7.4 times as long as wide and 3.1-3.9times the corresponding body diameter, occupying about two-fifths (36-41%) of total neck length.Pharyngeal gland nuclei obscure in the specimens examined.Nerve ring at 80-91 μm from anterior end or 32-38% of total neck length.Cardia rounded conoid, 5.0-6.5 x 7-10 μm.

Female
Genital system monodelphic-opisthodelphic: anterior branch absent and posterior one moderately developed, 76-125 μm long.Ovary 53-110 μm long, often reaching and surpassing the sphincter level; oocytes first in two or more rows, then in one row.Oviduct 39-63 μm long, or 1.5-2.4times the body diameter, consisting of a slender portion with prismatic cells and a moderately developed pars dilatata.Sphincter distinct between oviduct and uterus.Uterus a simple tube-like structure, 39-44 µm long or 1.4-1.8times the body diameter.Uterine egg 82-92 x 21-26 μm, 2.5-3.5 times as long as the corresponding body diameter.Vagina extending inwards 12-17 μm or one-half to three-fifths (50-60%) of body diameter; pars proximalis 7-9 x 6-12 μm, with parallel or distally convergent walls and enveloped by weak circular musculature; pars refringens with two small, rectangular or triangular pieces measuring 1 x 3 μm and with a combined width of 5-6 μm; and pars distalis 3-4 μm long.Vulva a nearly equatorial, about 5 μm long, transverse slit.Prerectum 2.3-4.1, rectum 0.8-1.2anal body diameters long.Tail short and rounded, visibly more convex dorsally; the cuticle at its level consisting of a thick inner layer, a distinct intermediate layer and thinner outer layer, occasionally bearing blister-like structures (saccate bodies).Two pairs of causal pores, one subdorsal, another sublateral, at the middle of the tail.

Male
Very rare, only one specimen found among several dozens of females.Genital system diorchic, with opposite testes.In addition to the ad-cloacal pair, situated at 6.5 µm from cloacal aperture, there is a series of five widely spaced, 10-20 µm apart, ventromedian supplements, the most posterior of which is located 9.5 µm from the ad-cloacal pair, within the range of spicules.Spicules dorylaimoid, about 5.0 times as long as wide and 1.2 times the body diameter.Lateral guiding pieces obscure.Caudal region similar to that of the female.

Remarks
The above description of Iberian populations perfectly fits the available information about this species, especially the contributions by Andrássy (1962) and Coomans & Geraert (1962), who provided detailed data and illustrations.Nevertheless, some relevant features such as the three-layered nature of the cuticle, the morphology of the lip region and the structure of the female genital system are here described for the first time.Besides, the range of some measurements (body length, tail length, etc.) and morphometrics (vulva position) are in general extended.

Material examined
Five females from one location, in various states of preservation.

Description Female
Moderately slender to slender nematodes of medium size, 1.15-1.56mm long.Habitus after fixation ventrally curved, especially in the posterior body region, C-or G-shaped.Body cylindrical, tapering towards the anterior end.Cuticle three-layered, especially perceptible at the tail, 1.5-2.5 μm thick in anterior region, 2.0-2.5 μm at mid-body and 4.0-5.0μm on dorsal side of tail; outer layer with very fine transverse striation, intermediate layer thicker than the outer and inner layers.Lateral chord 5.5-7.0 µm wide or occupying 12-19% of mid-body diameter, very granular, but lacking any differentiation.Lateral pores comparatively coarse, in two rows at the margins of lateral chord; ventral pores well perceptible along entire body (Fig. 1K), apparently connecting with sub-epidermal gland bodies.Lip region offset by constriction, 2.4-2.5 times as wide as high and ca.one-third (28-35%) of body diameter at neck base.Lips moderately angular and separate; labial and cephalic papillae distinct, but not especially protruding above cephalic contour.Amphid fovea cup-shaped, opening at level of cephalic constriction, occupying 6.5 μm or about one-half (52%) of lip region diameter.Cheilostome almost cylindrical, its walls distinctly thick and sclerotized at the anterior half; perioral refractive dots or platelets very prominent.Odontostyle 10.0-12.5 times as long as wide, 1.8-2.3times longer than lip region diameter or 1.5-2.1% of total body length; aperture 3.0-5.0μm long or 14-19% of its length.Odontophore rodlike, almost equal (0.9-1.1 times) to odontostyle.Guiding ring double, located at 15-16 μm or 1.2-1.5 times the lip region diameter from anterior end.Pharynx enlarging gradually; basal expansion 5.7-7.1 times as long as wide and 2.9-3.7 times the corresponding body diameter long, occupying about twofifths (39-43%) of total neck length.Pharyngeal gland nuclei located as follows: DO = 52, DN = 56-61, S 1 N 1 = 77, S 1 N 2 = 79, S 2 N = 87-90.Nerve ring at 120-128 μm from anterior end or 38% of total neck length.Cardia rounded conoid, 7-9 x 10-14 μm.Genital system didelphic-amphidelphic, with both branches well and equally developed, the anterior 169-182, the posterior 144-210 μm long.Ovaries 43-162 μm long; oocytes first in two or more rows, then in one row.Oviduct 82-118 μm long, or 2.2-2.9 times the body diameter, consisting of a slender portion with prismatic cells and a moderately developed pars dilatata.Oviduct-uterus junction marked by a distinct sphincter.Uterus 56-68 µm long or 1.4-1.6 times the body diameter, apparently tripartite, i.e., consisting of a proximal section with thick walls and distinct lumen, an intermediate narrowing, and a spheroid pars dilatata uteri.Vagina extending inwards 18-22 μm or about one-half (44-59%) of body diameter; pars proximalis almost as as wide, 13-18 x 12-16 μm, with convergent walls and enveloped by weak circular musculature; pars refringens not very distinct in the females examined, but two pieces might be present; pars distalis 5.5-7.0 μm long.Vulva a pre-equatorial, short transverse slit, which is preceded by a depression of body surface.Prerectum 2.9-4.2,rectum 0.9-1.1 anal body diameters long.Tail short and rounded conoid, somewhat more straight ventrally.Caudal pores two pairs, subdorsal, at the anterior half of tail.

Male
Unknown.

Distribution
This species was collected from one location in the southern Iberian Peninsula: the province of Granada, Sierra Nevada National Park, near road to Veleta summit (37°7' N -3°32' W), in association with Mediterranean brushwood Retama sphaerocarpa (Linnaeus, 1753) Boissier 1840 as dominant species.

Remarks
The specimens from the Iberian population of this species are nearly identical to those of the types as described by Altherr (1950a) as well as to the type material of P. alpinus Vinciguerra & Giannetto, 1984, the latter regarded as a junior synonym by Ahmad et al. (2000).Some morphological features, for instance the nature of the cuticle and the female genital system, are more accurately described here.

Material examined
Twenty females from two locations, in various states of preservation.

Male
Not found.

Distribution
This species has been collected from two locations in the southern Iberian Peninsula: the province of Jaén, Cazorla, Segura y Las Villas Natural Park, near the road to the Segura river source (38°17' N -2°39' W), in association with pine forest; and the province of Granada, Sierra Nevada National Park, near the road from Capileira to Veleta (36°57' N -3°21' W), in a pine forest and among herbaceous plants where the dominant species was Asphodelus albus Miller, 1768.

Remarks
The present description fits those available from the literature.It agrees especially well with the redescription of the species by Coomans & Geraert (1962).These authors regarded P. thornei Goodey, 1943 as a junior synonym of P. silvestris, an action that seems to be totally justified and herein accepted.