Descriptions of Pelodera scrofulata sp. nov. and Pelodera aligarhensis sp. nov. (Nematoda: Rhabditidae) with supplementary information on Pelodera teres (Schneider, 1866).

Pelodera scrofulata sp. nov. is characterized by cuticle with longitudinal lines and punctations; offset lip region with separate lips, wider in females, moderately developed cheilostom, presence of glandular bodies associated with uterus, tail conoid to spicate; males with spicules fused up to 60–68% from distal end; peloderan bursa with two precloacal papillae 9–12 μm apart and seven postcloacal pairs in the configuration of 1 + 1/4 + P + 2 + 1. Pelodera aligarhensis sp. nov. is characterized by sexual dimorphism in anterior body region; cuticle often with faint longitudinal lines; slightly offset lip region; tail cupola-shaped; males with spicules fused up to 20-25% from distal end; peloderan bursa; one precloacal pair at anterior level of cloaca; two precloacals shifted posteriad to the cloacal level; seven postcloacal pairs of genital papillae in 1/2 + (2 + P) + (3 + 1) configuration. Some new information is added to the description of Pelodera teres (Schneider, 1866) with special reference to labial structure. http://zoobank.org/urn:lsid:zoobank.org:pub:91E78F4F-08F2-45DE-B6A6-91F4E42011FF


Introduction
The genus Pelodera was raised by Schneider in 1866 with Pelodera strongyloides as the type species. Andrássy (1976) placed the genus within the subfamily Peloderinae. Earlier, Dougherty (1953) also gave Pelodera a generic rank and proposed within it two subgenera, Coarctadera and Cylindridera. Later, Sudhaus and Fitch (2001) considered Pelodera as a subgenus of Rhabditis under the subfamily Rhabditinae. However, Sudhaus in his later (2011) revision of Rhabditidae considered Pelodera as a genus and, following the cladistic approach, considered Pelodera as a clade including three major monophyletic species groups: the 'strongyloides', the 'coarctata' and the 'teres'. In the present paper, two new species of the genus, Pelodera scrofulata sp. nov. and Pelodera aligarhensis sp. nov. belonging respectively to strongyloides and coarctata groups, are described and illustrated, whereas Pelodera teres is redescribed.

Material and methods
Pelodera scrofulata sp. nov. and P. teres were obtained from fixed samples stored in 4% formaldehyde from previous surveys. Before fixation, nematodes were extracted from soil samples by Cobb's (1918) sieving, decanting and modified Baermann funnel technique. For light microscopy, fixed specimens were processed to anhydrous glycerin (Seinhorst 1959) and mounted on slides using the wax ring technique. The nematodes were measured with an ocular micrometer and illustrated using a drawing tube. Light microscopy photographs were taken with a Jenoptik digital camera 'ProgRes' attached to an Olympus BX-51 DIC microscope. For scanning electron microscopy, the specimens were fixed in 2% glutaraldehyde, post-fixed in 2% osmium tetroxide, dehydrated in an ethanol series and critical-point dried using CO 2 . The nematodes were mounted on a stub on double-sided adhesive tape and coated with 10 nm gold before observing at 10 kV under a scanning electron microscope model XL30 FEG. Pelodera aligarhensis sp. nov. was collected from a landfill area and was cultured on standard NGM plates seeded with Escherichia coli. To prepare the medium, 3 g NaCl, 17 g agar and 2.5 g peptone were mixed in 975 ml H 2 O and autoclaved for 50 min. The autoclaved mixture was cooled in a 55°C water bath for 15 min and 1 ml 1 M CaCl 2 , 1 ml 5 mg/ml cholesterol in ethanol, 1 ml 1 M MgSO 4 and 25 ml 1 M KPO 4 buffer were added. Finally the mixture was poured into Petri plates after swirling it well. Stock cultures were maintained from single gravid females. The nematodes were observed under an Olympus BX-51 DIC microscope and photographs were taken by a Jenoptik digital camera 'ProgRes' coupled to the microscope.

Cluster analysis
The morphological characters (Tables 1 and 2) were used to compare P. scrofulata sp. nov. with other species of the 'strongyloides' group and P. aligarhensis with other species of 'coarctata' group. All the characters were selected by experience as being taxonomically informative. The characters were ranked on the basis of the commonality principle. Character state '0' represented the most commonly occurring trait while a gradual increase in value represented relative rarity and presumed increasing deviation. Data matrices (Tables 3 and 4) were prepared, and analysed using Statistica-99 software including cluster analysis. The relationships were interpreted from the dendrograms obtained.

Remarks
Cluster analysis (Figure 4) based on 21 morphological characters (Tables 1 and 3) indicates a close affinity of P. cutanea with P. nidicolis. In fact, these two species have previously been identified as sibling species (Sudhaus and Schulte 1988).
Likewise, two other species of the group, P. strongyloides and P. orbitalis, showed a close relationship. These four species formed a more inclusive cluster from which P. termitis is excluded, at least at low distance values (< 0.6). In addition, P. scrofulata sp. nov. clearly stands out as the most morphologically divergent species considered in the cluster analysis.
The species of Pelodera constituting the 'strongyloides' group have largely been reported to be parasitic with third-stage juveniles (J3) having association with vertebrate or invertebrate hosts, namely chicken, wood mice, voles, horses and termites. The present species was collected from the muddy sample of a ditch. Unfortunately, juvenile specimens were not recorded from this sample and only the adults were preserved for morphological evaluation. The locality apparently indicates any association with termites, mice or voles to be unlikely. The new species is morphologically similar to P. strongyloides, P. termitis and P. punctata, but not convincingly enough to place it in any of the corresponding species. Table 6 gives a comparative account of the differences between P. scrofulata and other congeners of the strongyloides group. The species apparently resembles P. punctata with respect to the freshwater type habitat as well as the longitudinally striated and punctated body cuticle; however, a short conoid tail, and more slender spicules (including an angular dorsal profile), and widely spaced precloacal papillae are features that clearly differentiate the two species. With P. termitis, the species shows similarity with respect to lip morphology, female tail shape and spacing of precloacal papillae; however, the presence of longitudinal cuticular lines and punctations, as well as the shape and length of spicules and their trapezoid capitulum, the length of the gubernaculum and the continuous prerectum region are some of the prominent distinguishing features. Another interesting feature is their putative association with termites that were not found and were unlikely to be found in the ditch with mud and slurry around. Hence it is perhaps another sibling species in the group such as P. cutanea, P. nidicolis and P. orbitalis that have had its status confirmed through cross-breeding tests with P. strongyloides (Sudhaus and   ). It appears that the process of speciation in the group has led to different species adaptations for parasitism and switching over to a variety of hosts with very little change in morphology. This would have given rise to several cryptic sibling species. The present species seems to form one such example.

Description
Female. Body medium-sized, straight to slightly arcuate with tapering ends. Females are relatively obese with prominent and expanded lip region compared with their male counterparts. Cuticle very finely annulated, with very fine longitudinal lines and inconspicuous punctations. Lateral field with three ridges. Lip region expanded, slightly offset from adjoining body. Lips rounded, separate and distinct, outer labial sensilla slightly raised, inner labials slightly shifted outward. Amphid openings small and rounded, labial. Stoma well developed, fairly long, rhabditoid type 4.8-5 times longer than wide or 13.4-13.8% of pharyngeal length. Cheilostom cuticularized, appearing dot-like in lateral view; gymnostom not markedly conspicuous. Metastegostom strongly cuticularized, widened with each swelling bearing three setose denticles; telostegostom heavily cuticularized. Pharyngeal collar usually surrounding 50-55% of length of stoma as measured from base. Pharynx differentiated into a swollen corpus, slightly narrower isthmus and a muscular, ovoid (27-33 × 22-25 μm wide) basal bulb with well-developed grinder and a faintly two-chambered haustrulum. Nerve ring encircling anterior half of isthmus at 67.2-68.2% of the pharyngeal length. Secretory-excretory pore in close proximity with the nerve ring at 74.5-79.8% of pharyngeal length; cell bodies located at pharyngeal base 95-103% of pharyngeal length. Cardia (pharyngeal-intestinal valve) 5-9 μm long, often appearing as the extension of basal bulb. Intestine with wide lumen, often anteriorly dilated and distinguishable there as a sac. Posterior intestinal region with dilated lumen bounded by enlarged cells. Rectum 16-22 μm long, about three-quarters of anal body diameter, often with dilated lumen; rectal glands inconspicuous.
Reproductive system didelphic, amphidelphic, ovaries well-developed, paired, opposed and lateroventrally reflexed, often with distal end of posterior ovary extending further posterior to the vulva; anterior and posterior ovaries respectively on right and left lateral side of intestine, spermatheca and uterus not differentiated by sphincter. Intrauterine eggs about 42-48 × 30-38 μm with three or four eggs at different stages of embryonation within uterus. Vagina thick-walled, at right angle to longitudinal body axis. Vulva a transverse slit with protruded vulval lips. Vulva-anus distance 9.4-11.6 times that of tail length. Tail conical, often cupola-shaped with a small spike of one-half to one-third of tail length. Phasmid openings positioned at 50-55% of the broader conical part of the tail.
Male. Similar to female in general morphology except lip region structure, tail shape and greater curvature of the posterior region. Lip region in males narrow, continuous with adjoining body. Lips relatively smaller and amalgamated. Stoma very narrow in most individuals while relatively wider in a few; metacorpal swelling weak; basal bulb   relatively smaller than that of female. Secretory-excretory pore located at c.68-75% of the length of pharyngeal region with secretory-excretory gland cells visible near anterior region of isthmus. Cardia very narrow, often appearing as a postbulbar extension continuing into an equally narrow intestine. Testis single, dorsally reflexed, on right lateral side of intestine. Seminal vesicle separated from vas deferens by a constriction. Ejaculatory glands present; right lateral gland slightly anterior to left one. Tail conical, narrowing into a spike with fine terminus. Bursa well-developed peloderan, anteriorly closed, ovoid. Spicules fused up to 20-25% of length from distal end, provided with rounded capitula, narrow necks and slender shafts with velum terminating as a short spur. Gubernaculum slender, trough-shaped, about 40-45% of spicule length. Genital papillae comprising nine pairs in 1/2 + (2 + P) + (3 + 1) configuration. Two precloacal pairs shifted posteriorly: GP1 with base in precloacal region reaches the level of cloaca; GP2 and GP3 slightly posterior to GP1 opening posterior to cloacal level. GP4, GP5 in close proximity. Region surrounding phasmid openings relatively swollen relative to papillae, located between GP5 and GP6. GP6, GP7 and GP8 basally joined in close proximity to dorsally oriented GP9. Diagnosis and relationships Pelodera aligarhensis sp. nov. is characterized by gonochoristic individuals having cuticle with fine longitudinal striations and punctations; sexual dimorphism in anterior region with females having conspicuously dilated lips, large stoma; cupolashaped tail; males with stoma smaller than females, an elongated cardia and narrower anterior intestine; slender spicules fused up to 20-25% of length from distal end; anteriorly closed peloderan bursa; three precloacal papillae pairs with two other pairs shifted posterior to cloacal opening. The new species resembles Pelodera tretzeli (Sachs 1950) in most morphometric characteristics as well as by the presence of three pairs of precloacal papillae in males but differs in females by the smaller body (0.6-0.8 mm vs 1.1-1.3 mm) with presence (vs absence) of fine longitudinal lines and punctations; smaller b (3.9-4.6 vs 5.9-7.9) and relatively greater V (56.7-63.5 vs 56.2-57.7) values; intestine not overlapping (vs overlapping) the basal pharyngeal region; tail spike smaller (vs larger) than cupolashaped part of tail; males having relatively smaller (30-38 vs 35-47 μm) spicules and precloacal papillae positioned posterior (vs anterior) to cloacal opening in P.tretzeli apud Sachs (1950).
The new species resembles Pelodera par Andrássy, 1962 in most morphometric characteristics as well as by the presence of three pairs of precloacal papillae in males but differs in having (vs lacking) sexual dimorphism in lip region; presence (vs absence) of fine longitudinal lines and punctations on the cuticle; smaller b (3.9-4.6 vs 7.1) and relatively greater V (56.7-63.5 vs 59) values; presence of small-sized eggs (42-48 vs 58-64 μm); tail spike smaller (vs larger) than cupola-shaped part; males having smaller (30-38 vs 40-44 μm) spicules and two precloacal papillae pairs positioned posteriorly (vs anteriorly) in P. par apud Andrássy (1962).

Remarks
Sudhaus (2011) identified 10 species under the coarctata-group. The species of the group have been reported from dung, cow pats, moulds, horse droppings and in most cases, associated with beetles or mites. Pelodera aligarhensis sp. nov. was, however, reared in the laboratory on an E.coli culture at a temperature of 25 ± 2°C; the development was found to be normal without any dauer stage.
The species of the coarctata-group are characterized by obese females and relatively slender, smaller males. Most of them show sexual dimorphism in the lip region with lips more prominent, wide and offset in females and they have a wide stoma with heavily cuticularized metastegostom bearing conspicuous teeth compared with their male counterparts. The only species lacking sexual dimorphism in the group is stated to be P. par apud Andrássy (2006). The species P. tretzeli, P. voelki and P. kolbi in particular have males with an extremely reduced anterior region. In the present species males were observed to have a slightly reduced or extremely reduced labial region with amalgamated lips as well as a narrower stoma. Even the pharynx was found to be relatively cylindrical and the anterior intestinal region was considerably narrowed. On the contrary, the anterior end of the intestinal lumen was widened in the female similar to the bacterial pouch found in many entomopathogenic nematodes. The excretory cells in males were small and confined to the level of the middle of the isthmus in males while the females possessed relatively larger cells. Females of all the species of the coarctata group have a cupola-shaped tail with a terminal spike except in Pelodera cylindrica where the female tail is bluntly rounded. As a rule the males have an anteriorly closed peloderan bursa that appears sucker-shaped and often indented and lobed, however, variation has been observed in the number of precloacal genital papillae. Most of them possess two pairs of precloacals with the exception of P. tretzeli and P. par, which possess three precloacal pairs.
Cluster analysis (Figure 8) based on 20 morphological characters (Tables 2 and 4) indicates a close affinity of P. aligarhensis with P. tretzeli compared with other existing species of the group. Both species appears to form a more inclusive cluster with P. par and P. voelki. This larger cluster is related to two other species, Pelodera cystilarva and Pelodera serrata, and the resulting cluster is itself related to a cluster containing a sister relationship between P. cylindrica and P. isociensis which together are distantly related to P. kolbi. Pelodera coarctata maintains a separate identity and clearly stands out as the most morphologically divergent species showing an overall affinity with other species of the group. The species Pelodera operosa has not been included in the analysis because the species is represented by females only.
Some observations on Pelodera teres (Schneider, 1866) ( Figure 9) A few fixed specimens of P. teres described earlier by Hussain et al. (2006), were processed for scanning electron microscopy to observe labial details. Although the results were not particularly good, due to some debris adhering to the specimens, nevertheless some observations together with light microscope observations could be taken and the description summarized as follows.
The population of P. teres with 1:1 sex ratio, represented by ovoviviparous females with relatively wider lip region than males; low, apically compressed slightly offset lip region; heavily cuticularized inner labial margins; four to six fine bristles in each interlabial groove with the peripheral ones slightly longer than central ones; pharyngeal sleeve surrounding more than 50% of stoma; pharyngeal corpus moderately swollen, basal bulb with single-chambered haustrulum; intestinal cells compactly arranged at anterior and posterior extremities, intestinal lumen thick and refractive; reproductive system didelphic; large, conspicuous sphincter between spermatheca and uterus; males with dorsally reflexed testis; ejaculatory glands present; spicules massive with an indistinct neck; fused at distal end; bursa moderately developed, open peloderan with three pairs of precloacal genital papillae and six postcloacal pairs.
Pelodera has been considered a taxon with three monophyletic groups (Sudhaus and Fitch 2001;Sudhaus 2011). Some of the important features present among these groups include: sexual dimorphism in the lip region (most species though more pronounced in the coarctata group); buccal cavity relatively wide, expanding posteriorly with heavily cuticularized telostegostom in the coarctata group; pharyngeal sleeve lacking in strongyloides group but present in the other two groups; pharyngeal corpus usually swollen (few exceptions in the males of the species showing sexual dimorphism); female reproductive system usually with a sphincter between the oviduct and uterus, spermatheca conspicuous; lining of intestine lumen thickened intestinal cells most compactly arranged anteriad and posteriad, the latter forming  prerectum conspicuously seen in the strongyloides group; female tail conoid, cupolashaped to spicate; phasmid openings usually on the cupola part of tail or at 30-50% of tail length from anal opening in spicate tail; males with dorsally reflexed testis; ejaculatory glands often of unequal dimensions; spicules fused distally up to 70% of their length in strongyloides group and to lesser degrees in coarctata and teres groups respectively; bursa peloderan, anteriorly closed, sucker-shaped in coarctata group but anteriorly open in others; bursal velum smooth to striated or punctated; two pairs of precloacal genital papillae in all members of strongyloides group and in most species of the coarctata group with few exceptions whereas three pairs of precloacals present in members of teres group. Few members (P. teres and P. pseudoteres) in teres group are distinct from the rest in having markedly low, apically compressed lip region with heavily cuticularized inner labial margins and interlabial grooves bearing fine bristles.