Tripylella intermedia (Bütschli, 1873) Brzeski & Winiszewska-Ślipinska, 1993

(Figs 4‒6)

Measurements. See Table 1.

Description. Female. (n = 7). Body ventrally arcuate after heat relaxation (Figs 4 A; 5A). Cuticle annulated, and lacks somatic setae and cervical setae on the body. Labial region rounded, 17.5 ± 1.0 (16–19) Μm wide, slightly narrower than the rest region. SEM and LM micrographs show (Figs 5 F; 6A-C): three triangular lips, inner labial papillae short and conical, outer labial and cephalic setae in a single whorl; six longer setae (outer labial) 2.9 ± 0.2 (2.5–3.0) Μm long, 16–19% of head diameter long, more or less arcuate and directed anteriorly, four shorter setae (cephalic setae) 2.1 ± 0.2 (2.0–2.5) Μm, 11–13% of head diameter long, thinner than the long cephalic setae, more or less arcuate. Stoma with two chambers; dorsal tooth wedge-shaped, triangular and lying in posterior buccal chamber, one large subventral and a small subventral tooth, lying in posterior and anterior buccal chambers, respectively (Figs 4 C,D; 5C). Amphidial aperture oval-shaped, 9–12 Μm from anterior end. Pharynx cylindrical, strongly muscular. Cardiac glands large, composed of six fused cells (Fig. 5 F). Coelomocytes not seen. Female genital system amphidelphic, gonad lying ventro-lateral to intestine, 240.9 ± 35.8 (185–279) Μm long. Distance between posterior end of pharynx and vulva 2.2–2.4 times longer than pharynx. Vulval lips not sclerotized, vagina short, less than one third of body diameter, S-shaped and directed towards anterior (Fig. 5 I). Vulva–anus distance about 2.3–2.6 tail length. Rectum about as long as anal body diameter (Fig. 5 K). Tail 121–155 Μm long, ventrally bent, anterior half broad and gradually tapered, then suddenly narrowed and posterior half tapered to a narrow cylindrical part (Figs 5 J; 6F). Three caudal glands, spinneret small.

Male. Not found.

Habitat and locality. The studied specimens were extracted from mosses growing on alder trees in Guilan province, northern Iran (GPS coordinates: 37°40ʹ 06.0" N, 49° 00' 24.6" E). All specimens were collected by the first author during May-June 2013.

Remarks. Morphometrics and general morphology of our examined specimens agree well in all aspects with the description of Tripylella intermedia, expect for the location of the dorsal and subventral teeth in the stomal chambers; the dorsal tooth in the Iranian specimens is located in the posterior chamber but in the original description was located in the anterior chamber, and subventral teeth in the Iranian specimens lie in the posterior and anterior buccal chambers versus the posterior chamber in the original description. However, despite these differences, the present specimens are identified as Tripylella intermedia .

On tail shape, Tripylella intermedia is morphologically close to T. iucunda and T. subintermedia, but differs from the former species by longer body (881‒1067 vs 680‒750 Μm), form of cuticle (annulated vs smooth) and shape of tail (thicker in more than half vs in anterior third its length), and separates from the latter species by having a wider labial region (16‒19 vs 15‒16 Μm) and position of subventral teeth (posterior and anterior to dorsal tooth vs anterior to dorsal tooth). Also, Tripylella intermedia differs from T. maiuscula and T minuscula by tail shape (tail suddenly tapering near of its middle vs slowly tapering), length of cephalic setae (very short, about one tenth of labial width vs long, less than one third of labial width) and form of cuticle (annulated vs smooth). Other characters that differentiate the rest of Tripylella species from T. intermedia are listed in Table 3.

Tripylella intermedia was first described and illustrated by Bütschli (1873) from Germany and was reported as Tripyla intermedia . Brzeski (1964) erected a new genus Paratripyla, and moved two species Tripyla minuta and T. intermedia to the new genus. However, Brzeski & Winiszewska-Ślipinska (1993) based on the arrangement of cephalic seta on head, disregarded the genus Paratripyla in the family of Tripylidae, and moved Paratripyla minuta to Tripyla minuta (six and four cephalic seta in two whorls) and Paratripyla intermedia to new genus of Tripylella ( T. intermedia) (six and four cephalic seta in single whorl). This is the first record of Tripylella in Iran.

Molecular characterization and phylogenetic relationships. SSU molecular phylogenetic studies (Fig. 7), using Dorylaimus stagnalis as an outgroup, suggested that i) the orders Triplonchida and Enoplida are in two separate clades with a posterior probability (pp) of 100% support; ii) the genera Trischistoma, Tripylina and Trefusia form a monophyletic group with 100% pp support; iii) the genus Tripylella shares a more recent common ancestor with Tobrilus, Tripyla, Prismatolaimus, and two trichodorid genera ( Trichodorus and Paratrichodorus); i v) Tripylella is paraphyletic with the genus Tobrilus; v) T. abharensis n. sp. (KM658325) is in a 100%-supported monophyletic clade with other species of Trischistoma (AY284735, AY284736, JF480409, GQ503076, GQ503079, GQ503075, EU359038), and vi) T. intermedia (KM658324) is in a monophyletic clade with other species of Tripylella (AY284737, GQ433068, GQ433067, KP 010362 and FJ 040488 with 100% pp support).

TABLE 3. Comparative morphometrics of Tripylella species. All measurements are in Μm and in the range form.

Based on SSU molecular phylogenetic studies (Fig. 5), interspecific sequence variations of T. abharensis n. sp., compared with other Trischistoma species were: 4.2% (39 in 907 bp) for Trischistoma sp. 2 (AY284736); 6.5% (57 in 869 bp) for T. monohystera (AJ966509); 3.5% (31 in 869 bp) for T. waiotama 1 (GQ503076); 3.5% (31 in 869 bp) for T. waiotama 2 (GQ503075); 4.4% (40 in 904 bp, for Trischistoma sp. 1 (AY284735), 3.2 % (28 in 869 bp) for Trischistoma sp. CH 09c (GQ503079); and 51.4% (350 in 680bp) for T. veracruzensis (EU359038).

Also, based on SSU molecular phylogenetic studies with some Tripylella species, interspecific sequence variations of Tripylella intermedia were: Tripylella sp. (AY284737) by 1.2% (11 in 869 bp); T. subintermedia (KP 010362) by 3% (25 in 808 bp); Tripylella sp. (FJ 040488) by 1.8% (16 in 858 bp). The 18S sequence of T. intermedia from Iran is not comparable to Tripylella sp. (GQ433068) and Tripylella sp. (GQ433067) because they are short and from different directions.

A BlastN search of partial sequences of LSU for Trischistoma abharensis n. sp. (KM658323) revealed the highest matches were with some Trischistoma species: from Trischistoma sp. 2 (GQ503053), by 132 nucleotides (82% similarity, 620/752 identities) and 35 gaps (4%, 35/752); from Trischistoma sp. 1 (GQ503052), by 134 nucleotides (82% similarity, 618/752 identities) and 35 gaps (4%, 35/752); from T. otaika (JN673805), by 144 nucleotides (81% similarity, 613/757 identities) and 44 gaps (5%, 44/757); from Trischistoma triregium (JN673804), by 150 nucleotides (80% similarity, 603/753 identities) and 35 gaps (4%, 35/753). Also a BlastN search of T. intermedia (KM658322) on the LSU revealed the highest match was with Tripylella sp. (GQ503051), by 106 nucleotides (86% similarity, 666/772 identities) and 21 gaps (2%, 21/772).

On the basis of SSU rRNA sequences, van Megen et al. (2009) does not support the positioning of Enoplida and Triplonchida in Clade 1 as proposed by Holterman et al. (2006). The trees of the former phylogenetic analysis showed a major split among them with Trischistoma and Tripylina residing among the Enoplida, but Tripyla and Tripylella positioned among the Triplonchida . This analysis is supported by similarities in the morphology of the labial region, amphids, digestive system and male copulatory apparatus between Trischistoma and well-known enoplids such as Trefusia . It is also in agreement with results of another SSU rRNA-based phylogenetic study, where Zhao (2011) suggested transferring Trischistoma and Tripylina from Triplonchida and placing them in the family Trischistomatidae within Enoplida .

Results of the present work provide additional support for the conclusions from recent molecular phylogenetic studies of the genus Trischistoma (Zhao & Buckley 2009; Cid del Prado Vera et al. 2010; Zhao 2011; Cid del Prado Vera et al. 2012; van Megen et al. 2009), and suggest that Trischistoma is not closely related to Tripylidae, but has a closer affinity to the members of Alaimidae and Trefusiidae within Enoplida . For Tripylella, the current analysis points to its close relations to other sequences of the genus and to Tripyla, the type genus of the family Tripylidae . It seems to support the presence of a muscular pouch around the male spicule as a main morphological characteristic in the order Triplonchida (De Ley & Blaxter 2002, 2004; Zullini 2006).