Morphology of Cissophyllus leytensis Tubangui & Villaamil, 1933 (Figs. 1, 2, 3, 4, Table 2)
General. Medium-sized, whitish nematodes. Body cylindrical, maximum width at about region of middle body. Cuticle with fine transverse striations. Lateral alae absent. Oral aperture dorsoventrally elongate, surrounded by three small chitinized lips (Figs. 1a, c, 2a). Dorsal lip with one pair of large double papillae, one pair of small triangular cuticular projections (inner ridge armed with 3–5 acuminate denticles (lamellae), single quadrate cuticular plate and large trilobed tooth plate (Figs. 2a, 3b). Subventral lips each with single large double papillae, small papilla and amphid; inner ridge of each subventral lip armed with three clusters of acuminate denticles (lamellae) (smallest cluster with 6–9 denticles, largest cluster with about 80 denticles, medium one with 12–15 denticles) (Figs. 1b, c, 2a). Esophagus divided into short chitinized pharynx with three flabellate pharyngeal plates (Figs. 3a, b, 4a, b), cylindrical corpus, slightly inflated isthmus (Figs. 3a, 4a) (isthmus also nearly as wide as corpus in some specimens) and ovoid posterior bulb with valves (Figs. 3a, 4a). Nerve-ring situated at about 1/3 of total esophageal length. Position of excretory pore varied from anterior edge of isthmus to level of middle of esophageal bulb (Figs. 3a, 4a). Deirids not observed. Tail of both sexes conical, with blunt tip (Figs. 1e, 2b, c, 3e, f, i, j, 4d–f).
Male (based on 10 specimens). Body 14.0–18.0 (16.6) mm long, maximum width 976–1220 (1068). Esophagus 2.00–2.39 (2.13) mm in total length, representing 11.1–17.1 (13.0) % of body length; pharynx + corpus + isthmus 1.70–2.04 (1.83) mm long, size of bulb 250–350 (305) × 260–400 (357). Nerve ring 522–807 (672) and excretory pore 1.82–2.00 (1.94) mm from anterior extremity, respectively. Posterior end of body slightly curved ventrally. Precloacal sucker absent. Spicules alate, equal in length, 600–900 (749) long, distal end sub-pointed, representing 3.89–5.63 (4.55) % of body length (Figs. 3g, j, 4e, f). Gubernaculum present, triangular, 149–248 (206) long (Figs. 3h, 4f). Caudal papillae 10 pairs in total, distributed as: six pairs of precloacal papillae (last three pairs close to each other), one pair of paracloacal papillae and three pairs of postcloacal papillae (one pair ventrolateral, two pairs ventral) (Figs. 2b–g, 3i, j). Single median ventral papilla present (Figs. 2b, c, 3i). Tail 150–260 (228) long, with rounded tip (Figs. 2b, c, 3i, j, 4e, f). Phasmids present, between last two pairs postcloacal papillae (Figs. 2b, 3i, j).
Female (based on five specimens). Body 14.0–18.0 (17.0) mm long, maximum width 976–1293 (1112). Esophagus 2.15–2.49 (2.28) mm in total length, representing 12.1–15.3 (13.5) % of body length; pharynx + corpus + isthmus 1.78–2.07 (1.92) mm long, size of bulb 293–415 (361) × 341–463 (390). Nerve ring 604–894 (753) and excretory pore 1.70–1.96 (1.86) mm from anterior extremity, respectively. Vulva transverse slit, 9.40–12.3 (11.1) mm from anterior extremity, representing 63.0–68.2 (65.3) % of body length (Figs. 1d, 3c, 4c). Vagina muscular (Figs. 3c, 4c); egg oval, with smooth surface, 97–111 (105) × 53–63 (57) (n = 20) (Fig. 3d). Tail 250–350 (296) long, with rounded or more or less finger-like tip (Figs. 1e, 3e, f, 4d). Phasmids present (Figs. 1e, f, 3e, f).
Taxonomic summary
Host and locality
Philippine sailfin lizard H. pustulatus (Eschscholtz, 1829) (Reptilia: Squamata) in a zoo in Tangshan, Hebei Province, China.
Level of infection
Single lizard infected with 15 nematodes.
Voucher specimen deposition
Ten males (HBNU–N-2021R0013L), five females (HBNU–N-2021R0014L), College of Life Sciences, Hebei Normal University, Hebei Province, China.
Genetic characterization
Partial 18S region
Two 18S sequences of C. leytensis obtained herein were both 1749 base pairs (bp) in length, with no nucleotide divergence detected. In the Kathlaniidae, the 18S sequence data are available in GenBank for Megalobatrachonema terdentatum (MG594352–MG594364), Megalobatrachonema wangi (MW325957), Cruzia americana (U94371), Cruzia tentaculata (MN873564–MN873566, MN873570), Cruzia sp. (MT809125–MT809126), Falcaustra ararath (MT160412), Falcaustra araxiana (KM200715), Falcaustra catesbeianae (AB818380), Falcaustra sp. (MN727387, MN727389, MN727390) and Spectatus spectatus (KR139827). Pairwise comparison of 18S sequences of C. leytensis and these 10 kathlaniid species displayed 4.77% (F. araxiana) to 9.61% (C. tentaculata) nucleotide divergence.
Partial ITS (ITS1–5.8S-ITS2) region
Three ITS sequences of C. leytensis obtained herein were all 837 bp in length, with no nucleotide divergence detected. In the Kathlaniidae, the ITS sequence data are available in GenBank for M. terdentatum (MN444703–MN444704), Megalobatrachonema hainanensis (MH545567–MH545569), M. wangi (MN245657–MN245659), Falcaustra sinensis (MF061681), Falcaustra sp. (MN727388, MN727391, MN727392) and Cruzia sp. (MT809125). Pairwise comparison of ITS sequences of C. leytensis and these six kathlaniid species displayed 12.1% (Cruzia sp.) to 34.8% (F. sinensis) nucleotide divergence.
Partial 28S region
Two 28S sequence of C. leytensis obtained herein were both 725 bp in length, with no nucleotide divergence detected. In the Kathlaniidae, the 28S sequence data are available in GenBank for M. terdentatum (MN444705–MN444706), M. wangi (MN245660–MN245662), M. hainanensis (MH545569–MH545570), F. sinensis (MF094270), Falcaustra sp. (LC605539–LC605541) and C. americana (U94757). Pairwise comparison of 28S sequences of C. leytensis and these six kathlaniid species displayed 12.5% (C. americana) to 20.0% (M. hainanensis) nucleotide divergence.
Partial cox1 region
Two cox1 sequences of C. leytensis obtained herein were both 384 bp in length, with 0.52% of nucleotide divergence detected. In the Kathlaniidae, the cox1 sequence data are available in GenBank for M. terdentatum (MN444709–MN444710), M. wangi (MN245668–MN245670), F. sinensis (MF113223), Falcaustra sp. (MN729570–MN729572) and C. tentaculata (MN842776–MN842778). Pairwise comparison of cox1 sequences of C. leytensis and these five kathlaniid species displayed 12.3% (Falcaustra sp.) to 53.8% (C. tentaculata) nucleotide divergence.
Partial cox2 region
Two cox2 sequences of C. leytensis obtained herein were both 501 bp in length, with no nucleotide divergence detected. In the Kathlaniidae, the cox2 sequence data are available in GenBank for C. americana (AF179911) and F. sinensis (MF120240). Pairwise comparison of cox2 sequences of C. leytensis and these two kathlaniid species displayed 16.6% (F. sinensis) to 22.0% (C. americana) nucleotide divergence.
Partial 12S region
Two 12S sequences of C. leytensis obtained herein were both 469 bp in length, with no nucleotide divergence detected. In the Kathlaniidae, the 12S sequence data are available in GenBank for M. terdentatum (MN444707–MN444708), M. hainanensis (MN245666–MN245667), M. wangi (MN245663–MN245665) and F. sinensis (MF140337). Pairwise comparison of 12S sequences of C. leytensis and these four kathlaniid species displayed 24.7% (F. sinensis) to 28.6% (M. terdentatum) nucleotide divergence.
Phylogenetic analyses (Figs. 5, 6)
The phylogenetic results of ML and BI trees using 18S + 28S sequence data were more or less identical, with both showing the representatives of the superfamily Cosmocercoidea divided into four large clades (Fig. 5). The species of Cosmocerca + Aplectana + Cosmocercoides formed clade I, which represents the family Cosmocercidae. The species C. americana formed clade II, which represents the subfamily Cruzinae in the Kathlaniidae. The species of Falcaustra + Cissophyllus + Megalobatrachonema formed clade III, which represents the subfamily Kathlaniinae in the Kathlaniidae. The species of Orientatractis + Rondonia formed clade IV, which represents the family Atractidae. Cissophyllus and Falcaustra formed a sister group in the ML tree in clade III, but Cissophyllus clustered together with Falcaustra + Megalobatrachonema in the BI tree (Fig. 5). By contrast, the phylogenetic results of ML and BI trees using ITS sequence data showed the representatives of the superfamily Cosmocercoidea divided into three large clades, due to the lack of available ITS data for atractid species (Fig. 6). The genus Cruzia (clade II) is at the base of the ML and BI trees, and the genus Cissophyllus showed a closer relationship to Megalobatrachonema than Falcaustra with weak support (Fig. 6).