Infection rate/status

Type host: Phrynocephalus axillaris Blanford, 1875 (Lacertilia: Agamidae)

Type locality: Alehui Town, Toksun County, Turpan City, China

Site of infection: Stomach and intestines

Prevalence: 18.89% (17/90)

Range of infections: 1–25 parasites per analyzed hosts

Mean intensity: 8.41 ± 1.82 parasites per analyzed hosts

Morphological characteristics

Microscopy findings (light and SEM) indicated that the nematode specimens in this study were consistent with P. phrynocephali in terms of their key identifying features. These features included the head structure, positions of the nerve ring, excretory pores, vulva, tail morphology of males, and uterine branching patterns of females. Given these similarities, they were confirmed to belong to the same species. The following is a detailed morphological description:

General: The body was white and linear, thin at both ends, gradually thickening toward the center, with a thick cuticle featuring fine transverse stripes (Fig. 2A, F). The anterior cuticle was dilated, forming a ring-like head collar (Fig. 2B, G). The head had two symmetrical pseudolabials (Figs. 3A–D, 4A, 5A) and two pairs of cephalic papillae arranged symmetrically on the pseudolabials (Figs. 3D, 4A, 5A), with a rounded raised small amphid between each pair of cephalic papillae (Figs. 4A, 5A). The inner margin of the pseudolabials had a triangular conical tooth in the middle, with a divergent denticle within the conical tooth (Figs. 3B, 4A, 5A) and a variable number of closely spaced denticles on each side of the base of the conical tooth, 8–14 in males (Fig. 4A) and 7–9 in females (Fig. 5A). The esophagus was divided into a short anterior muscular portion and a long posterior glandular portion (Fig. 3A, C). The nerve ring encircled the posterior muscular esophagus (Fig. 3A, C), and the cervical papillae was located slightly posterior to the deirids (Fig. 3A, 5B). Sexual dimorphism was evident, with females being larger than males.

Males (based on six samples): body length 5.58–9.95 mm (7.65 mm), maximum width 0.37– 0.45 mm (0.40 mm), esophagus length 1.54–1.85 mm (1.70 mm), 18.16%–28.23% (22.03%) of body length; muscular esophagus length 0.20–0.26 mm (0.24 mm); glandular esophagus 1.23–1.62 mm (1.42 mm) long; ratio of length of muscular to glandular esophagus 13.76%–21.71% (17.33%); nerve ring, deirids and excretory pore 0.18–0.24 mm (0.22 mm), 0.37–0.53 mm (0.46 mm), and 0.37–0.65 mm (0.51 mm) from cephalic extremity, respectively. Tail length 0.48–0.66 mm (0.56 mm). Tail with two unequal spicules, elongate whip-like with pointed, left spicule 0.51 mm (0.48–0.53 mm) long, right spicule 0.21 mm (0.19–0.23 mm) long (Figs. 2J, 3H, 4D). Male body with curled caudal end, cuticle fused ventrally to form well-developed caudal bursa (Fig. 2I), with 21 caudal papillae (Figs. 2H, 3E, 4B), including four pairs of pedunculate papillae (Fig. 2H), six pairs of sessile papillae, and one large unpaired papilla (Fig. 3E). Of the four pairs of pedunculate papillae, one pair was located anterior to the cloaca, one pair was located on either side of the horizontal line of the center of the cloaca, two pairs were located posteriorly to the cloaca; and a large unpaired papilla was located at the anterior margin of the cloaca (Fig. 4E). Of the six pairs of sessile papillae, one pair was located on either side of the central horizontal line of the cloaca, one pair was located on the posterior margin of the cloaca, and four pairs are located posterior to the cloaca; one pair was located close to the cloaca, and two pairs of small unpaired papillae were located posteriorly, and the last pair was small and located close to the caudal tip (Figs. 3E, 4B).

Females (based on 10 samples): body length 6.56–12.57 mm (9.05 mm), maximum width 0.44–0.61 mm (0.52 mm), esophagus length 1.55–2.29 mm (1.95 mm), 17.26%–25.48% (21.95%) of the body length; muscular esophagus length 0.18–0.30 mm (0.24 mm); glandular esophagus length 1.46–2.06 mm (1.71 mm); the ratio of the lengths of the muscular esophagus and the glandular esophagus was 9.69%–18.49% (14.27%); nerve ring and deirids 0.16–0.27 mm (0.21 mm), 0.46–0.55 mm (0.50 mm) from cephalic extremity, respectively. The vulva was located in the anterior part of the body, 1.25–2.67 mm (2.18 mm) from the end of the head, accounting for 17.19%– 30.22% (21.85%) of the body length (Figs. 2C, 3F, 5C). The vagina was long and well-developed, and the uterus was divided into six branches. The tail of the female worm was in the form of a small mucronate conical shape with caudal phasmids at the base of the tail tip (Figs. 2D, 3G, 5D), and the distance between the anus and the tail end was 0.29–0.41 mm (0.35 mm). The eggs were in the form of an elongated ellipsoid shape (Figs. 2E, 3I, 5E), with thick shells and smooth surfaces, and were internally embryonic smooth, 30–38 × 46–64 μm in length and width, and internally embryonated.

Comparative morphological analysis

Tao (1985) described P. phrynocephali type specimens collected from the stomach and coelomic cavity of P. axillaris in Yecheng County, Tarim Basin, Xinjiang Uygur Autonomous Region. However, the morphological characterization diverges from our findings. Notably, the length of the male copulatory spicule (left/right: 0.32–0.53/ 0.18–0.23 mm) differs substantially from Tao’s measurements (left/right: 0.14–0.24/ 1.714 –2.355 mm), potentially attributable to specimen preservation artifacts, technical limitations (e.g., measurement errors from low-resolution light microscopy), and intraspecific variation influenced by host physiology. Morphological discrepancies extended to caudal papillary architecture. Tao identified two pairs of precloacal and two pairs of postcloacal pedunculated papillae, with 13 sessile papillae distributed as: three isolated precloacal papillae forming an inverted “pin” configuration, four postcloacal papillae in two adjacent rows, and three paired caudal papillae. Conversely, our specimens exhibited one pair of precloacal papillae, one pair flanking the cloacal midline, and two postcloacal pairs, with sessile papillae comprising one unpaired precloacal papilla, one cloacal-level pair, two postcloacal pairs, and three caudal pairs. Additional variations included pseudolabial dentition: Tao reported 11 apical tooth-associated denticles in males and 5–9 in females, versus our counts of 8–14 (males) and 7–9 (females). Vulval positioning also differed, with Tao’s specimens showing anterior placement at 21.32%–23.99% body length versus 17.19%–30.22% (mean 21.85%) in our study.

The genus Physalopteriata was established by Sobolev and subsequently reassigned to Pentadentoptera by Shakhnazarova (1949). P. phrynocephali exhibits marked morphological distinctions from both the type species P. schulzi and other congeners within Pentadentoptera. Body size: P. phrynocephali is notably smaller than the others. Apical and small tooth distribution: P. phrynocephali has only one large apical tooth, whereas P. schulzi, P. mustelae, and P. citelli have three apical teeth. Beneath the apical tooth of P. phrynocephali is a small bifurcated tooth, and the outer edge of its mouth is lined with a relatively large number of small teeth (5–11). Conversely, P. schulzi, P. mustelae, and P. citelli have either two or no teeth along the outer edge of their mouth. Uterine branches: The uterus of P. phrynocephali has six branches, whereas the uteri of P. schulzi, P. mustelae, and P. citelli have two branches. Based on these significant morphological discrepancies, we propose that P. phrynocephali should not be classified within Pentadentoptera.

In 1920, Travassos separated Abbreviata based on the number, pattern, and initial position of the uterus. Subsequent research efforts have summarized the following key morphological characteristics of Abbreviata: the mouth is encircled by two symmetrically arranged pseudolabias. Each pseudolabium is equipped with one lateral apical tooth and one medial bifurcated tooth. In addition, there are two pairs of labial papillae and one pair of amphids. The entire perimeter of the pseudolabia is fringed with small teeth. The male tail is characterized by caudal alae. It typically has four pairs of stalked papillae, with two pairs located anterior and two pairs posterior to the cloaca. The number of sessile papillae is variable. Typically, there are three pairs anterior to the cloaca, two pairs surrounding the cloaca, and three, four, or five pairs on the ventral side of the tail. The copulatory spicules generally have unequal lengths. The female vulva is situated within the anterior third of the body. The uterus can have two, four, or more than four (multiple) branches. The eggs are typically oval, smooth in texture, and have a thick shell (Morgan 1945; Jones 1983; Jones 1986; Jones 2013). These features are consistent with the morphological descriptions of the nematode species investigated in this study. Therefore, the nematode specimens in this study were identified as belonging to Abbreviata.

Comparison revealed pronounced morphological disparities between the nematode specimens in this study and those of previously reported species in Abbreviata. Specifically, the male tails of the specimens in this study possessed four pairs of stalked papillae and 13 sessile papillae, whereas the female uterus had six branches. Conversely, the male tail of A. adonisi Sulahian, 1968 has three pairs of stalked papillae, A. anomala Jones, 1986 and A. baltazardi Chabaud, 1953 have five pairs, and A. hastaspicula Jones, 1979 has seven pairs. Regarding the number of sessile papillae on the male tail, A. levicauda Jones, 1983, A. bancrofti Irwin-Smith, A. hastaspicula, A. perenticola Mus, 1985, A. tumidocapitis Jones, 1983 and A. glebopalmae Jones, 1988 have 7; A. adonisi has 8, A. borneensis Schad, 1959 and A. baltazardi have 11, and A. achari Mirza has 12. In terms of female uterine branches, the uterus of A. pilbarensis has one branch, whereas the uteri of the other Abbreviata nematodes have four branches. Thus, considering these species identification traits and incorporating the comparative morphological data in Table 2, the nematode specimens in this study were identified as belonging to Abbreviata, however, were distinct from previously reported species.

......continued on the next page BL body length, MW maximum width, ML length of muscular esophagus, GL length of glandular esophagus, NC distance of nerve ring to cephalic end, EPC distance of excretory pore to cephalic end, DC distance of deirids to cephalic end, SL length of left spicule, SR length of right spicule, TL length of tail, ES size of eggs, PP number of pedunculate papillae, PSP number and arrangement of caudal sessile papillae, and UB number of uterine branches. * This species was included for comparison with Abbreviata spp. Please refer to the main text for the proposed taxonomic revisions.

BL body length, MW maximum width, ML length of muscular esophagus, GL length of glandular esophagus, NC distance of nerve ring to cephalic end, EPC distance of excretory pore to cephalic end, DC distance of deirids to cephalic end, SL length of left spicule, SR length of right spicule, TL length of tail, ES size of eggs, PP number of pedunculate papillae, PSP number and arrangement of caudal sessile papillae, and UB number of uterine branches.

In summary, the key morphological characteristics of P. phrynocephali closely aligned with the identification criteria of Abbreviata, strongly indicating its classification within this genus. However, a comparison with the currently documented Abbreviata nematodes revealed marked disparities in several crucial features used for identification. These include the length and morphological traits of the male spicules, quantity and distribution patterns of the caudal papillae, location of the female vulva, and number and structural characteristics of the uterine branches. Given these distinct differences, this study proposes rectifying the taxonomic status of Pentadentoptera phrynocephali and reclassifying it as Abbreviata (Spirurida: Physalopteridae).

Phylogenetic analysis of Abbreviata phrynocephali comb. nov.

The mitochondrial COI sequences of A. phrynocephali comb. nov. (400–421 bp) exhibited 0% intraspecific nucleotide divergence, while showing substantial genetic differentiation from congeneric species in GenBank: A. caucasica (accession number MT231307.1) (53.55%, 217/404) and A. kazachstanica (accession number MK57875 2.1) (52.10%, 210/403). BI phylogenetic analysis revealed that A. phrynocephali clustered with A. kazachstanica and A. caucasica within a monophyletic clade (posterior probability>0.95), demonstrating significant branch-length divergence from congeners (Fig. 6). This clade was phylogenetically distinct from outgroup genera (Physaloptera and Spauligodon), with no intergeneric sequence admixture observed. The combined evidence of pronounced nucleotide divergence and robust nodal support conclusively validated both the classification of A. phrynocephali within Abbreviata and its genetic distinctiveness from related taxa.