Dugesia cylindrica Chen & Dong sp. nov.

Figs 1, 4–8

Collection site and habitat.

On 4 December 2021, the specimens were collected from a small pool formed by a stream flowing down from the southern Taihang Mountains at an altitude of 346 m above sea level (a. s. l.). At the time of collection, the air temperature was 16 ° C and the water temperature was 13.7 ° C (Figs 1, 4 A, B). The water was clear and flowed slowly, and it was used as drinking water by local residents.

Material examined.

Holotype: • ZMHNU - LMG 3, Laomugou village (35°44'28"N, 114°4'34"E; alt. 350 m a. s. l.), Hebi City, Henan Province, China, 4 December 2021, coll. G-W Chen, Z-M Dong and co-workers, sagittal sections on 36 slides.

Paratypes: • ZMHNU - LMG 4 -6, ibid., sagittal sections on 41, 37, 40 slides, respectively; • RMNH.VER.22726.1, ibid., sagittal sections on 46 slides; • RMNH.VER.22726.2, ibid., sagittal sections on 45 slides; • ZMHNU - LMG 8, ibid., horizontal sections on 13 slides; • ZMHNU - LMG 7, 9, and 10, ibid., transverse sections on 24, 32 and 36 slides, respectively.

Other material: • ZMHNU - LZSXG 1, 5, 6, Shibanyan village (36°9'59"N, 113°42'51"E; alt. 1050 m a. s. l.), Linzhou City, Henan Province, sagittal sections on 12, 69, and 57 slides, respectively; • ZMHNU - LZSXG 2, 7, ibid., horizontal sections on 25 and 9 slides; • ZMHNU - LZSXG 4, 8, ibid., transverse sections on 29 and 28 slides, respectively. • RMNH.VER.22727.1, ibid., sagittal sections on 32 slides.

• ZMHNU - BJHR 2-4, Huairou village (40°35'37"N, 116°34'48"E; alt. 510 m a. s. l.), Beijing City, sagittal sections on 37, 59, and 18 slides, respectively. • RMNH.VER.22728.1, ibid., sagittal sections on 56 slides.

• ZMHNU - XDH 1, 2, 5, Xiaodianhe village (35°36'53"N, 114°0'1"E; alt. 250 m a. s. l.), Weihui City, Henan Province, sagittal sections on 13, 19, and 21 slides, respectively; • ZMHNU - XDH 4, ibid., horizontal sections on 21 slides; • ZMHNU - XDH 3, ibid., transverse sections on 27 slides, respectively. • RMNH.VER.22729.1, ibid., sagittal sections on 24 slides.

Diagnosis.

Dugesia cylindrica is characterized by the presence of the following features: a sac-shaped copulatory bursa that is located immediately posterior to the pharyngeal pocket; stubby, cylindrical penis papilla with a blunt tip; symmetrical openings of the oviducts into the bursal canal at the point where it communicates with the common atrium; vasa deferentia separately and symmetrically opening into the mid-lateral portion of the seminal vesicle; relatively large and pointed diaphragm; a long connecting duct between seminal vesicle and diaphragm; slightly ventrally displaced ejaculatory duct with a terminal opening. Anatomically, D. cylindrica is very similar to D. patula, but in the latter the copulatory bursa is located at some distance behind the pharyngeal pocket, whereas in the former the bursa lies immediately posterior to the pharyngeal pouch.

Etymology.

The specific epithet is derived from the Latin cylindrus, cylindrical, and alludes to the blunt and cylindrical penis papilla.

Karyology.

Eight intact specimens were randomly selected to prepare metaphase plates. In total, 168 metaphase plates were examined, in which 160 plates exhibited diploid chromosome complements of 2 n = 2 x = 16, while the remaining eight plates could not be determined due to either a lack of well-dispersed chromosomes or over-dispersed sets of chromosomes. All eight specimens exhibited diploid chromosome complements, and all chromosomes were metacentric. Karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 2. Chromosomal plates and the idiogram are shown in Fig. 5.

Description.

Living asexual specimens measured 10–14 mm in length and 1.2–2.1 mm in width, while sexual specimens were 15–19 mm in length and 1.5–2.2 mm in width. The low-triangular head is provided with two blunt auricles and with two eyes, which are located in pigment-free patches. Each pigmented eyecup houses numerous light-sensitive cells. The dorsal surface of the body is dark brown, with a pale, broad margin. The ventral surface is lighter in color than the dorsal one (Fig. 4 C).

The pharynx is located at ca. 2 / 5 of the body length (as determined from the anterior margin), measuring approximately 1 / 6 of the body length (Fig. 4 C). The mouth opening is located at the posterior end of the pharyngeal pocket. Outer pharyngeal musculature is composed of a subepithelial layer of longitudinal muscles, followed by a thin layer of circular muscles; an extra inner layer of longitudinal muscles was not observed. The inner pharyngeal musculature consists of a subepithelial and thick layer of circular muscle, followed by a thin layer of longitudinal muscle.

In all specimens in which the ovaries could be examined (except LMG- 6), the gonads were not hyperplasic, occupying 1 / 3 of the dorsoventral space. The ovaries are located at a considerable distance behind the brain, i. e., at approximately 1 / 4 th of the distance between the brain and the root of the pharynx. The oviducts extend from the postero-dorsal wall of the ovaries towards the level of the genital pore along the ventral side of the body, where they curve towards the dorsal body surface to open symmetrically into the bursal canal at the point where the canal communicates with the common atrium. The oviducts are lined with a columnar, infranucleated epithelium. Cyanophil shell glands discharge their secretion into the vaginal region of the bursal canal around the oviducal openings. (Fig. 6 A, E).

A large, sac-shaped copulatory bursa is situated immediately posterior of the pharyngeal pocket and occupies most of the dorso-ventral space. The bursa is lined by a stratified, columnar, vacuolated, and nucleated epithelium; it is devoid of any surrounding musculature; some bursae contain large clumps of sperm, located within a spermatophore (LMG- 1 and 4). The bursal canal runs from the mid-posterior wall of the bursa in caudal direction, slightly to the left side of the male copulatory apparatus. The canal has a width of approximately 1 / 7 to 1 / 5 of the dorso-ventral space. At the level of the gonopore, the bursal canal curves ventrally to open into the common atrium. The bursal canal is lined with a ciliated epithelium with basal nuclei and shows a smooth inner wall and has a uniform diameter. The bursal canal is surrounded by three layers of muscle, a subepithelial, thin layer of longitudinal muscles, followed by a thick layer of circular muscle, which is bounded by a thin layer of longitudinal muscles, forming the ectal reinforcement layer. This ectal reinforcement musculature extends from the atrium to 3 / 4 of the bursal canal.

The small and rounded testes are located on the dorsal side, occupying 1 / 3 of the dorso-ventral space. Testicular follicles extend from the posterior level of the ovaries to the posterior end of the body. Mature spermatozoa are present in the testes.

The vasa deferentia expand to form spermiducal vesicles at the level of the pharynx and are filled with mature spermatozoa. At the level of the penis bulb, the spermiducal vesicles curve dorso-medially, decrease considerably in diameter, forming narrow ducts, and subsequently penetrate the penis bulb to open symmetrically into the mid-lateral section of the seminal vesicle (Fig. 6 D). The sperm ducts are lined with a nucleated epithelium and surrounded by a layer of circular muscle.

The sac-shaped seminal vesicle occupies a major portion of the penis bulb (Fig. 6 A, C) and is lined by a flat, nucleated epithelium. The vesicle is surrounded by a well-developed coat of irregularly, crosswise arranged muscle fibers. A well-developed and long duct extends from the posterior wall of seminal vesicle and opens into the ejaculatory duct via a diaphragm. This connecting duct is lined with an infranucleated epithelium, which is underlain by a subepithelial layer of intermingled muscle fibers. The diaphragm is located at approximately 2 / 3 of the length of the penis papilla and receives the openings of erythrophil penis glands (Fig. 6 A). The relatively large and pointed diaphragm opens into a very broad as well as short ejaculatory duct, which is lined with a cuboidal, infranucleated epithelium and is devoid of any discernible musculature. The short ejaculatory duct has an extremely broad opening at the blunt tip of the penis papilla. In several specimens, a spermatophore protruded from the opening of the ejaculatory duct at the tip of the penis papilla. With respect to the stubby, cylindrical penis papilla, the slightly ventrally displaced ejaculatory duct causes a slight asymmetry, with the dorsal lip being only marginally larger than the ventral lip. The penis papilla is covered with a nucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibers (Fig. 6 A – C).

The genital atrium is divided into the common atrium and the male atrium, connected through a canal. The common atrium communicates with a gonoduct and leads to the gonopore, which is lined by a columnar epithelium and receives the openings of erythrophil cement glands (Fig. 6 A – C).

Reproduction.

The worms were collected at 16 ° C, and approximately one-third of all the worms were sexually mature in the field. After 3 months of laboratory culturing (at 16 ° C), the asexual specimens continuously sexualized and then produced infertile cocoons, as no juveniles hatched from these cocoons. Asexual reproductive behavior with broken tail regeneration was observed in asexual specimens.

Discussion.

Evidently, D. cylindrica is anatomically very similar to D. patula. However, the copulatory bursa of D. patula is located at a considerable distance (approximately 400–600 μm) behind the pharyngeal pocket, while its length is approximately twice the dorsoventral diameter of the body, i. e., measuring approximately 600–800 μm. This contrasts with the sac-shaped copulatory bursa located immediately posterior to the pharynx in D. cylindrica. In view of the fact that the position of the bursa appears to be different in D. patula and the molecular information suggests that it is different from D. cylindrica, the two species should be considered sibling species.

In addition, Dugesia cylindrica exhibits a combination of features that sets it immediately apart from other congeners, in particular, the presence of a ventrally displaced short ejaculatory duct with a terminal opening, as well as a duct between the seminal vesicle and diaphragm. The specimens from the three other localities are consistent with those from the type site (Fig. 7). Many species of Dugesia show similar anatomical features. However, there are only three Chinese species with a diaphragm located near the tip of the penis papilla, viz., D. tumida, D. postica, and D. patula (Chen et al. 2022; Wu et al. 2025). However, D. cylindrica cannot be synonymized with any of these species. Dugesia tumida possesses a large, symmetrical penial valve from the middle of which arises the small, distal section of the penis papilla; such a valve is absent in D. cylindrica. Dugesia postica possesses a large and long penis papilla with a small, dorsal bulge; such a bulge is absent in D. cylindrica. In addition, the small diaphragm of D. postica is different from the relatively large and pointed diaphragm of D. cylindrica.

The karyotype of D. cylindrica exhibits a diploid set (2 n = 2 x = 16), with a basic chromosome number of 8. In Dugesia species from China, chromosome portraits similar to that of D. cylindrica have been documented for D. constrictiva and D. musculosa. Evidently, the two species are anatomically dissimilar to D. cylindrica. Dugesia constrictiva has a laterally compressed seminal vesicle, and D. musculosa has a bursal canal provided with a strong, thick layer of circular muscle, which extends from the copulatory bursa to the common atrium and gonoduct, which is absent in D. cylindrica. However, the karyotype of D. musculosa exhibits a complex diploid set (2 n = 2 x = 16 and 16-7 th - 8 th), while aneuploidy is not observed in D. cylindrica.

Although D. cylindrica, D. patula, and D. postica belong to a relatively small clade in the phylogenetic tree (Fig. 2) and D. cylindrica has the lowest genetic distance with D. patula, notably, each species occupies its own branch, while they are anatomically different, albeit only marginally in the case of D. cylindrica and D. patula. The separate species status of D. cylindrica is also supported by the molecular species delimitation results.