Dugesia elongata Chen & Dong sp. nov.

Figs 1, 9–12

Collection site and habitat.

On 25 October 2021, the specimens were collected from a small pool formed by a stream flowing down from Baiyun Mountain at an altitude of 1100 m a. s. l., an air temperature of 8 ° C, and a water temperature of 6 ° C (Figs 1, 9 A, B). The water was clear and flowed slowly. At collection, individual worms were large, while mayfly larvae were present in the water.

Material examined.

Holotype: • ZMHNU - JJM 8, Baiyun Mountain in Song village (33°54'43"N, 111°83'49"E; alt. 1100 m a. s. l.), Luoyang County, Henan Province, China, 25 October 2021, coll. G-W. Chen, Z-M. Dong and co-workers, sagittal sections on 36 slides.

Paratypes: • ZMHNU - JJM 1, 2, 4, 8, ibid., sagittal sections on 39, 55, 65, and 55 slides, respectively; • ZMHNU - JJM 6, 7, 10, ibid., horizontal sections on 32, 16, and 29 slides; • ZMHNU - JJM 3 and 11, ibid., transverse sections on 49 and 51 slides, respectively. • RMNH.VER.22730.1, ibid., sagittal sections on 41 slides; • RMNH.VER.22730.2, ibid., sagittal sections on 32 slides.

Other material: • ZMHNU - SYXKZ 1-3, Shuiyunxuan pub Yangcheng village (35°21'29"N, 112°11'1"E; alt. 1150 m a. s. l.), Jincheng City, Shanxi Province, sagittal sections on 15, 14, and 15 slides, respectively. • RMNH.VER.22731.1, ibid., sagittal sections on 10 slides.

Diagnosis.

Dugesia elongata is characterized by the presence of the following features: relatively large worms; plump, barrel-shaped penis papilla; presence of an elongated, dumb-bell-shaped seminal vesicle; a well-developed connecting duct extending from the seminal vesicle and communicating with a low-conical diaphragm; ventrally displaced ejaculatory duct opening at the tip of the penial papilla, albeit at its ventral portion; asymmetrical openings of the oviducts, with the left oviduct opening into the ventral portion of the common atrium and the right oviduct opening into the bursal canal; copulatory bursa located at a short distance (approximately 487–696 μm) behind the pharyngeal pocket.

Etymology.

The specific epithet is derived from the Latin elongatus, having been stretched out, extended, and alludes to the long and dumb-bell-shaped seminal vesicle.

Karyology.

Eight intact specimens were randomly selected to prepare metaphase plates. A total of 185 metaphase plates were examined, in which 83 plates exhibited diploid chromosome complements of 2 n = 2 x = 16, while in 102 plates, chromosome complements were triploid with 2 n = 3 x = 24 chromosomes. All eight specimens exhibited mixoploid chromosome complements, and all chromosomes were metacentric. Karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 3. Chromosomal plates and the idiogram are shown in Fig. 10.

Description.

The animals were rather large, and most of them were sexually mature. The asexual animals measured 15–30 mm in length and 1.8–3.8 mm in width, while the sexual worms were 25–38 mm in length and 2.8–3.9 mm in width. The low-triangular head is provided with two blunt auricles and two eyes, which are placed in pigment-free patches (Fig. 9 C). Each pigmented eyecup houses numerous photoreceptor cells. The dorsal surface is black-brown, except the pale, broad margin of the body; the ventral surface shows a brown hue, which is paler than the dorsal colouration.

Pharynx situated in the posterior region of the body, measuring approximately 1 / 7 th of the body length (Fig. 9 C). Mouth opening located at the posterior end of the pharyngeal pocket. Outer pharyngeal muscle layer is composed of a subepithelial, thin layer of longitudinal muscles, followed by a thin layer of circular muscles; no extra inner layer of longitudinal muscles was observed. The inner pharyngeal musculature consists of a subepithelial, thick layer of circular muscle, followed by a thin layer of longitudinal muscle.

In all specimens in which the ovaries could be examined (JJM- 5, 8, 9), the gonads were hyperplasic, occupying 1 / 3–2 / 3 of the dorsoventral space The oviducts extend from the dorsal wall of the ovaries and run to the posterior part of the body, and when posteriorly to the gonopore, they curve dorsally and also recurve and then open asymmetrically into the copulatory apparatus (Fig. 11 B). The right oviducal opening is higher than the left one, in that it is located at the point where the bursal canal communicates with the common atrium. The left oviduct opens into the lower, ventral portion of the common atrium. Cyanophil shell glands discharge their secretion into the vaginal region of the bursal canal, at the level of the oviducal openings.

The large and sac-shaped copulatory bursa is situated at a short distance behind the pharyngeal pocket, ranging between approximately 487–696 μm in all specimens in which it could be measured (JJM- 2, 4, 5, 8, 9). The bursa is a large and oblong, sac-like structure that occupies the entire dorso-ventral space, and is twice as long as it is high. Its lining consists of columnar, vacuolated cells with basal nuclei.

The bursal canal arises from the postero-dorsal wall of the bursa and then expands in diameter to become a wide duct. At the level of the gonopore the bursal canal makes a knee-shaped bend towards the ventral body surface to connect with the common atrium. The bursal canal is lined with a ciliated epithelium with basal nuclei. It is surrounded by three layers of muscle, the innermost layer being a thin layer of longitudinal muscles, the middle layer a slightly thicker layer of circular muscle, followed by a thin outermost layer of longitudinal muscles, which forms the ectal reinforcement layer and extends from the atrium to approximately 4 / 5 of the length of the bursal canal. The circular muscle layer is relatively thick along the ventral wall of the bursal canal but much thinner along its dorsal wall.

In all specimens in which the testes could be examined (except JJM- 4), the well-developed, albeit small, follicles are located on the dorsal side and extend from directly behind the ovaries to the posterior end of the body. At the level of the pharyngeal pocket, in several specimens (except JJM- 4), the vasa deferentia expand to form spermiducal vesicles, which are packed with sperm.

The barrel-shaped penis papilla is asymmetrical, in that the ejaculatory duct is ventrally displaced, opening at the tip of the papilla. 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. A major portion of the dorsal wall of the penis papilla receives the openings of erythrophil glands, while a smaller part on its ventral wall is penetrated by openings of similar glands.

The penis bulb is lopsided, with its antero-ventral portion being near the ventral epidermis. That portion of the penis bulb houses the ventro-anterior portion of the seminal vesicle, which at its dorsal side narrows into a canal that runs anterodorsally and opens into another, again expanded portion of the seminal vesicle. Consequently, the seminal vesicle has the shape of a dumb-bell. The seminal vesicle is lined by a flat, nucleated epithelium and is surrounded by a layer of intermingled muscle fibers. The dorsally located portion of the dumb-bell symmetrically receives at its mid-ventral section the separate openings of the highly narrowed vasa deferentia, which are surrounded by a layer of circular muscles.

The anterior wall of the dorsally located portion of the seminal vesicle continues into a relatively long connecting duct that curves antero-ventrad and opens into a low-conical diaphragm, which opens into the ejaculatory duct. The diaphragm and the ejaculatory duct receive the abundant secretion of erythrophil penis glands (Figs 11 B, 13).

The genital cavity is divided into a common atrium and a male atrium, which communicate through a relatively narrow canal. The common atrium communicates with the bursal canal and also leads to the gonopore, which receives the openings of a large number of cement glands (Figs 11 B – D, 13).

In several specimens, a spermatophore protruded from the opening of the ejaculatory duct at the tip of the penis papilla.

Reproduction.

After 4 months of laboratory culturing, the asexual specimens continuously sexualized and produced cocoons. After approximately 20 days, five juveniles hatched from these cocoons. However, from then on, none of the worms produced any more cocoons.

Discussion.

The specimens from the other locality are consistent with those from the holotype site (Fig. 12). The coexistence of a long, dumb-bell-shaped seminal vesicle, a diaphragm, and a duct is found in only two Dugesia species, D. saccaria and D. verrucula. However, D. elongata cannot be synonymized with either of these two species. Dugesia saccaria has a dumbbell-shaped, muscularized hump located just anterior to the knee-shaped bend in the bursal canal. In addition, the ejaculatory duct opens terminally through the dorsal portion of the blunt tip of the penis papilla (Zeng et al. 2024), which contrasts with the ventral opening in D. elongata. Dugesia verrucula also has a dumbbell-shaped seminal vesicle surrounded by distinct musculature, like D. elongata. Unfortunately, this was not properly described by Wang et al. (2021 a), and it was also incorrectly described that in D. verrucula, the ejaculatory duct arises from the dorsal portion of the seminal vesicle. However, the ejaculatory duct always arises after, i. e., distally to the diaphragm. There is a difference, in that in D. verrucula, the vasa deferentia open into the proximal portion of the dumb-bell, whereas in D. elongata, the ducts open into the distal part of the dumb-bell-shaped seminal vesicle. However, the most characteristic feature of D. verrucula is the permanent dorsal bump on its penis papilla, which is absent in D. elongata (Wang et al. 2021 a).

In addition, D. elongata, D. verrucula, and D. constrictiva also belong to a small clade in the phylogenetic tree (Fig. 2). However, D. elongata and D. constrictiva are anatomically rather different (Wang et al. 2022). Dugesia constrictiva has a laterally compressed seminal vesicle and a cuboidal copulatory bursa, which are different from the elongated, dumb-bell-shaped seminal vesicle and the oblong, sac-like copulatory bursa of D. elongata. Furthermore, D. verrucula and D. constrictiva exhibit a diploid chromosome complement of 2 n = 2 x = 16, with a haploid number of n = 8, in contrast to the chromosome portrait of D. elongata, which shows a mixoploid karyotype with diploid complements of 2 n = 2 x = 16 and triploid sets of 2 n = 3 x = 24. The lowest COI and ITS-1 distance values between D. elongata and its congeners were 12.02 % (D. elongata with D. constrictiva) and 1.66 % (D. elongata with D. verrucula), respectively. Therefore, D. elongata is well separated from its congeners, which further supports its separate specific status as suggested by the anatomical and karyological analyses.