Dugesia saccata Chen & Dong, 2025

Wang, Lei, Chang, Yi-Fang, Sun, Xin-Xin, Sluys, Ronald, Liu, De-Zeng, Dong, Zi-Mei & Chen, Guang-Wen, 2025, Two new species of freshwater planarian from Hainan Island and Leizhou Peninsula, southern China (Platyhelminthes, Tricladida, Dugesiidae), ZooKeys 1233, pp. 289-313 : 289-313

Dugesia saccata Chen & Dong sp. nov.

Figs 1 , 2 , 3 , 4 , 5 , 6

Collection site, habitat, and reproduction.

On 24 February 2018, specimens were collected from a freshwater stream in the Yingge Mountains, Hainan Island (Figs 1 , 3 A ), which is located within a national Nature Reserve at an altitude of 430 m a. s. l.; air temperature was 24 ° C and water temperature was 21 ° C. In the population of D. saccata , all worms were asexual at collection in the field, and under laboratory conditions were fissiparous. During a period of ~ 4 months, each of the ten specimens sexualised; in the first month only two sexualised individuals were found, while at the end of the fourth month ten sexual animals were present. After 18 months of culturing, sexualised worms produced> 10 cocoons. The spherical cocoons (1.3 mm in diameter) were dark brownish and provided with a stalk. Thus far, none of the cocoons hatched, most likely infertile. During laboratory culturing, the sexualised worms sometimes lost their copulatory apparatus and, subsequently, returned to the asexual mode of reproduction.

Material examined.

Holotype • ZMHNU -YZCB 1 , Yongzhong village (18°46'6"N, 109°38'42"E), alt. 430 m a. s. l., Wuzhishan City, Hainan Province, China, 24 February 2018, coll. G-W Chen, D-Z Dong and co-workers, sagittal sections on 28 slides . Paratypes • RMNH.VER.22249.1 , ibid., sagittal sections on 12 slides • RMNH.VER.22249.2 , ibid., sagittal sections on 20 slides • ZMHNU -YZCB 2 , 3, 6, ibid., sagittal sections on 19, 18, and 26 slides, respectively • ZMHNU -YZCB 5 , ibid., horizontal sections on 16 slides • ZMHNU -YZCB 8 , ibid., transverse sections on 35 slides .

Diagnosis.

Dugesia saccata is characterised by the presence of the following features: symmetrical openings of the oviducts into the most proximal section of the bursal canal, near the point where the latter communicates with the atrium; a sac-shaped expansion at the knee-shaped bend of bursal canal; vasa deferentia opening symmetrically into posterior portion of the seminal vesicle; ventrally displaced ejaculatory duct with subterminal opening; a duct between diaphragm and seminal vesicle; mixoploid karyotype, with diploid chromosome portraits of 2 n = 2 x = 14, and triploid complements of 2 n = 3 x = 21, with all chromosomes being metacentric.

Karyology.

Seven intact specimens were randomly selected to prepare metaphase plates. In a total of 157 metaphase plates that were examined, 42 plates exhibited diploid chromosome complements of 2 n = 2 x = 14, while in 104 plates chromosome complements were triploid with 2 n = 3 x = 21 chromosomes; chromosome complements on the remaining 11 plates could not be determined, due to either lack of well dispersed chromosomes or over-dispersed sets of chromosomes. All seven specimens exhibited mixoploid chromosome complements, with all chromosomes being metacentric. Karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 2 . Chromosomal plates and an idiogram are shown in Fig. 4 .

Morphology.

In sexualised living specimens, the body measured 14–22 mm in length and 1.3–1.6 mm in width. Triangular head provided with two blunt auricles and two eyes, which are placed in pigment-free spots. Each pigmented eyecup houses numerous photoreceptor cells. The dorsal surface is taupe, the ventral surface is paler in colour than the dorsal one (Fig. 3 C ).

Pharynx situated at the mid-region of the body, measuring ~ 1 / 5 of the body length. Mouth opening located at posterior end of the pharyngeal pocket. The outer pharyngeal musculature is composed of a subepithelial layer of longitudinal muscles, followed by a layer of circular muscles. The inner pharyngeal musculature consists of a thick subepithelial layer of circular muscles, followed by a thin layer of longitudinal muscles.

The hyperplasic ovaries are located at 1 / 3–1 / 5 of the distance between the brain and the root of the pharynx, occupying ~ 1 / 2 of the dorso-ventral space, with several scattered masses. The oviducts arise from the dorsal wall of the ovaries, then turn to the ventral side and run in a caudal direction to the level of the genital pore, after which they curve dorso-medially to open separately and symmetrically into the bursal canal, near the point where the latter communicates with the atrium (Fig. 5 A ). Cyanophil shell glands discharge their secretion into the vaginal region of the bursal canal, at the level of the oviducal openings.

The small, dorsally located testes are poorly developed and provided with only a few mature spermatozoa. As a consequence, we found spermatozoa to be present in the vasa deferentia only in specimens YZCB 3, 5, 6, and 8, as well as in RMNH.VER.22249.2 . Testicular follicles are arranged on either side of the midline of the body in four or five longitudinal zones, extending from the posterior level of the ovaries to almost the posterior end of the body.

At the level of the penis bulb, the sperm ducts curve towards the dorsal body surface, then penetrate the ventral wall of the penis bulb to open separately into the seminal vesicle. The precise approach of the ducts to the seminal vesicle differs somewhat between specimens. In the holotype one sperm duct exhibits a short dorso-ventral recurvature before opening into the proximal section of the duct that leads from the seminal vesicle to the diaphragm; the other duct opens at the same position but has a much more direct approach (Fig. 6 ). In paratype YZCB- 6 there is an even more distinctly asymmetrical approach of the sperm ducts, with one duct having a dorsal approach, after a well-developed dorso-ventral recurvature, opening in the antero-lateral portion of the seminal vesicle, again close to the duct leading to the diaphragm. The other sperm duct does not show the recurvature and opens directly into the proximal section of the connecting duct between seminal vesicle and diaphragm. In contrast, in paratype YZCB- 8 both ducts exhibit a well-developed recurvature before symmetrically opening into the latero-dorsal portions of the seminal vesicle, close to the point where the connecting duct opens into the seminal vesicle (Fig. 5 B ).

The sperm ducts are lined with nucleated cells and surrounded by a layer of circular muscles. The oval-shaped, rather large seminal vesicle is lined by a flat, nucleated epithelium and is surrounded by a layer of irregularly crosswise arranged muscle fibres. The postero-ventral section of the seminal vesicle gives rise to a duct that is lined by an infranucleated epithelium, which is underlain by a subepithelial layer of intermingled muscle fibres and via a small diaphragm opens into the ejaculatory duct (Figs 5 D , 6 ). The small diaphragm is located at approximately the root of the penis papilla and receives the abundant secretion of erythrophil penis glands (Fig. 5 D ). The ejaculatory duct, which is lined with a cuboidal, infranucleated epithelium, is devoid of any discernible musculature and follows a ventrally displaced course through the penis papilla, opening subterminally at its tip (Figs 5 D , 6 ). Cyanophil penis glands discharge abundant secretion into the central and distal portion of the ejaculatory duct.

Because of the ventrally displaced course of the ejaculatory duct, the penis papilla is asymmetrical, with its dorsal lip being considerably larger than the ventral one (Figs 5 D , 6 ). The cylindrical penis papilla has an oblique, ventro-caudal orientation and is covered by a nucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres (Figs 5 D , 6 ).

The copulatory bursa is a large sac-shaped structure that may occupy the entire dorso-ventral space (paratype YZCB- 6), while in other specimens it extends well over the central longitudinal axis of the body but does not fully occupy the dorso-ventral space (e. g., holotype YZCB- 1; Fig. 6 ). The bursa is lined by a vacuolated epithelium with basal nuclei and is almost devoid of any surrounding musculature. The bursal canal arises from the postero-dorsal wall of the bursa, after which it runs in a caudal direction to the left side of the male copulatory apparatus (Figs 5 C , 6 A ). The rather broad bursal canal occupies ~ 1 / 4 of the dorso-ventral space. At the level of the gonopore it decreases somewhat in diameter, whereafter it gives rise to a saccate posterior extension (Figs 5 C , 6 ). The antero-ventral, knee-shaped bend in the bursal canal arises from the ventral wall of the saccate portion and communicates with the common atrium (Figs 5 C , 6 ).

The bursal canal is lined with a ciliated epithelium with basal nuclei. Particularly the dorsal wall of the canal may be thrown into several folds. It is noteworthy that the ventral wall of the bursal canal is lined with cylindrical cells, whereas the dorsal wall is composed of cuboidal or even flat cells; the saccate expansion is also lined with a low epithelium. The bursal canal is surrounded by a subepithelial layer of longitudinal muscles, followed by a layer of circular muscle that is particularly well developed on the ventral wall of the canal; an extra outer layer of longitudinal musculature, forming the ectal reinforcement, extends from the atrium to 2 / 3 on the bursal canal.

The common atrium communicates with a gonoduct, which is lined by a columnar epithelium and receives the openings of erythrophil cement glands (Fig. 6 ).

Etymology.

The specific epithet is derived from the Latin noun saccus, bag, and alludes to the sac-shaped expansion at the knee-shaped bend of the bursal canal.

Discussion.

There is one character that immediately sets D. saccata apart from all of its known congeners, the sac-shaped expansion on the posterior section of the bursal canal, near the knee-shaped bend of the canal. This is slightly reminiscent of a situation in Dugesia aethiopica Stocchino, Corso, Manconi & Pala, 2002 , in which the posterior section of the bursal canal, immediately before receiving the separate openings of the oviducts, is expanded in lateral direction and gives rise to several large folds ( Sluys 2007). However, this is merely a superficial resemblance to the situation in D. saccata , and these two species also differ in many other features. For example, D. aethiopica shows a horizontal approach of the bursal canal to the atrium, which represents a rare feature among species of Dugesia and is also absent in D. saccata . In Dugesia arabica Harrath & Sluys, 2013 the bursal canal is considerably expanded as well as highly folded in the region of the oviducal openings ( Harrath et al. 2013). However, this situation differs from that in D. saccata in that the expansion sits near the oviducal openings, whereas in D. saccata the sac-shaped expansion occurs dorsally, or entally to the openings of the oviducts into the bursal canal. Another difference between these two species concerns the presence of a duct between seminal vesicle and diaphragm in D. saccata and absence of such a duct in D. arabica .

Two characteristic features of D. saccata may be found also in other species of Dugesia , a ventrally displaced ejaculatory duct with subterminal opening and the presence of a duct between the seminal vesicle and the diaphragm. Besides D. saccata , these two character states are also expressed, among others, in the three Chinese species D. majuscula , D. umbonata Song & Wang, 2020 , and D. verrucula Chen & Dong, 2021 ( Song et al. 2020; Wang et al. 2021 a, b). However, in contrast to D. saccata , in both D. majuscula and D. umbonata the ejaculatory duct has a subterminal dorsal opening at the tip of the penis papilla, while D. verrucula exhibits a permanent dorsal bump near the root of the penis papilla, which is absent in D. saccata . Another difference concerns the presence of a large muscularised hump on the dorsal surface of the bursal canal of D. umbonata , which is absent in D. saccata . Although these three species ( D. umbonata , D. verrucula , and D. saccata ) belong to the same clade, they are molecularly well-differentiated. On the other hand, while D. saccata , D. pendula , D. tumida , and D. majuscula belong to the same small clade, they are anatomically well differentiated.

In Dugesia species, the basic chromosome number concerns three types, 7, 8, and 9. Previous studies have shown that in China number 8 is the most frequent type, while 7 is much rarer ( Wang et al. 2024). In that respect, it is noteworthy that the basic chromosome number in D. saccata is n = 7, which is shared only with D. pendula , D. hepta Pala, Casu, & Vacca, 1981 , D. batuensis Ball, 1970 , and D. ryukyuensis Kawakatsu, 1976 ( Kawakatsu et al. 1976; Pala et al. 1981; Khang et al. 2017; Wang et al. 2024). However, D. pendula exhibits an aneuploid plus mixoploid karyotype, with diploid (2 n = 2 x = 14 + 0-1 B-chromosome) and triploid (2 n = 3 x = 21 + 0-1 B-chromosome) sets, while D. batuensis exhibits six metacentric chromosomes and one subtelocentric chromosome, and D. ryukyuensis shows six metacentric chromosomes and one submetacentric chromosome, which is the case also in D. hepta . In contrast, D. saccata exhibits a mixoploid karyotype with diploid (2 n = 2 x = 14) and triploid (2 n = 3 x = 21) sets, with all chromosomes being metacentric, thus contrasting with the chromosome complements of the other species.

In fact, D. saccata produced infertile cocoons and only showed asexual reproduction by means of fission, which corresponds with its poorly developed or hyperplasic ovaries and the triploid chromosome complement. It has been established that in such abnormal ovaries the oocytes are anomalous, thus preventing regular oogenesis ( Harrath et al. 2014).

Figures

Figure 1. Collection sites of Dugesia in Hainan Island and Leizhou Peninsula.

Figure 2. Topology of the molecular phylogenetic tree obtained from Bayesian analysis of the concatenated dataset (dataset III). Numbers at nodes indicate support values (pp / bs). New species indicated by asterisks. Scale bar: substitutions per site.

Figure 3. Habitat and external appearance of Dugesia saccata A, B sampling site and habitat C sexually mature, live individual. Abbreviations: coa: copulatory apparatus; e: eye; ho: hyperplasic ovary; ph: pharynx. Scale bar: 2 mm.

Figure 4. Dugesia saccata A 1, B 1 metaphase plate and karyogram of triploid complement A 2, B 2 metaphase plate and karyogram of diploid complement C idiogram. Scale bars: 5 μm.

Figure 5. Dugesia saccata. Photomicrographs of transverse (A, B) and sagittal (C, D) sections. A Paratype ZMHNU - YZCB 8, showing symmetrical openings of oviducts into the bursal canal B paratype ZMHNU - YZCB 8, showing symmetrical openings of vasa deferentia into the seminal vesicle C holotype ZMHNU - YZCB 1, showing copulatory bursa and bursal canal D holotype ZMHNU - YZCB 1, showing penis papilla, vasa deferentia, seminal vesicle, diaphragm, and ejaculatory duct. Abbreviations: bc: bursal canal; cb: copulatory bursa; d: diaphragm; ed: ejaculatory duct; lod: left oviduct; ma: male atrium; od: oviduct; pp: penis papilla; rod: right oviduct; se, sac-shaped expansion of bursal canal; sv: seminal vesicle; vd: vas deferens. Scale bars: 100 μm.

Figure 6. Dugesia saccata. Sagittal reconstruction of the copulatory apparatus of holotype YZCB 1 A female copulatory apparatus B male copulatory apparatus. Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; cg: cement glands; cpg: cyanophil penial glands; d: diaphragm; ed: ejaculatory duct; epg: extrabulbar penial glands; go: gonopore; lod: left oviduct; ma: male atrium; mo: mouth; pb: penis bulb; pp: penis papilla; rod: right oviduct; se, sac-shaped expansion of bursal canal; spv: spermiducal vesicles; sg: shell glands; sv: seminal vesicle; vd: vas deferens. Scale bars: 100 μm.