Dugesia majuscula Chen & Dong, 2021

Wang, Lei, Dong, Zi-mei, Chen, Guang-wen, Sluys, Ronald & Liu, De-zeng, 2021, Integrative descriptions of two new species of Dugesia from Hainan Island, China (Platyhelminthes, Tricladida, Dugesiidae), ZooKeys 1028, pp. 1-28 : 1

Dugesia majuscula Chen & Dong sp. nov. Figures 8 , 9 , 10 , 11 , 12

Material examined.

Holotype: ZMHNU-YHYQ 4, sagittal sections on 36 slides, Yingge mountain , Qiongzhong County, Hainan Province, China (19°3'24"N, 109°33'49"E), 24 February 2018, altitude 584 m a.s.l., coll. GW Chen and co-workers.

Paratypes: ZMHNU-YHYQ1, ibid. sagittal sections on 45 slides; ZMHNU-YHYQ2, ibid., sagittal sections on 18 slides; ZMHNU-YHYQ3, ibid., sagittal sections on 24 slides; ZMHNU-YHYQ5, ibid., sagittal sections on 32 slides; ZMHNU-YHYQ 6, horizontal sections on 18 slides; ZMHNU-YHYQ 7, horizontal sections on12 slides; ZMHNU-YHYQ8, ibid., transverse sections on 24 slides; ZMHNU-YHYQ9, ibid., sagittal sections on 15 slides; ZMHNU-YHYQ10, ibid., sagittal sections on 19 slides.

Diagnosis.

Dugesia majuscula is characterized by the following features: oviducts opening asymmetrically into female reproductive system; hyperplasic ovaries; expanded posterior section of bursal canal; vasa deferentia separately opening into mid-dorsal portion of seminal vesicle; asymmetrical penis papilla due to ventral course of ejaculatory duct, which has subterminal and dorsal opening at tip papilla; mixoploid chromosome complement diploid (2n=16) and triploid (3n = 24), with metacentric chromosomes.

Etymology.

The specific epithet is derived from the Latin adjective majusculus, somewhat larger, and alludes to expanded portion of the bursal canal.

Habitat and reproduction.

Animals were collected from a freshwater stream in the Yingge Mountains, at a water temperature of 19 °C and an altitude of 584 m a.s.l. (Fig. 8A, B ). Approximately 40 individuals were collected in February 2018, none of which was sexually mature. During the first period of 80-90 days (March to May) in the laboratory culture, worms only showed asexual reproduction by means of fission. However, during the following period of 40-60 days, seven individuals sexualized, while after another 180 days, 1/3 of the animals sexualised, although thus far they have not produced any cocoons.

Karyology.

Each of the five, randomly selected specimens exhibited mixoploid chromosome complements. In a total of 100 metaphase plates examined, 75 exhibited diploid chromosome complements of 2n = 2x = 16, while in 18 plates chromosome complements were triploid with 2n = 3x = 24 chromosomes (Fig. 9 ); chromosome complements of the remaining seven plates could not be determined, due to either lack of well-dispersed chromosomes or over-dispersed sets of chromosomes. All chromosomes were metacentric; karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 3 . The first pair of chromosomes is clearly larger than the others, being 1.86 times larger than the shortest chromosome. Chromosomal plates and idiogram are shown in Fig. 9 .

Description.

Body size of living asexual specimens is 5-8 mm in length and 0.65-0.78 mm in width, while in sexualized specimens the body measures 10-16 mm in length and 1.05-1.36 mm in width. Head of low triangular shape, provided with two blunt auricles and two eyes located in pigment-free patches (Fig. 8C ). Each pigmented eye cup is provided with numerous retinal cells. Dorsal surface brown with numerous dark spots (Fig. 8C ); ventral surface paler than dorsal one and provided with only scattered small, dark spots.

Pharynx positioned in the mid-region of the body and measuring ~ 1/6th of the body length (Fig. 8C ); the mouth opening is situated at the hind end of the pharyngeal pocket. The pharyngeal outer musculature is composed of an outer, subepithelial layer of longitudinal muscles, followed by a layer of circular muscles. An extra longitudinal muscle layer internally to the circular muscles is absent (Fig. 10A ). Inner pharyngeal musculature composed of an outer layer of longitudinal muscles and a subepithelial layer of circular muscles, the latter being thicker than the outer layer (Fig. 10A ).

Ovaries hyperplasic, with several scattered masses at a short distance behind the brain, extending backwards over a distance of at least 800 μm (Fig. 10B ) and occupying the entire dorso-ventral space. In live animals, the hyperplasic ovaries are visible through the dorsal body surface as two short, transparent stripes (Fig. 8C ). From the ovaries, the oviducts run ventrally in a caudal direction to the level of the genital pore where they turn dorso-medially to open separately and asymmetrically into the female reproductive system. In point of fact, the right oviduct opens either into the most postero-ventral part of the expanded portion of the bursal canal or somewhat more ventrally (YHYQ-5), while the left oviduct opens into the common atrium, a distinct female atrium being absent (Figs 10C , 12 ).

The well-developed testes are situated dorsally and extend from the level of the ovaries to the posterior end of the body (Fig. 10D ). Upon approaching the level of the penis bulb, composed of intermingled muscle fibers, the vasa deferentia curve dorso-mediad and near the postero-dorsal wall of the penis bulb the sperm ducts abruptly bend ventrad to penetrate the wall of the penis bulb. Within the bulb, the sperm ducts open separately and symmetrically into the mid-dorsal portion of a large reniform seminal vesicle (Figs 10E, F , 12 ). The sperm ducts are lined with nucleated cells and are surrounded by a layer of circular muscles. The seminal vesicle opens into the ejaculatory duct via a very small diaphragm positioned at a level that corresponds with the root of the penis papilla (Figs 11A , 12 ). The ejaculatory duct runs ventrally through the penial papilla and opens to the exterior by means of subterminal dorsal opening at the tip of the papilla (Figs 11A-C , 12 ). The ejaculatory duct is lined by a cuboidal epithelium; we were unable to discern any musculature around the ejaculatory duct.

The cylindrical penis papilla is covered by an epithelium that is underlain with a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibers. As a result of the ventral course of the ejaculatory duct, the penis papilla is asymmetrical, with its dorsal lip being much larger than the ventral one. The basal part of the penis papilla has an oblique, ventro-caudal orientation, after which the penis papilla makes a bend, so that the rest of the papilla has a more or less horizontal orientation (Figs 11A-C , 12 ). The papilla almost completely occupies the male atrium, which communicates with the common atrium via a more or less pronounced constriction (Fig. 12 ). In turn, the latter communicates with a gonoduct, lined with a columnar epithelium and receiving the openings of abundant cement glands, which leads to the ventral gonopore.

The large sac-shaped copulatory bursa, which occupies the entire dorso-ventral space, is lined by a vacuolated epithelium provided with basal nuclei. There is hardly any musculature surrounding the bursa (Figs 11A-C , 12 ). From the postero-dorsal wall of the bursa, the bursal canal runs in a caudal direction to the left side of the male copulatory apparatus. Dorsally to the male atrium the bursal canal expands, particularly in dorso-ventral direction, to form a spacious posterior section that communicates with the common atrium. The bursal canal is lined with cylindrical, infranucleated, and ciliated cells and is surrounded by a subepithelial layer of longitudinal muscles, followed by a well-developed layer of circular muscle and a thin layer of longitudinal muscles. The outer longitudinal muscles constitute the ectal reinforcement layer, which extends from the vaginal region to approximately halfway along the bursal canal (Figs 11D , 12 ). Erythrophil shell glands open into the vaginal region of the bursal canal around the oviducal openings.

Discussion.

Our present task of comparing the features of D. majuscula with already known species of Dugesia is made considerably easier by the fact that it exhibits the unusual character of a subterminal dorsal opening of the ejaculatory duct at the tip of the penis papilla, which thus far has been reported for only one other species, viz., D. umbonata from southwest China ( Song et al. 2020). In view of the sister-group relationship between these two species (Fig. 2 ), this subterminal, dorsal opening of the ejaculatory duct may be hypothesized to represent a synapomorphy for D. umbonata and D. majuscula .

Although D. umbonata exhibits also asymmetrical oviducal openings and a ventrally displaced ejaculatory duct, as in D. majuscula , it differs from the latter notably in the presence of a large, muscularized hump on its bursal canal, among other differences. A subterminal opening of the ejaculatory duct is not uncommon among species of Dugesia , but in most cases the openings are ventral, in contrast to the dorsal opening in D. majuscula and D. umbonata . The only exception is D. hepta Pala et al., 1981 but in this species the ejaculatory duct exits at the dorso-lateral tip of the penis papilla, while in this species the ejaculatory duct is dorsally displaced, in contrast to the ventral course in D. majuscula and D. umbonata . Furthermore, in D. hepta the oviducts open symmetrically into the bursal canal ( Stocchino et al. 2005), whereas in D. majuscula and D. umbonata the oviducal openings are asymmetrical.

Figures

Figure 2. Phylogenetic tree obtained from Bayesian analysis of the concatenated dataset. Numbers at nodes indicate support values (posterior probability / bootstrap). ∗: Bootstrap value not applicable to the node, because of different topologies of trees obtained by BI and ML methods. Scale bar: substitutions per nucleotide position.

Figure 8. Habitat and external appearance of Dugesia majuscula A sampling locality B habitat C sexually mature, living individual (anterior is to the up). Abbreviations: coa: copulatory apparatus; e: eye; ph: pharynx. Scale bar: 1 mm.

Figure 9. Dugesia majuscula A idiogram B 1 metaphasic plate of diploid cell B 2 karyogram of diploid cell C 1 metaphasic plate of triploid cell C 2 karyogram of triploid cell.

Figure 10. Dugesia majuscula A transverse section of paratype YHYQ 8, showing longitudinal and circular muscles in outer and inner pharyngeal musculature B horizontal section of paratype YHYQ 7, showing hyperplasic ovaries C transverse section of paratype YHYQ 8, showing opening of the left oviduct D sagittal section of paratype YHYQ 10, showing dorsal testes E transverse section of paratype YHYQ 8, showing course of a vas deferens F sagittal section of paratype YHYQ 3, showing opening of a vas deferens into seminal vesicle. The anterior is to the front in A, C, E and anterior is to the right in B, D, F. Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; cm: circular muscles; go: gonopore; ho: hyperplasic ovaries; lm: longitudinal muscles; lod: left oviduct; ma: male atrium; pp: penis papilla; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bars: 100 μm.

Figure 11. Dugesia majuscula A sagittal section of holotype YHYQ 4, showing seminal vesicle, ejaculatory duct, small diaphragm, penis papilla, and copulatory bursa B Sagittal section of holotype YHYQ 4, showing seminal vesicle, ejaculatory duct, penis papilla, and copulatory bursa C sagittal section of paratype YHYQ 5, showing terminal and dorsal opening of ejaculatory duct D sagittal section of paratype YHYQ 9, showing bursal canal and its musculature. The anterior is to the right in A, B, C, D. Abbreviations: bc: bursal canal; cb: copulatory bursa; cm: circular muscles; d: diaphragm; ed: ejaculatory duct; in: intestine; lm: longitudinal muscles; ma: male atrium; pp: penis papilla; sv: seminal vesicle; vd: vas deferens. Scale bars: 100 μm.

Figure 12. Dugesia majuscula. Sagittal reconstruction of the copulatory apparatus of the holotype (anterior is to the right). Abbreviations: bc: bursal canal; cb: copulatory bursa; cg: cement glands; d: diaphragm; ed: ejaculatory duct; go: gonopore; lod: left oviduct; pp: penis papilla; rod: right oviduct; sg: shell glangs; sv: seminal vesicle; vd: vas deferens. Scale bar: 100 μm.