Dugesia verrucula Chen & Dong
Wang, Lei, Chen, Jin-zi, Dong, Zi-mei, Chen, Guang-wen, Sluys, Ronald & Liu, De-zeng, 2021, Two new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the tropical monsoon forest in southern China, ZooKeys 1059, pp. 89-116: 89
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|Dugesia verrucula Chen & Dong|
Dugesia verrucula Chen & Dong sp. nov.
Holotype. ZMHNU-ZJYA5, sagittal sections on 27 slides, Shiwan Dashan Mountain National Natural Reserve (21°53'40"N, 107°54'30"E; alt. 520 m a.s.l.), Shangsi County, Guangxi Province, China, 1 January 2019, coll. G-W Chen , D-Z Dong and co-workers GoogleMaps . Paratypes. ZMHNU-ZJYA1, ibid., sagittal sections on 25 slides; ZMHNU-ZJYA2, ibid., sagittal sections on 36 slides; ZMHNU-ZJYA3, ibid., sagittal sections on 38 slides; RMNH VER. 19975.a, ibid., sagittal sections on 34 slides; ZMHNU-ZJYA6, ibid., horizontal sections on 24 slides; ZMHNU-ZJYA7, ibid., transverse sections on 30 slides; RMNH VER. 19975.b, ibid., sagittal sections on 24 slides; ZMHNU-ZJYA9, ibid., horizontal sections on 19 slides; ZMHNU-ZJYA10, ibid., sagittal sections on 32 slides; ZMHNU-ZJYA11, ibid., sagittal sections on 30 slides.
Dugesia verrucula is characterised by the presence of the following features: large size of the live worm, usually exceeding 3.5 cm in length; asymmetrical openings of the oviducts into the bursal canal; subterminal opening of the ventrally displaced ejaculatory duct; vasa deferentia symmetrically opening into the postero-lateral portion of the seminal vesicle; well-developed duct between seminal vesicle and diaphragm; single dorsal bump near root of penis papilla; bursal canal with pleated wall and spacious posterior section; unstalked cocoons; chromosome complement diploid with 16 metacentric chromosomes.
The specific epithet is derived from the Latin Dugesia verrucula , small wart, and alludes to the dorsal bump on the penis papilla; the specific epithet is treated as an adjective.
Habitat and reproduction.
The average annual temperature of the tropical monsoon rain forest in the Shiwan Dashan Mountain National Natural Reserve ranges between 20 and 25 °C, while in the coldest month (January), the average temperature is ca. 10-3 °C. The worms were collected from a pond under a waterfall on the Shiwan Dashan Mountain, with a water temperature of 8.4 °C, while air temperature was 5.5 °C (Fig. 9A, B View Figure 9 ). It is noteworthy that the water temperature is lower than the coldest average temperature of the tropical monsoon rain forest and that, thus, the worms live under relatively harsh climatic conditions, as compared to that of the forest itself. At collection, ten specimens were sexually mature. After ca. 30 days of culturing under laboratory conditions, the animals produced unstalked spherical cocoons (approx. 1.5 mm in diameter) that were firmly attached to the glass wall of the containers. Newly laid cocoons at first were red but turned dark brown after two or three days. After 10-20 days, 8-11 juveniles hatched from each cocoon. Juvenile planarians were light brown, after ca. 2 days, measuring 1.5-2.0 mm in length and 0.18-0.21 mm in width. After approximately 50 days, the animals measured 15-20 mm in length and 1.4-1.8 mm in width and were sexual (Fig. 9C View Figure 9 ). Fission was not observed in the laboratory culture.
Each of the six, randomly selected specimens exhibited diploid chromosome complements. In a total of 100 metaphase plates examined, 85 chromosome complements were diploid with 2n = 2 × = 16 chromosomes, with all chromosomes being metacentric (Fig. 4 View Figure 4 ); chromosome complements of the remaining 15 plates could not be determined, due to either lack of well-dispersed chromosomes or over-dispersed sets of chromosomes. The first pair of chromosomes is clearly larger than others, being 2.21 times larger than the shortest chromosome. Karyotype parameters, including relative length, arm ratio, and centromeric index are given in Table 3, while a chromosomal plate and idiogram are shown in Fig. 10 View Figure 10 .
The body of live, sexual specimens measures 2.8-3.9 mm in length and 1.8-2.8 mm in width. Low-triangular head provided with two blunt auricles and two eyes, which are located in the centre of the head and placed in pigment-free spots and house numerous retinal cells (Fig. 9C View Figure 9 ). Dorsal surface brown, excepting the pale body margin and an accumulation of pigment following the outline of the pharynx. Furthermore, there is a dark brown mid-dorsal stripe, extending from the head to the posterior end of the body. The ventral surface is light brown.
Pharynx situated in the mid-region of the body and measuring ca. 1/5th of the body length (Fig. 9C View Figure 9 ). Mouth opening located at the posterior end of the pharyngeal pocket. Outer pharyngeal musculature composed of a subepidermal layer of longitudinal muscles, followed by a layer of circular muscles; inner pharyngeal musculature composed of a thick subepithelial layer of circular muscles, followed by a layer of longitudinal muscles (Fig. 11A View Figure 11 ).
Ventral ovaries located at a short distance behind the brain, occupying ca. 1/6th of the dorso-ventral space (Fig. 11B View Figure 11 ). The oviducts run ventrally in a caudal direction posteriorly to the genital pore, after which they curve dorsally to open separately and asymmetrically into the ventral portion of the bursal canal. The right branch opens dorsally to the left one, the latter actually opening into the common atrium (Figs 11C View Figure 11 , 13 View Figure 13 , 14 View Figure 14 ).
The large, sac-shaped copulatory bursa occupies the entire dorso-ventral space, and is lined by a vacuolated epithelium with basal nuclei (Fig. 11E, F View Figure 11 ). From the dorso-posterior wall of the bursa, the bursal canal runs in a caudal direction to the left side of the copulatory apparatus. The anterior section of the bursal canal is narrow, but dorsally to the male copulatory apparatus the canal expands in dorso-ventral direction and also presents a distinctly folded wall, formed by numerous pleats, particularly in its dorsal wall. In particular the posterior portion of the bursal canal expands to form a spacious chamber that almost imperceptibly grades into the equally spacious common atrium (Figs 11D View Figure 11 , 13 View Figure 13 , 14B View Figure 14 ). The ventral portion of the latter communicates with the gonoduct (Figs 12D, G View Figure 12 , 13 View Figure 13 , 14B View Figure 14 ).
The bursal canal is lined with columnar, nucleated, ciliated cells and is surrounded by a thin subepithelial layer of longitudinal muscles, followed by a thicker layer of circular muscle. An ectal reinforcement layer of longitudinal muscles runs from the vaginal region to ca. halfway along the bursal canal (Figs 11D View Figure 11 , 13 View Figure 13 , 14B View Figure 14 ). Shell glands discharge their erythrophil secretion into the vaginal region of the bursal canal, near the oviducal openings.
The well-developed testes are situated dorsally and provided with mature spermatozoa (Fig. 12A View Figure 12 ). On either side of the midline of the body, testicular follicles are arranged in five or six longitudinal zones and extend from the posterior level of the ovaries to almost the posterior end of the body.
The vasa deferentia expand to form large spermiducal vesicles, filled with sperm (Figs 12C View Figure 12 , 13 View Figure 13 , 14A View Figure 14 ). At the level of the penis bulb, the sperm ducts curve medio-dorsad and considerably decrease in diameter before penetrating the lateral wall of the penis bulb, and, subsequently, separately and symmetrically open into the ventro-lateral or mid-lateral portion of the relatively large, rounded seminal vesicle (Figs 12B View Figure 12 , 13 View Figure 13 , 14A View Figure 14 ). The latter is lined by a flat, nucleated epithelium and is surrounded by a layer of intermingled muscle fibres.
The ejaculatory duct arises from the antero-dorsal wall of the seminal vesicle and, subsequently, curves downwards towards the ventral root of the penis papilla. Near the ventral root of the papilla the ejaculatory duct is provided with a small diaphragm and changes its more or less vertical orientation by turning towards the tip of the penis papilla, having a subterminal opening at its tip (Figs 12C-H View Figure 12 , 13 View Figure 13 , 14A View Figure 14 ). The ejaculatory duct is lined by an infranucleated epithelium and is hardly surrounded by any musculature. The diaphragm receives the secretion of erythrophil glands, while the ejaculatory duct also receives the abundant secretion of extrabulbar penis glands (Figs 12E-H View Figure 12 , 14A View Figure 14 ).
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 lip. The conical or sub-cylindrical penis papilla has an oblique, ventro-caudad orientation and is covered by an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres. Near its dorsal root, the penis papilla is provided with a pronounced bump (Figs 12D-H View Figure 12 , 13 View Figure 13 , 14A View Figure 14 ).
The most characteristic feature of D. verrucula is the permanent dorsal bump on its penis papilla, a similar character being known only from Dugesia gibberosa Stocchino & Sluys, 2017. However, the latter is provided with two dorsal bumps on its penis papilla instead of one, while its ejaculatory duct opens terminally at the tip of the papilla ( Stocchino et al. 2017), in contrast to the subterminal opening in D. verrucula .
Apart from the penial bump and the subterminal opening of the ejaculatory duct, other characteristic features of D. verrucula are the asymmetrical penis papilla with ventrally displaced ejaculatory duct (character 1, state 1 in Sluys et al. 1998; see also Stocchino et al. 2017), and the presence of a duct between the seminal vesicle and the diaphragm (character 5, state 1 in Sluys et al. 1998). Besides D. verrucula , these two character states are also expressed in the following species: D. andamanensis (Kaburaki, 1925), D. austroasiatica Kawakatsu, 1985, D. batuensis , D. bengalensis , D. borneana Kawakatsu, 1972, D. burmaensis (Kaburaki, 1918), D. deharvengi Kawakatsu & Mitchell, 1989, D. gibberosa , D. hymanae (Sivickis, 1928), D. indonesiana Kawakatsu, 1973, D. japonica , D. lindbergi De Beauchamp, 1959, D. mertoni (Steinmann, 1914), D. naiadis Sluys, 2013, D. notogaea Sluys & Kawakatsu, 1998, D. novaguineana Kawakatsu, 1976, D. ryukyuensis Kawakatsu, 1976, D. siamana Kawakatsu, 1980, D. tamilensis Kawakatsu, 1980, D. majuscula , D. umbonata (see Sluys et al. 1998; Sluys et al. 2013; Stocchino et al. 2017; Song et al. 2020; Wang et al. 2021). However, these species all differ from D. verrucula in the gross anatomy of the copulatory apparatus or in detailed character states, such as the openings of the oviducts (common oviduct or symmetrical openings, in contrast to the asymmetrical oviducal openings in D. verrucula ), opening of the ejaculatory duct at the tip of the penis papilla (terminal, in contrast to the subterminal opening in D. verrucula ), or presence of a penial valve (absent in D. verrucula ).It is noteworthy that the cocoons of D. verrucula are unstalked, since usually in species of Dugesia the egg capsules are provided with a pedicel ( Sluys and Riutort 2018), the only other exception being D. bifida from Madagascar ( Stocchino et al. 2014). However, in other aspects of their reproduction D. verrucula and D. bifida are rather different, in that in the latter cocoons were produced by ex-fissiparous, sexualised specimens, which had developed hyperplasic ovaries; the juveniles emerging from these fertile cocoons gave rise to new fissiparous clones. This reproductive strategy differs much from the fully sexual life cycle of D. verrucula .
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