Integrative descriptions of two new species of Dugesia from Hainan Island, China (Platyhelminthes, Tricladida, Dugesiidae) Author Wang, Lei College of Life Science, Henan Normal University, Xinxiang, 453007 Henan, China & Medical College, Xinxiang University, Xinxiang 453003, China Author Dong, Zi-mei https://orcid.org/0000-0002-6416-590X College of Life Science, Henan Normal University, Xinxiang, 453007 Henan, China Author Chen, Guang-wen https://orcid.org/0000-0001-8723-523X College of Life Science, Henan Normal University, Xinxiang, 453007 Henan, China chengw0183@sina.com Author Sluys, Ronald https://orcid.org/0000-0002-9776-3471 Naturalis Biodiversity Center, Leiden, the Netherlands Author Liu, De-zeng College of Life Science, Henan Normal University, Xinxiang, 453007 Henan, China text ZooKeys 2021 2021-04-05 1028 1 28 http://dx.doi.org/10.3897/zookeys.1028.60838 journal article http://dx.doi.org/10.3897/zookeys.1028.60838 1313-2970-1028-1 A5EF1C8A805B4AAEACEBC1CACB691FCA 3A44C7AE33E45C6DB6B86AFB136AEFCE Dugesia semiglobosa Chen & Dong sp. nov. Figures 3 , 4 , 5 , 6 , 7 Material examined. Holotype : ZMHNU-JWT5, Jiuwentang village ( 19°23'10"N , 110°19'42"E ; alt. 80 m above sea level (a.s.l.), Anding County , Hainan Province , China , 24 February 2018 , coll. GW Chen and co-workers, sagittal sections on 17 slides. Paratypes : ZMHNU-JWT1, ibid., sagittal sections on 17 slides; ZMHNU-JWT2, ibid., sagittal sections on 14 slides; ZMHNU-JWT3, ibid., sagittal sections on 16 slides; ZMHNU-JWT4, ibid., horizontal sections on 7 slides; ZMHNU-JWT6, ibid., transverse sections on 33 slides; ZMHNU-JWT7, ibid., transverse sections on 30 slides; ZMHNU-JWT8, ibid., sagittal sections on 49 slides; ZMHNU-JWT9, ibid., sagittal sections on 30 slides. Diagnosis. Dugesia semiglobosa is characterized by the following features: hemispherical, asymmetrical penis papilla with ventrally displaced ejaculatory duct opening terminally at tip of penis papilla; absence of duct intercalated between seminal vesicle and diaphragm; vasa deferentia separately opening into mid-dorsal portion of intrabulbar seminal vesicle; two diaphragms in the ejaculatory duct; symmetrical openings of oviducts into bursal canal; copulatory bursa formed by expansion of bursal canal, lined with complex stratified epithelium, which projects through opening in bursa towards intestine, without having open communication with the gut; mixoploid chromosome complement diploid (2n = 16) and triploid (3n = 24); chromosomes metacentric. Etymology. The specific epithet is derived from the Latin semis , half, and globosus , spherical, and alludes to the hemispherical penis papilla. Habitat and reproduction. Specimens were collected from Jiuwentang volcano spring at an altitude of 80 m a.s.l. and with a water temperature of 23 °C. This spring is the third largest volcano spring in China, while it is also its largest selenium-rich spring (Fig. 3A, B ). None of the animals was sexually mature at collection. However, after having been kept under laboratory conditions for ~ 150 days, the animals sexualized and laid cocoons. Newly laid cocoons are yellow, but turn dark brown after 2 to 3 days. Cocoons are spherical in shape (1 mm in diameter) and provided with a stalk. Thus far, none of the cocoons hatched, thus, most likely being infertile. Figure 3. Habitat and external appearance of Dugesia semiglobosa A sampling site B habitat C sexually mature, living individual (anterior is to the up). Abbreviations: coa: copulatory apparatus; e: eye; ph: pharynx. Scale bar: 1 mm. Karyology. Each of the five, randomly selected specimens exhibited mixoploid chromosome complements. In a total of 100 metaphase plates examined, 67 exhibited diploid chromosome portraits of 2n = 2x = 16, while in 20 plates chromosome complements were triploid with 2n = 3x = 24 chromosomes (Fig. 4 ); chromosome complements of the remaining 13 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 2 . The first pair of chromosomes is clearly larger than others, being 1.8 times larger than the shortest chromosome. Chromosomal plates and idiogram are shown in Fig. 4 . Table 2. Karyotype parameters (mean values and standard deviations) of Dugesia semiglobosa ; m: metacentric.

Chromosome	Relative length	Arm ratio	Centromeric index	Chromosome type
1	17.43 +/- 0.93	1.12 +/- 0.09	47.31 +/- 1.83	m
2	14.71 +/- 0.46	1.19 +/- 0.09	45.88 +/- 1.69	m
3	13.69 +/- 0.45	1.30 +/- 0.18	43.86 +/- 3.53	m
4	12.93 +/- 0.46	1.34 +/- 0.14	43.21 +/- 2.54	m
5	11.78 +/- 0.26	1.29 +/- 0.11	43.82 +/- 2.12	m
6	10.80 +/- 0.17	1.27 +/- 0.06	44.33 +/- 1.35	m
7	9.92 +/- 0.53	1.28 +/- 0.10	44.24 +/- 1.97	m
8	8.72 +/- 0.64	1.21 +/- 0.05	45.40 +/- 1.02	m

Figure 4. Dugesia semiglobosa A idiogram B1 metaphasic plate of diploid cell B2 karyogram of diploid cell C1 metaphasic plate of triploid cell C2 karyogram of triploid cell. Description. Body of living asexual specimens is 4-6 mm in length and 0.72-0.85 mm in width, while in sexualized animals the body is 8-12 mm in length and 1.25-1.51 mm in width. Two eyes located in the center of the head, being situated in pigment-free patches. Each pigmented eye cup houses numerous photoreceptor cells. Head of low triangular shape and provided with two blunt auricles. Body light brown dorsally, excepting the pale body margin and accumulations of pigment following the outline of the pharynx. Ventral surface is paler than the dorsal one (Fig. 3C ). Pharynx situated in the mid-region of the body, measuring ~ 1/5th of the body length (Fig. 3C ). 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 thick layer of circular muscles; extra inner layer of longitudinal muscles is absent (Fig. 5A ). The inner pharyngeal musculature consists of a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle, the former being thicker than the latter (Fig. 5A ). Figure 5. Dugesia semiglobosa A transverse section of paratype JWT 6, showing longitudinal and circular muscles in outer and inner pharyngeal musculature B transverse section of paratype JWT 7, showing hyperplasic ovaries. C sagittal section of holotype JWT 5, showing testes and small ovary D transverse section of paratype JWT 7, showing symmetrical openings of the oviducts E transverse section of paratype JWT 6, showing spermiducal vesicles F transverse section of paratype JWT 7, showing openings of the vasa deferentia into the seminal vesicle. The anterior is to the front in A, B, D, E, F , and anterior is to the right in C . Abbreviations: bc: bursal canal; ca: common atrium; cm: circular muscles; in: intestine; lm: longitudinal muscles; lod: left oviduct; ov: ovary; rod: right oviduct; spv: spermiducal vesicle; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bars: 100 μm . In specimen JWT-7 the ventrally placed ovaries are clearly hyperplasic and fused to form a single mass that extends into the lateral regions of the body (Fig. 5B ). In other specimens examined (JWT-2, JWT-3, JWT-5, JWT-6, JWT-10), the gonads are generally atypical, in that they are very small (Fig. 5C ). Only in specimen JWT-8 the ovaries are more or less of normal size. In general, ovaries are situated at a short distance behind the brain. From the ovaries the oviducts run ventrally in a caudal direction to the level of the genital pore, after which they curve dorso-medially to open separately and symmetrically into the ventral portion of the bursal canal, close to its communication with the atrium (Figs 5D , 7 ). The small, dorsally located testes are well developed and provided with mature spermatozoa (Fig. 5C ). Testicular follicles are arranged on either side of the midline of the body in nine or ten longitudinal zones, extending from the posterior level of the ovaries to almost the posterior end of the body. The vasa deferentia, filled with spermatozoa, expand to form spermiducal vesicles at the level of the pharynx that occupy <1/3rd of the dorso-ventral space (Fig. 5E ). At the level of the penis bulb the vasa deferentia decrease in diameter and bend sharply towards the dorsal body surface and upon recurving ventrad they penetrate the dorso-lateral wall of the penis bulb to open separately and symmetrically into the mid-dorsal portion of the seminal vesicle (Figs 5F , 6A , 7 ). The sperm ducts are lined with nucleated cells and are surrounded by a layer of circular muscles. The reniform seminal vesicle is lined by a flat, nucleated epithelium and is surrounded by intermingled muscle fibers (Figs 5F , 6A ,). The seminal vesicle opens into the ejaculatory duct via a small, pointed diaphragm. A second, rather large and blunt diaphragm is located in the proximal portion of the ejaculatory duct (Figs 6A , 7 ). The ejaculatory duct is lined by an infranucleated epithelium; we were unable to discern any musculature around the duct. The ejaculatory duct follows a noncentral, ventrally displaced course through the penis papilla, opening at its tip, thus resulting in an asymmetrical penis papilla in which the dorsal lip is much larger than the ventral one (Figs 6A , 7 ). Figure 6. Dugesia semiglobosa A sagittal section of holotype JWT 5, showing reniform seminal vesicle, two diaphragms in the ejaculatory duct, ventral course of the ejaculatory duct, and hemispherical penis papilla B sagittal section of holotype JWT 5, showing copulatory bursa. C transverse section of paratype JWT 7, showing copulatory bursa D sagittal section of holotype JWT 5, showing bursal canal and its surrounding musculature E sagittal section of holotype JWT 9, showing small copulatory bursa, and mouth F horizontal section of paratype JWT 4, showing hemispherical penis papilla. The anterior is to the right in A, B, D, E, F and anterior is to the front in C . Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; cm: circular muscles; d: diaphragm; ed: ejaculatory duct; in: intestine; lm: longitudinal muscles; m: mouth; ma: male atrium; pp: penis papilla; sv: seminal vesicle; vd: vas deferens. Scale bars: 100 μm . The complete penis, comprising papilla and bulb, is nearly spherical, with the penis papilla being a hemispherical structure that is covered with an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres (Fig. 6A ). The penis papilla almost completely occupies the male atrium, the latter communicating with the common atrium via a slight constriction (Figs 6E, F , 7 ). The common atrium opens to the exterior via a gonoduct, which is lined by a columnar epithelium and receives the openings of abundant cement glands. Figure 7. Dugesia semiglobosa . 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; in: intestine; m: mouth; od: oviduct; pp: penis papilla; sg: shell glangs; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bar: 100 μm . From its point of communication with the common atrium, the bursal canal gradually expands in diameter, meanwhile curving anteriad, while running on the left side of the male copulatory apparatus (Figs 6D , 7 ). The bursal canal is lined with columnar, nucleated, ciliated cells and is surrounded by a subepithelial layer of longitudinal muscles, followed by a layer of circular muscle (Figs 6D , 7 ). An ectal reinforcement layer of longitudinal muscles runs from the vaginal region to approximately halfway along the bursal canal. Shell glands discharge their erythrophil secretion into the vaginal region of the bursal canal, near the oviducal openings. More or less dorsally to the penis bulb, the bursal canal first decreases somewhat in diameter but thereafter greatly expands to give rise to a more or less globular structure immediately in front of the male complex. This globular structure may be called a copulatory bursa since it occupies the same position as in other species of Dugesia , or freshwater planarians in general. The bursa is lined with a complex type of stratified epithelium. The basal portion of this epithelium consists of more or less cuboidal, nucleated cells and is basically a continuation of the lining epithelium of the rest of the bursal canal, albeit that there the cells are columnar. This basal layer is followed by a thick zone of stratified, non-nucleated squamous epithelium, leaving very little room for any lumen within the bursa. The cells of this squamous layer have an irregular shape, while those in the top zone, near the lumen, are vacuolated and provided with granular, cyanophil inclusions. The bursa is surrounded by a layer of longitudinal muscles (Figs 6C , 7 ). However, at one point this muscle layer is interrupted because of the presence of an opening in the bursa. This opening is located, more or less, at the antero-dorsal wall of the bursa. A portion of the squamous inner lining of the bursa projects through the opening and approaches and/or touches portions of the gut that are in its proximity. However, in none of the specimens examined we discerned an open connection between bursa and intestine. Discussion. The curious copulatory bursa of D. semiglobosa is unparalleled among species of Dugesia , or freshwater planarians in general. Generally, copulatory bursae are lined with an epithelium consisting of tall columnar, vacuolated, and nucleated cells, while they are surrounded by only a very weak musculature. The structure of the bursa of D. semiglobosa differs considerably from this ground-plan condition, as it is basically an expanded continuation of the bursal canal, albeit with a simpler coat of muscles and a more complex lining epithelium. Dugesia semiglobosa exhibits a combination of three characteristic features (ventrally displaced ejaculatory duct, absence of duct intercalated between seminal vesicle and diaphragm, terminal opening of ejaculatory duct) that is found in only nine congeners, viz., D. annandalei Kaburaki, 1918, D. damoae De Vries, 1984, D. didiaphragma De Vries, 1988, D. elegans De Vries, 1984, D. gibberosa Stocchino & Sluys, 2017, D. maghrebiana Stocchino et al. 2009 , D. malickyi De Vries, 1984, D. naiadis Sluys, 2013, and D. sinensis. Among these nine species, D. semiglobosa most closely resembles D. didiaphragma and D. maghrebiana in that these species also possess two diaphragms, in contrast to all other species mentioned. Presence of two diaphragms is a rare condition among species of Dugesia and is only known from three other species, viz., D. bijuga Harrath & Sluys, 2019, D. machadoi de Beauchamp, 1952, and D. mirabilis De Vries, 1988. However, in these three last-mentioned species the ejaculatory duct runs a central course through the penis papilla, in contrast to the ventral trajectory in D. semiglobosa . Further, there are ample other features that preclude assignment of our specimens to either of these Afrotropical species. Neither is it possible to assign our animals to D. didiaphragma or D. maghrebiana as they lack the large seminal vesicle enclosed by a highly muscularized, elongated penis bulb of the former and the knob-like extension on the penis papilla of the latter. It is interesting to note that in all species in possession of two diaphragms, the small proximal diaphragm basically is formed by a non-glandular constriction of the seminal vesicle, while the true diaphragm is a larger structure and receives the secretion of penial glands, as usual for the diaphragm of species of Dugesia . The same situation applies to the two diaphragms in D. semiglobosa . It is noteworthy that in D. mirabilis both the proximal and distal diaphragm are glandular ( De Vries 1988 ).