An integrative analysis and account of two new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the Hengduan Mountains, southwest China, with reflections on the historical biogeography of Eastern Palearctic / Oriental lineages Author Wu, Fan 0000-0001-6443-7955 College of Life Science, Henan Normal University, Xinxiang, 453007, China Author Wang, Lei 0000-0003-2328-1530 College of Life Science, Henan Normal University, Xinxiang, 453007, China Author Sluys, Ronald 0000-0002-9776-3471 Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, Netherlands Author Sun, Xin-Xin 0000-0001-8167-1495 College of Life Science, Henan Normal University, Xinxiang, 453007, China Author Liu, De-Zeng College of Life Science, Henan Normal University, Xinxiang, 453007, China Author Dong, Zi-Mei 0000-0002-6416-590X College of Life Science, Henan Normal University, Xinxiang, 453007, China Author Chen, Guang-Wen 0000-0001-8723-523X College of Life Science, Henan Normal University, Xinxiang, 453007, China text Zoosystematics and Evolution 2025 2025-08-19 101 4 1513 1529 journal article 311846 10.3897/zse.101.156742 6de266e1-4272-4944-983e-936bb81858ef 5B7A906E-8485-44B6-A20E-DBC0BAAF93FD Dugesia postica Chen & Dong sp. nov. Habitat and reproduction. Specimens were collected from Butterfly Spring, Dali City, at an elevation of 1980 m and a water temperature of 15 ° C, where the planarians inhabit small pools formed by spring water and a large number of free-roaming planarians could be observed (Figs 1 , 6 A, C ). In the field, sexually mature individuals were observed, as well as one yellow cocoon attached to a stone, which turned from yellow to black within 2 to 3 days. Under laboratory conditions, after approximately 60 days of culturing, 6 ex-fissiparous individuals sexualized and were capable of laying cocoons. The cocoons were spherical in shape (about 1 mm in diameter) and provided with a stalk (Fig. 6 D, E ). However, to date, neither cocoons collected from the wild nor those produced in the laboratory have hatched, suggesting that they are infertile. Figure 6. Habitat and external appearance of Dugesia postica . A. Sampling site and habitat; B. Sexually mature, live individual; C. Free-roaming worms; D. A yellow cocoon (indicated by a red arrow) attached to a stone; E. A cocoon with a stalk. Scale bars: 2 mm ( A, B ); 1 mm ( C – E ). Figure 7. Dugesia postica . A. Transverse section of paratype ZMHNU –NJWS 11, showing symmetrical openings of oviducts into bursal canal; B. Transverse section of paratype ZMHNU –NJWS 8 , showing vasa deferentia and seminal vesicle; C. Sagittal section of paratype ZMHNU –NJWS 3 , showing the location where the bursal canal connects to the copulatory bursa and the copulatory bursa directly behind the pharynx; D. Sagittal section of paratype ZMHNU –NJWS 1 , showing bump on penis papilla, diaphragm, and ejaculatory duct; E. Sagittal section of holotype ZMHNU –NJWS 4 , showing bump on penis papilla, diaphragm, and seminal vesicle; F. Sagittal section of paratype ZMHNU –NJWS 3 , showing diaphragm and ejaculatory duct. Scale bars: 100 μm Material examined. Holotype : ZMHNU –NJWS 4 , Butterfly Spring , Dali City , Yunnan Province , China ( 25°54'33"N , 100°5'41"E ), 6 October 2019 , coll. G-W Chen , Z-M Dong , and coworkers, sagittal sections on 14 slides . Paratypes : ZMHNU –NJWS 1 , 2, 5, 6, ibid., sagittal sections on 32, 51, 14, and 38 slides, respectively ; RMNH .VER.22273.1 , ibid., sagittal sections on 12 slides ; RMNH .VER.22273.2 , ibid., sagittal sections on 16 slides ; ZMHNU –NJWS 8 and 10, ibid., transverse sections on 32 and 41 slides ; ZMHNU –NJWS 7 , ibid., horizontal sections on 7 slides . Etymology. The specific epithet is derived from the Latin adjective posticus , behind or placed after, referring to the diaphragm being displaced towards the terminal part of the penis papilla. Diagnosis. Dugesia postica is characterized by the following features: symmetrical openings of the oviducts into the bursal canal; comparatively long bursal canal; broad penis papilla; generally with a slightly asymmetrical penis papilla, with a somewhat larger dorsal lip as compared with the ventral one; penis papilla with a small, dorsal bulge; a long connecting duct between the seminal vesicle and diaphragm; small diaphragm displaced towards the tip of the penis papilla; short ejaculatory duct opening subterminally through the ventral wall of the penial papilla; posterior portion of male atrium forming a relatively long and broad duct that connects with the common genital atrium. Description. Living, sexual animals measured 12.1–14.5 mm in length and 2.3–3.8 mm in width; asexual worms measured 9.7–11.6 mm in length and 1.6–1.9 mm in width. The head is bluntly triangular and provided with two short auricles and two eyes located within pigment-free spots. The dorsal colour is dark charcoal, with the edges being lighter, while the ventral surface is light grey (Fig. 6 B ). The pharynx is positioned in the middle of the body, measuring approximately one-sixth of the body length (Fig. 6 B ). The outer pharyngeal musculature is composed of a subepithelial layer of longitudinal muscles, followed by a thin layer of circular muscles. The inner pharyngeal musculature consists of a subepithelial and thick layer of circular muscle, followed by a thin layer of longitudinal muscle. The mouth opening is situated at the hind end of the pharyngeal pocket. The ovaries are oval, occupying about one-third of the dorso-ventral space. The ovaries are located at a considerable distance behind the brain, i. e., at approximately 1 / 4 th of the distance between the brain and the root of the pharynx. From the ovaries, the infranucleated oviducts originate at the postero-dorsal wall of the ovaries and run ventrally in a caudal direction to the level of the gonopore. Hereafter, the oviducts curve dorso-medially to open symmetrically into the anterior portion of that part of bursal canal where the canal communicates with the common genital atrium (Fig. 7 A ). Cyanophil shell glands discharge their secretion into the vaginal region of the bursal canal, immediately ventrally to the oviducal openings. A moderately sized, sac-shaped copulatory bursa is situated immediately posterior to the pharynx and usually at some distance anterior to the penis bulb; the bursa occupies the entire dorso-ventral space (Fig. 7 C, E ). The bursa is lined by a stratified, columnar, vacuolated epithelium provided with basal nuclei and is devoid of any surrounding musculature. This rather anterior position of the bursa, combined with the posterior portion of male atrium that forms a relatively long and broad duct, which connects with the posteriorly displaced common genital atrium, makes the bursal canal comparatively long. It runs backwards, more or less parallel to the body surfaces, while at the level of the gonopore it bends downwards to open into the common atrium. The bursal canal is lined with cylindrical, nucleated, ciliated cells and surrounded by a subepithelial layer of longitudinal muscles, followed by a layer of circular muscle. There is an extra outer layer of longitudinal musculature extending from the atrium to halfway along the bursal canal, forming the ectal reinforcement. The mature testes, provided with numerous spermatozoa, are situated dorsally and extend from the rear of the ovaries to the posterior end of the body. At the level of the copulatory bursa, the vasa deferentia expand to form spermiducal vesicles, which are packed with sperm. At the level of the penis bulb, the vasa deferentia curve dorso-medially, decrease considerably in diameter, forming narrow ducts, and, subsequently, penetrate the penis bulb to open symmetrically into the posterior portion of the seminal vesicle (Fig. 7 B ). The sperm ducts are lined with nucleated cells and surrounded by a layer of circular muscles. The penis bulb occupies about one-half of the dorso-ventral space (Figs 7 D – F , 8 , 9 ) and houses a small, oval-shaped or globular seminal vesicle, which is lined by a flat, nucleated epithelium and surrounded by a layer of irregularly crosswise arranged muscle fibers (Fig. 7 D – F ). From the posterior wall of the seminal vesicle arises a comparatively broad and long connecting duct that opens into a rather small and stubby diaphragm (Fig. 7 D ). This connecting duct is lined with a nucleated epithelium and surrounded by several layers of thin circular muscles. Seminal vesicle and connecting duct receive the openings of orange-staining penial glands. The diaphragm points into the short ejaculatory duct, which curves abruptly downwards to open subterminally through the ventral wall of the penis papilla. The diaphragm receives the abundant secretion of erythrophil penis glands (Fig. 7 D – F ). The ejaculatory duct is lined with a cuboidal, infranucleated epithelium and is devoid of any discernible musculature. Cyanophil penis glands discharge their secretion into the ejaculatory duct (Fig. 7 D, E ). A spermatophore is present in the ejaculatory duct and / or the male atrium of the specimens NJWS 1, NJWS 2, NJWS 4, and NJWS 6. Figure 8. Dugesia postica . Sagittal reconstruction of the copulatory apparatus of holotype ZMHNU –NJWS 4 . A. Male copulatory apparatus; B. Female copulatory apparatus. Scale bars: 100 μm Figure 9. Dugesia postica . Sagittal reconstruction of the copulatory apparatus of paratype ZMHNU –NJWS 1 . A. Male copulatory apparatus; B. Female copulatory apparatus. Scale bars: 100 μm The penis papilla is a rather broad structure, which is somewhat asymmetrical in that it has a slightly larger dorsal lip as compared with the ventral one. This condition is due to the slightly ventrally displaced position of the seminal vesicle and the equally displaced course of the connecting duct. The asymmetrical condition is only weakly expressed in the paratypes ZMHNU – NJWS 1 and NJWS 2 but more strongly present in holotype NJWS 4 and paratype NJWS 6 (Figs 8 , 9 ). The papilla is characterized by a bulge or bump on its dorsal wall, where erythrophilic glands discharge their secretion to the exterior. The penis papilla is lined with a nucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibers (Figs 7 D , 8 , 9 ). The penis papilla fills almost the entire anterior portion of the male atrium, but the posterior portion of male atrium forms a relatively long and broad duct that connects with the common genital atrium. The male atrium is lined with a cuboidal, nucleated epithelium. The common atrium communicates with the gonoduct, which leads to the ventral gonopore; the gonoduct is lined by a tall, columnar epithelium and receives the openings of abundant cement glands (Figs 7 F , 8 , 9 ). Discussion. A centrally or ventrally displaced course of the ejaculatory duct and a terminal or subterminal opening of the ejaculatory duct at the tip of the penis papilla form important taxonomic characters in the identification of species of Dugesia and for resolution of phylogenetic relationships within the genus (cf. Sluys et al. 1998 ; Stocchino et al. 2017 ). Another important character concerns the absence or presence of a duct intercalated between the seminal vesicle and the diaphragm (character state 5– 1 in Sluys et al. 1998 ). With respect to these three characters, D. postica exhibits the combination of character states concerning a ventrally displaced ejaculatory duct (i. e., an asymmetrical penis papilla), a subterminal ventral opening of the ejaculatory duct, and a duct (either long or short) connecting the diaphragm with the seminal vesicle. In this context it is important to realize that a subterminal opening of the ejaculatory duct does not necessarily imply that the penis papilla is asymmetrical, since the latter condition is defined by the ventrally or dorsally displaced course of the ejaculatory duct at the level of the root of the penis papilla (cf. Stocchino et al. 2017 ). The combination of these three character states (ventrally displaced ejaculatory duct, subterminal opening ejaculatory duct, and interconnecting duct between diaphragm and seminal vesicle) is also present in the following species: D. adunca Chen & Sluys, 2022 ; D. ancoraria Zhu & Wang, 2024 ; D. andamanensis (Kaburaki, 1925) ; D. austroasiatica Kawakatsu, 1985 ; D. batuensis Ball, 1970 ; D. bengalensis ; D. deharvengi Kawakatsu & Mitchell, 1989 ; D. gemmulata ; D. japonica Ichikawa & Kawakatsu, 1964 ; D. lindbergi De Beauchamp, 1959 ; D. novaguineana Kawakatsu, 1976 ; D. tamilensis Kawakatsu, 1980 ; and D. verrucula Chen & Dong, 2021 ( Sluys et al. 1998 ; Wang et al. 2021 ; Zhu et al. 2024 ; Chen et al. 2022 ; Liu et al. 2022 b ; Ichikawa and Kawakatsu 1964 ; Kawakatsu et al. 1985 ; Kawakatsu and Mitchell 1989 ). However, D. postica differs from all of these congeners in several respects. For example, the symmetrical openings of the oviducts into the bursal canal in D. postica contrast with the asymmetrical openings in D. adunca , D. ancoraria , D. gemmulata , and D. verrucula . Further, in D. andamanensis and D. deharvengi the oviducts fuse to form a common oviduct that opens into the bursal canal, in contrast to the separate openings in D. postica , while in D. lindbergi the oviducts (which open symmetrically into the bursal canal) may sometimes unite to form a very short common oviduct. Dugesia batuensis possesses a penial valve, which is absent in D. postica . In D. japonica , the vaginal region is surrounded by a hump of mesenchymal tissue traversed by several rows of longitudinal and circular muscle fibers, which is absent in D. postica . In D. bengalensis the vasa deferentia open through the roof of the seminal vesicle, while the ectal reinforcement around the bursal canal extends up to the copulatory bursa, both conditions being different in D. postica . The ectal reinforcement layer of D. lanzai also extends up to the bursa, while it also has an elongated seminal vesicle that differs from the small and rounded vesicle in D. postica . Dugesia novaguineana shows the following features, which are all different from the situation in D. postica : (a) large, hemispherical penis bulb; (b) large, ovoid seminal vesicle; (c) stubby penis papilla; and (d) the posterior half of the bursal canal being provided with a thick coat of muscles. Interestingly, D. ancoraria , D. austroasiatica , D. tamilensis , and D. verrucula all have a kind of dorsal bump on the penis papilla, as is the case also in D. postica . In the above it was already shown that D. postica anatomically differs from D. ancoraria and D. verrucula , notably with respect to the oviducal openings into the bursal canal, despite the presence of a penial bump in these three species. However, anatomical differences between D. postica on the one hand and D. austroasiatica and D. tamilensis on the other hand are more difficult to discern. In both D. austroasiatica and D. tamilensis the ectal reinforcement muscle layer around the bursal canal does not reach up to the bursa, as is the case also in D. postica . It could be argued that in D. tamilensis (a) the seminal vesicle is considerably larger, (b) the connecting duct between seminal vesicle and diaphragm is shorter (and thus the diaphragm not displaced towards the tip of the penis papilla), and (c) the ejaculatory duct has a much more extremely subterminal opening, situated near the root of the penis papilla, all being conditions that differ from those in D. postica . In D. austroasiatica the seminal vesicle is indeed also rather large, in contrast to the small vesicle in D. postica . A striking similarity between D. postica and D. austroasiatica concerns the fact that in the latter the penial bump or fold also receives the openings of many erythrophilic gland ducts, while its ejaculatory duct also receives abundant secretion of penial glands (cf. Kawakatsu et al. 1985 , Fig. 3 A, D ; Kawakatsu et al. 1986, Fig. 3 ). It is noteworthy that Kawakatsu et al. (1985 , 1986) presumed that D. austroasiatica was introduced in Japan from some Southeast Asian country. However, none of the species mentioned above in the comparative discussion, including D. austroasiatica , shows the characteristic posteriorly displaced position of the diaphragm, nor the long bursal canal or the long and broad duct connecting male and common atrium, as present in D. postica . Unfortunately, for not all of the Dugesia species discussed above is molecular information available, and therefore they could not be incorporated into our analyses. However, D. adunca , D. ancoraria , D. batuensis , D. bengalensis , D. japonica , D. gemmulata , D. verrucula , and D. deharvengi could be included in our phylogenetic and molecular distance analyses, which revealed that none of these species is similar to or genetically closely related to D. postica (Fig. 2 ; Suppl. materials 2 , 3 ).