A new species of the genus Bothrioplana (Platyhelminthes: Bothrioplanida: Bothrioplanidae) and a new species of the genus Pentacoelum (Tricladida: Bdellouridae) from southern China Author He, Yi Author Zhao, Jia-Qi Author Ning, Wan-Ru Author Zhuang, Jie-Yi Author Zhang, Yu Author Wang, An-Tai text Zootaxa 2016 4179 2 209 224 journal article 10.11646/zootaxa.4179.2.2 4b5b78d6-60aa-4671-aa40-d5a18fb647cd 1175-5326 260533 7CA48761-3F3D-404F-B8D8-476F0EB06293 Pentacoelum sinensis Wang & Zhao , n. sp. ( Figs. 6–10 ) Type material. Holotype: PLA-Pe0070, mounted specimens. Paratype: PLA-Pe0071–PLA-Pe0078 (PLA- Pe0071–PLA-Pe0072, mounted specimens; PLA-Pe0073–PLA-Pe0078, serial section). Collection locality. In a wetland in Shenzhen Sea Waterlands , located in the west coast of Shenzhen , Guangdong , China ( 22°43′16″N , 113°46′03″E ). Etymology. The species is named because it was discovered in China . Description. The body of mature individual is transparent, flat and elliptical in shape, without pigment on the surface. When moving, it is in an elongated shape ( Fig. 6 B). The length of the specimen is 500–770 µm. The width of the middle part of the body is 350–530 µm ( Figs. 6 A and 8). A pair of black and round eyes (e), 26 µm in diameter, is located at the anterior 1/6 position of the body ( Figs. 6 A–B, E). The distance between the two eyes is 40 µm. The distance from eye to body side is 130 µm. FIGURE 6. Pentacoelum sinensis n. sp. : A–B. living specimens; C–D. eggs (C. egg just laid; D. three developing embryos in the egg). E–G. ventral view (E. body in whole; F. posterior end; G. penis). Scale bars: A–E=100 µm, F=20 µm, G=10 µm. Digestive system. The mouth (m) is situated median-ventrally at the posterior 1/5 part of the body. The plicate pharynx (p) is located in the posterior part of the body and constitutes 1/4 of the body length. Three intestinal branches connect with the base of the pharynx. One of them extends forwards along the median line to the anterior part between the two eyes. This intestine (i) is relatively slender, has no branch and stops at 1/2 position from eyespots to the anterior end. The other two branches extend aside, forming the intestinal branches along the body. Each intestine intestinal branch has 9–12 shorter branches perpendicular to the body edge. Among the 180 living specimens examined, we did not find any individual with side intestines merge at the tail end ( Figs. 6 A–B, 8). Reproductive system. Hermaphrodite. A pair of elliptical-shaped testes (t) lies in the base between the anterior intestinal branches and the mid-intestinal branches. The cavities of testes are filled with sperm (s) ( Figs. 6 E, 8). The vasa deferentia (vd) extend separately from the inner sides of testes, go backward along the two sides of the pharynx and finally enter the seminal vesicle (sv) respectively ( Fig. 8 ). The vas deferens at each side of pharynx swells obviously ( Fig. 8 ). The spherical seminal vesicle at the base of penis bulb (pb) occupies 1/2 of the volume of penis bulb and is connected with the ejaculatory duct (ed). There is a very obvious cavity in the middle part of the ejaculatory duct ( Figs. 6 F–G, 9). An obvious penis papilla (pp) can be found at the end of penis bulb. Epidermis of penis and penis papilla are composed of a monolayer of flat and nucleated epithelial cells, while the internal part is a muscle layer. The peripheral space of the penis is male atrium (ma) ( Fig. 9 A). A pair of ovaries (o) is situated between the testes and the brain (b) ( Fig. 6 E, Fig. 8 ). Two oviducts (ov) go further back laterally to male atrium and then enter the two uteri (u) respectively. Note that the uteri of the new species localize at similar position as those of the other species of Pentacoelu m. The spherical uteri, 50–55 µm in diameter, lie at both sides of the male atrium. The peripheral part of the uterus is a 4 µm thick muscular layer, while the interior part is composed of a monolayer of epithelial cells ( Figs. 7 D–E). An obvious receptaculum seminalis (rs), which is filled with foreign sperm, is situated ventrally and posterior to each uterus. A muscular vagina externa (ve), 20–25 µm in length and 3.0–3.5 µm in thickness, is located at the ventral side of each receptaculum seminalis and goes externally ( Figs. 7 D–E). An egg could be found in the uterus ( Figs. 7 F–H). The left and right oviducts coming out from the two uteri merge as a common oviduct dorsally to male atrium ( Fig. 9 B). The common oviduct enters the male atrium caudal-ventrally and shares a common gonopore. FIGURE 7. Pentacoelum sinensis n. sp. : A–C. sagittal section of penis; D–G. sagittal section of uterus; H. dorsal view of the posterior end of the mounted specimen. Scale bars: 20 µm. Habits and reproduction. The specimens in the evaporating dish seldom move and live gregariously. The digestive and reproductive systems are readily detected under stereoscopic microscope. Copulation usually takes place in darkness, during which the mature individual slowly slides on the back of another individual. Their tail ends met together and uplifted slightly. Their penises stretched out almost simultaneously and then inserted into the vagina externa of each other for fertilization. The whole copulatory process usually lasts for 10 min. After copulation, they separated and retracted their penises in a few seconds ( Figs. 10 A–B). Eggs were found in the uteri ( Figs. 7 F–H). Newly produced eggs expanded greatly when laid in water. The light yellow eggs were about one-third as big as the mature worm and had already underwent the first cleavage ( Figs. 6 C–D, Figs. 10 C–D). The next day, the egg shell became brown-red, thinner and transparent. Three embryos with paired black eyespots were observed inside the egg ( Fig. 6 D). Observation shows that after oviposition, the adult usually hibernates aside the eggs, suggesting that this species has the habit of protecting eggs. FIGURE 8. Pentacoelum sinensis n. sp. : body in whole. Scale bar: 100 µm. FIGURE 9. Pentacoelum sinensis n. sp. : A. side view of reproductive system; B. ventral view of reproductive system. Scale bars: A=50 µm, B=100 µm. Habitat. The samples were collected from a wetland in Shenzhen Sea Waterlands , The salinity and pH of the ambient water was 12‰ and 9.0 2. The water depth was between 0.6 to 1.2 meters. The sampling site was an artificial seawater mariculture lake constructed in 2000, which is not only abundant in fishes, shrimps and crabs, but also in benthic animals such as polychaetes. In April 2016, 20 more individuals were also found in the protected natural mangrove forest in Shenzhen , China. Discussion. To date, 4 species of Pentacoelum were recorded in the world, namely P. fucoideum ( Westblad, 1935 ) , P. punctatum ( Brandtner, 1935 ) , P. kazukolinda ( Kawakatsu & Mitchell 1984 ) and P. hispaniense ( Sluys 1989 ) . However, a recent study shows that P. hispaniense should be called P. k a z u ko l i n da and that the species has a worldwide distribution ( Sluys et al. 2015 ). Therefore, there are actually only three species within this genus. In P. fucoideum ( Westblad 1935 ) , the external morphology and the localization of the reproductive organ are similar to those of P. s i ne n s i s n. sp. However, the eyespots of P. fucoideum have less pigment and are usually invisible in living specimens. The ejaculatory duct is in straight tubular shape, without obvious swelling cavity. As for P. sinensis n. sp. , the black round-shaped eyespots are readily detected from both the living and mounted specimens. In addition, there is an obvious cavity in the middle part of the ejaculatory duct. As for P. punctatum , the type specimen was found in Lake Pontchartrain , New Orleans , Louisiana , USA (Sluys & Bush 1988). The dorsal surface of the body has dense pigment. The pharynx is located in the middle of the body and occupies about 1/6 to 1/4 of the body length. Two intestinal branches meet at the tail end. Each side of the body has 5–6 small testes, respectively. A testis can also be found in the middle of the two hindguts. Before entering seminal vesicle, the vasa deferentia merge as a common vas deferens. FIGURE 10. Pentacoelum sinensis n. sp. : reproduction process and egg. A–B. copulation (penises enter the vagina externa of each other. The arrows indicate the direction of the penises); C–D. egg (the egg expands within seconds after being laid and the diameter increases about five times. The soft shell appears in light yellow and becomes hard 2-3 hours later.) Scale bars: A- D=150 µm. In P. k a z u ko l i n da , the type specimen was found in Algemesi, Valenciana, Spain ( Sluys 1989 ). Except for the body edge, the mid-dorsal stripe and the area anterior to the eyespots, the dorsal body surface shows a dense brown pigmentation. The pharynx occupies about 1/7–1/6 of the body length. There are 15–20 testes on each side of the body. The two vasa deferentia merge as a common vas deferens before entering the penis bulb. The ovaries lie at 1/ 4 to 1/3 of the distance between the brain and the base of the pharynx. The female copulatory apparatus contains a bursa copulatrix. For P. sinensis n. sp. , the body is transparent and devoid of any pigmentation. The eyespots are black and round with 3 retinal cells each. The pharynx occupies about 1/4 of the body length and is located in the posterior half of the body. Two caudal intestinal branches do not merge at the posterior end of the body. There is only one pair of big testes within the body. The vasa deferentia at both sides merge before entering the seminal vesicles. There is no bursa copulatrix behind the male atrium, but a pair of uteri, receptaculum seminalis and vagina externa. It is worth to note that lateral bursae of P. sinensis n. sp. might have gained a new evolutionary function as copulatory bursae and uteri. The two round cavities at the tail of Pentacoelum species are usually called lateral bursae ( Sluys 1989 ). In this contribution, the cavities of P. s i n e ns i s n. sp. (called uteri) were found to be connected with oviducts. All specimens were examined carefully, among which three were found to have egg in uterus. During copulation, the two individuals inserted their penis into the vagina externa instead of the common gonopore of each other. The vagina externa is connected with receptaculum seminalis and uterus. Oviducts from the paired uteri merge at the dorsal side behind the male atrium. Importantly, there are shell glands on the surface of the oviducts. To summary, the morphology of the reproductive system and the copulatory behavior of this new species are distinct from those of the above reported species. In view of this, the P. s i n e ns i s n. sp. is identified as a new species of the genus of Pentacoelum . Further studies on the phylogenetic position of this new species will enable a more thorough understanding of the evolutionary relation of P. s i n e ns i s n. sp. and the closely related species.