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The complete mitochondrial genome of a marine triclad Miroplana shenzhensis (Platyhelminthes, Tricladida, Maricola)

Paucumara falcata Wang & Li, sp. nov. Material examined. Holotype, PLA-Pa001, eastern beach of Shenzhen City, Guangdong Province, China, 22°28′N, 114°31′E, 29th April 2017, coll. Wei-Xuan Li, sagittal sections on 9 slides. Paratypes: PLA-Pa002, ibid., one sagittal sections on 4 slides; PLA-Pa003, ibid., horizontal sections on 5 slides; PLA-Pa004, ibid., whole mount on 1 slide; PLA-Pa005, ibid., whole mount on 1 slide; PLA-Pa006, ibid., whole mount on 1 slide. All specimens are deposited in IZCAS. Observations were made on both live and preserved specimens. Etymology. The specific epithet is derived from the Latin adjective falcatus, sickle-shaped, and alludes to the curved, chitinized tip of the musculo-parenchymatic organ. Diagnosis. Paucumara falcata is characterized by: three pale yellow transverse pigmentation bands, one at the anterior body margin, another immediately behind the eyes and a third one across the tail end of the body, while a brown band extends anteriorly from the level of the eyes; 8–11 testicular follicles on either side of the body, extending from immediately behind the ovaries to half-way along the pharyngeal pocket; a musculo-parenchymatic organ provided with a sickle-shaped chitinized, sclerotic tip or stylet. Description. Live specimens with low-triangular head and sharply pointed hind end, their length ranging between 3.8–4.1 mm and their width between 0.48–0.58 mm (n=5) (Figs. 7 A–B, 8A, 10A). A pair of black, kidney-shaped eyes is located at a position measuring 1/8 of the total body length, as determined from the anterior margin; the eyes are set rather close together, the distance between them ranging between 37–43 µm (n=5). Each eye cup has a large semi-circular lens (Fig. 8A). The dorsal body surface shows conspicuous pale yellow stripes as well as a region with brownish pigmentation. Pale-yellow transverse stripes occur at the anterior margin and immediately behind the eyes, and across the tail end of the body (Fig. 7B). The transverse band at the anterior margin is 300–330 µm in width, while the stripe behind the eyes measures 100–120 µm in width, while the one on the tail is 140–180 µm wide (n=5). Further there is a pale yellow longitudinal stripe that runs from the transverse pale band behind the eyes to the base of the pharynx that is 600–680 µm long and has a width of 90–100 µm (n=5). In addition to these pale stripes there are snowflake-like pale yellow specks scattered on the dorsal surface. A broad band of brown pigment extends anteriorly from the level of the eyes for a distance of about 180–200 µm (Figs. 7 A– B). The mouth opening is located at a distance of about 1/3 of the body length, as measured from the posterior body margin; the mouth is situated at the posterior end of the pharyngeal cavity (Fig. 10). The cylindrical pharynx is 840–950 µm in length and 190–220 µm in width (n=5) (Figs. 7B, 8A, 10A). The anterior intestinal ramus extends anteriorly to the eyes, while the two posterior gut trunks unite in the hind end of the body (Figs. 7A, 10A). The anterior gut trunk gives rise to a pair of lateral intestinal branches that extends anteriorly well beyond the eyes and also gives off 4–6 pairs of short lateral post-ovarial branches (Fig. 10A). Each posterior intestinal ramus gives off 10–12 short lateral branches. Mature individuals have 8–11 testes on either side of the body, the ventrally located follicles extending from directly behind the ovaries up to about half-way along the pharyngeal cavity (Figs. 7C, 9A, 10A). Immediately posterior to the mouth opening the vasa deferentia converge towards the midline of body, meanwhile turning towards the dorsal body surface, after which they separately penetrate the penis and then immediately unite to form a short common vas deferens (Figs. 7E, 9C, 10, 11A). The latter opens into a spacious, oval-shaped seminal vesicle that is situated at the base of the penis papilla and communicates with the ejaculatory duct (Figs. 9C, 10). The seminal vesicle is lined with a simple, cuboidal, nucleated epithelium and covered with a well-developed layer of muscles. The ejaculatory duct is rather broad and lined with a nucleated epithelium and surrounded by a welldeveloped layer of circular muscles; it opens at the tip of the penis papilla. The latter is a sausage-shaped structure that lies dorsally in the male atrium. The penis papilla is lined with a nucleated epithelium that is underlain by circular and longitudinal muscles (Figs. 9B, 10, 11A). A musculo-parenchymatic organ projects from the anterior wall of the male atrium, ventrally to the root of the penis papilla. The entire organ consist of a broad, muscular base to which is attached a sclerotic, hollow tip or stylet that is curved like a scimitar and that measures 76–80 µm length. The broad base of the musculo-parenchymatic organ consists of a folded epithelium, which is underlain by well-developed circular and longitudinal muscles (Figs. 9B, 11A). A pair of ovaries is situated at a short distance behind the brain (Figs. 7C, 8B, 10A). The oviducts arise from the antero-lateral wall of the ovaries. At the level of the gonopore the oviducts turn medially to open separately into the bursal canal (Figs. 9E, 10). The latter is lined with a cuboidal, nucleated epithelium, bearing well-developed cilia, and is surrounded by circular muscle. The bursal canal receives the secretion of shell glands ectally, that is, ventrally to the oviducal openings and is connected with a sac-shaped copulatory bursa, the latter being lined with vacuolated cells (Figs. 9D, 10B, 11A). Feeding and reproduction. Under laboratory conditions, P. falcata seldom moves when starved, and usually assembles in groups that hide from light. When the animals are in search of food, firstly their head touches the food lightly, after which they move their tail end towards the prey item and extend their pharynx, which starts to engulf portions of the prey. After feeding, the animals group together again, away from direct light. We observed that 4 or 5 mature individuals of P. falcata may feed on a prey specimen of Dugesia sinensis of about 10 mm in length. When not starved, copulations may occur. During copulation the two bodies intertwine (either clockwise or counter-clockwise, depending on the copulants), with their heads pointing downwards and the tails pointing upwards (Fig. 11B). The process of copulation, during which the tails quiver, lasts for approximately 10 minutes, after which the partners separate. During cocoon laying, a small amount of mucus is discharged from the common atrium, with which the capsules are being attached tightly to the substrate. The spherical cocoon, which is devoid of a pedicel, is brownred, measuring 340–400 µm in diameter (Fig. 7F). From a single cocoon hatches 1–2 young worms (n=5 cocoons observed). Discussion. When the features exhibited by Paucumara falcata are fed into the key to the genera in Sluys (1989a) the identification leads to the genus Vatapa Marcus, 1948. This is due to the fact that also Vatapa possesses a musculo-parenchymatic organ (mpo) and the key only offers the choice between mpo present or absent. However, in Vatapa the mpo consists of a large indentation of the posterior wall of the common atrium (see Sluys 1989a) and thus differs greatly from the mpo of P. falcata. Other features also point to the fact that P. falcata belongs to the genus Paucumara and not to Vatapa. The genus Paucumara was diagnosed as follows: " Ectoplaninae in which the vasa deferentia unite just within the penis bulb to form a more or less curved common vas deferens. Ventral testes, extending to half-way along the pharyngeal pocket or to the level of the mouth. Bursal canal receives the secretion of numerous unicellular glands" (Sluys 1989a, p.191–192). All of these features apply to P. falcata, while there are a few other characters that point also to a close relationship between P. falcata and P. trigonocephala. Both species exhibit an attenuated head, formed by a narrowing of the body width anterior to the eyes, as well as whitish specks or transverse bands on the dorsal body surface. The eyes are set close together and are provided with a lens, while the posterior gut trunks unite in the hind end of the body. Paucumara falcata shows one great difference with P. trigonocephala as the latter does not possess the musculo-parenchymatic organ (mpo). However, accessory reproductive structures resembling the musculoparenchymatic organ of P. falcata are present in Pacifides psammophilus Holmquist & Karling, 1972 and P. gladiatoris Sluys, 1989 (see Sluys 1989a). But in the two last-mentioned species these organs concern musculoglandular organs (mgo). In particular the mgo of P. gladiatoris, with its sickle-shaped, sclerotic tip, resembles very much the mpo of P. falcata. Because of this gross resemblance one may initially be inclined to assign P. falcata to the genus Pacifide s. However, upon further reflection it becomes clear that other characters preclude such a taxonomic assignment and point to the fact that P. falcata falls in the genus Paucumara, thus illustrating convergent evolution of such accessory reproductive organs. Sluys (1989a) synonymized Fovia graciliceps Stimpson, 1857 from Hong Kong, China with P. trigonocephala, albeit with some reservations. Now that we know that another species with an acute triangular head and a pointed tail occurs on Chinese coasts close to Hong Kong, China, i.e., P. falcata, it may be that F. graciliceps actually is a junior synonym of P. falcata. Unfortunately, there is no way to ascertain what actually holds true and therefore the epithet graciliceps should remain suppressed. Paucumara trigonocephala has been reported from Japan, Australia, the Bismark Archipelago, and South Korea, where it was always collected from low-salinity biotopes, which during ebb tide even may be completely fresh (Yang et al. 2018). This resembles the habitat of P. falcata, which was collected from 19‰–20‰ salinity biotopes, while in the laboratory animals can also survive in freshwater.

Nerpa fistulata Wang & Chen, sp. nov. Material examined. Holotype: PLA-N001, eastern beach of Shenzhen City, Guangdong Province, China, 22°28′N, 114°31′E, 29 th April 2017, coll. Jia-Jia Chen, sagittal sections on 2 slides. Paratypes: PLA-N002, ibid., sagittal sections on 3 slides; PLA-N003, ibid., sagittal sections on 2 slides; PLA- N004, ibid., horizontal sections on 1 slides; PLA-N005, ibid., whole mount on 1 slide; PLA-N006, ibid., whole mount on 1 slide. All material is deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS). Etymology. The specific epithet is derived from the Latin adjective fistulatus, provided with pipes, and alludes to the characteristic intestinal commissures in this species. Diagnosis. Nerpa fistulata is characterized by: transparent body; semi-circular lens in each eye cup; pentamerous intestine with three distinct commissures, one in the posterior end of the body and the other located between the ovaries and the testes; two very large, prepharyngeal testis follicles; distal end of the penis papilla with a chitinized, pointed stylet; two rounded lateral bursae. Description. Live animals transparent, without any pigmentation. Body shape varies from elongate to broadly oval-shaped, with a broadly rounded posterior end and a blunt anterior end. Length and width of active specimens varies between 800–1,180 µm (n=5) and 210–435 µm (n=5), respectively (Figs. 2 A–B, 3A, 6A). A pair of black and elliptical eyes is located at 1/5 of the body length, measured from the anterior margin (Figs. 2 A–B, 3A, 4A, 6A). The eyes are situated rather close together, the distance between the eye cups being about 25 µm (n=5) and the distance to the body margin being 90–135 µm (n=5). Each eye cup has a large semi-circular lens. The mouth opening is situated median-ventrally at 1/5 of the body, measured from the posterior body margin; the mouth opening is located at the posterior end of the pharyngeal cavity (Fig. 6A). The cylindrical pharynx measures 200–300 µm in length and 130–150 µm in width (n=5) (Figs. 2 A–B, 3A, 6A). The principally pentamerous intestine shows three distinct commissures, one uniting the two posterior gut trunks in the hind end of the body, another immediately behind the testes, and a third commissure connecting the central part of the anterior intestinal trunk with the two lateral and forwards directed branches; the latter commissure runs between the ovaries and the testes. This results in the situation that the gut forms three rings, with one pair each encircling the large testes follicles and another, larger ring encircling the pharyngeal pocket and the copulatory apparatus. The median, anterior intestinal trunk extends forwards anteriorly to the eyes and the brain, which holds true also for the forwards directed lateral branches (Fig. 6A). Each of the major gut trunks gives rise to 12–16 short lateral diverticula. A pair of large, ellipsoidal testes, 350–375 µm in length and 180–190 µm in width, are situated immediately in front of the pharynx (n=5) (Figs. 2A, 3A, 6A). The follicles are packed with sperm; mature spermatozoa measure 58–60 µm in length and are biflagellate, the flagella being 55–56 µm long (n=5) (Fig. 2D). The vasa deferentia originate from the ventro-medial wall of the testis follicles and extend posteriorly alongside the pharynx as greatly swollen spermiducal vesicles, which decrease in diameter before opening separately into the intrapenial seminal vesicle (Figs. 2A, 4 E–F, 5C, 6A–B). The proximal, anterior section of the seminal vesicle is separated from the smaller posterior section by means of a shallow constriction. The posterior section of the seminal vesicle communicates with the ejaculatory duct, which opens at the tip of penis papilla. Penis glands discharge their secretion into the proximal section of the ejaculatory duct (Figs. 3C, 5C, 6B). The tip of the penis papilla is provided with a sclerotic stylet, which is about 50 µm long (n=5) (Fig. 3C). The penis papilla is lined with a nucleated epithelium and is surrounded by well-developed layers of circular and longitudinal muscles. The vitellaria are well developed and lie between the lateral diverticula (Fig. 3A). A pair of ovaries is situated immediately behind the brain and the eyes (Figs. 2A, 3A, 4A, 5 A–B, 6A). The ovaries are oval-shaped and have an oblique orientation, as they tilt forwards. After mating, the anterior end of the oviducts extends to form an ampulla, which houses the allosperm (Figs. 3B, 5E). From the ovaries each oviduct runs posteriorly and at the level of the posterior portion of the male atrium it gives rise to two branches, one of which communicates with the lateral bursa, while the other opens into the bursal canal (Figs. 2C, 3B, 6 A–B). From its point of communication with the atrium, the bursal canal curves anteriad and opens into a rounded copulatory bursa, which lies dorsally to the male atrium. The bursal canal, surrounded by circular muscles, is lined with a nucleated epithelium and receives the openings of shell glands ventrally to the oviducal openings, (Figs. 5C, 6B). Two rounded lateral bursae, about 45 µm in diameter, are located on either side of the body, more or less at the level of the gonopore (Figs. 2C, 5D, 6 A–B); a duct connects each lateral bursa with the ventrally located lateral gonopore. Ample sperm is present in the lateral bursae (Fig. 5D). The bursae and the last-mentioned connecting ducts are lined with a single layer of nucleated epithelium and are surrounded by circular muscle.

Order Tricladida Lang, 1884 Suborder Maricola Hallez, 1892, Published as part of Chen, Jia-Jia, Li, Wei-Xuan, Sluys, Ronald, Wu, Ming-Qi, Wang, Lei, Li, Shuang-Fei & Wang, An-Tai, 2019, Two new species of marine flatworm from southern China facilitate determination of the phylogenetic position of the genus Nerpa Marcus, 1948 and the histochemical structure of the nervous system in the genus Paucumara Sluys, 1989 (Platyhelminthes, Tricladida, Maricola), pp. 149-167 in Zootaxa 4568 (1) on page 153, DOI: 10.11646/zootaxa.4568.1.9, http://zenodo.org/record/2599316