Girardia sinensis Chen & Wang, 2015

Chen, Xiao-Min, Chen, Yu-Hui, Wu, Cheng-Chen & Wang, An-Tai, 2015, A new species of the genus Girardia (Tricladida: Dugesiidae) from China, Zoological Systematics 40 (2), pp. 166-178 : 168-174

Girardia sinensis Chen & Wang , sp. nov. ( Figs 1–21 )

Locality. The animals were collected from the ventral part of stones underwater (about 1.2–1.5 m in depth) in Xinghu Lake , Zhaoqing , Guangdong Province, China (23°04 ′ 33 ″ N, 112°28 ′ 43 ″ E). Xinghu Lake appears karst topography, and the lake area is 8.23 km 2. There are many granite hills and karst caves in the lake, which forms lake wetland naturally. No aquatic plants were found nearby, and the lake belongs to the freshwater habitats. The shoreside is the urban residential area, where domestic sewage discharging into the lake.

Materials. PLA-0080, serial section vertically; PLA-0081, serial section horizontally; PLA–0082 –89, the whole body is fixed in 10% formaldehyde.

Etymology. The specific name is referring to its type locality, China.

Description. External morphology. Body size of living individuals ranges from 14.2 to 15.2 mm in length and 3.4 to 4.2 mm in width. The animal appears flat, and is covered by massive black and white spots dorsally, similar to granite pattern. Both sides of triangle-shaped head have triangle-shaped auricular flanges, which can stand up ranging from 15 to 100 degrees, which are unobvious in fixed specimens ( Fig. 3 ). Posterior back of auricular flange have obvious oval unpigmented area ( Figs 1–2 ). A pair of kidney-shaped eyes lies between the auricular flanges. Fan-shaped unpigmented area placed at the lateral part of eyes ( Figs 1–2 ). Mouth lies near the middle of ventral side of the body. Gonopore locates behind mouth at 1/3 rear part of the body ( Fig. 3 ). Both sides of the body have obvious cement glands thar ventrally distributed, when in stationary state, pseudopodium appear at both sides, and vary in shape ( Fig. 4 ).

Habits. Animals moves slowly, wriggles on the wall or close to the bottom of culture dishes when foraging. They prefer to cluster in the underside of stones. Also, the species have strong pollution-resistance ability, which can adapt to nitrogen and phosphorus eutrophic waters, and were fed with pork liver under artificial cultivation. When prey, they swim to the upward side of foods, darted out its pharynx from ventral side of body to encase food and ingest, then drift away or adsorbed on the surface food.

Reproduction. Collected individuals were cultivated indoor under room temperature (25±1°C). They showed sexual reproduction after a week, then copulatory apparatus disappeared 2 months later. After 9 months, they appeared sexual reproduction again, and conducted transverse fracture asexual reproduction during other periods. Mature individuals clustered in the ventral side of underwater stones to lay eggs. When laying eggs, gonopore excretes mucus to the surface of stones, and raised up gradually, then the mucus converted into egg stalk (0.43–1.00 mm in length). Finally, eggs are attached to the egg stalk, which looks similar to cherry shape as a whole. The size of spherical-shaped eggs differs from each other produced by different individuals (0.77–1.36 mm in diameter). The just-laid eggs appear light red, then bright red few days later, and dark red at last ( Fig. 5 ). The specimens stop eating after laying eggs, and incubate around egg stalk about 18 d (18±3d, n =36) until the larvae hatched ( Fig. 6 ). Each egg hatches one larva, and its appearance is similar to mature individual, uniformly distributing black and white oval spots dorsally ( Fig. 7 ).

Digestive system. Digestive system consists of mouth, pharynx and intestines. Mouth opens at the middle part ventrally. Cylinder-shape pharynx (18 mm × 0.33 mm) with thick pharyngeal musculature lies in the central part ventrally, elongated longitudinally to the rear. The surface of pharynx is covered by brown pigmentation. The base of pharynx connects to 3 intestinal branches, and the middle one stretches to the anterior part of eyes, the other two extend to the posterior end of the body.

Reproductive system. Hermaphrodite, with one gonopore. Female reproductive systems are composed of ovaries, oviducts, copulatory bursa, bursa stalk and common atrium. A pair of oval ovaries (80 μ m×130 μ m) lies in the anterior inner side of ventral nerve cord ( Figs 8–9 ). Extending from ovaries, oviducts elongate backward to gonopore dorsally, then join in the junction of bursa stalk and common atrium ( Figs 15–18 ). Sphere-shaped copulatory bursa placed right behind pharynx, and joints common atrium through bursa stalk ( Figs 11–18 ). Pipe-shaped bursa stalk (740 μ m×55 μ m) lies above seminal vesicle, extending to common atrium dorsally. Common atrium connect with the outside surroundings through gonopore ( Figs 15 , 18 ). Male reproductive systems consist of testes, vas deferens, seminal vesicle, penis, male atrium. More than 20 pairs of oval testes (60 μ m×90 μ m) stretch longitudinally along both sides of the body ( Fig. 10 ). Each testis extends out vas deferens, and elongate backward to seminal vesicle. Irregular net-shaped seminal vesicle locates right behind copulatory bursa and under bursa stalk, and the solid inner side is filled with spermatozoa. An obvious penis muscle cord lies below seminal vesicle, incline to body ventral part, and link to ventral muscle. Penis muscle cord give out 4–5 muscle fiber bundles around seminal vesicle. Each muscle fiber bundle gives out numerous muscle fibers, interconnecting into a mesh structure ( Figs 11–18 ). Penis consists of ejaculatory duct and penis papilla. Extending from seminal vesicle, ejaculatory duct opens to male atrium through penis papilla, and joins with common atrium. Nipple-shaped penis papilla locates just behind seminal vesicle, and connects with the outside surroundings through gonopore. The periphery of penis is male atrium. The rear muscle behind common atrium sink backward to form an obvious posterior diverticulum. The terminal joints of common atrium and bursa stalk are distributed with shell glands densely ( Figs 15–16 , 18 ).

AChE + nerve structure. AChE+ nerve structure appeared copper red after histochemical localization, and it consists of cerebral ganglion, ventral nerve cord, lateral nerve cord and transverse nerves ( Figs 19–21 ). The control specimens showed a negative reaction. Cerebral ganglion are formed by two-sided arc-rod-shaped nerves, they joint in the anterior end and appear reverse V shape. Both sides of cerebral ganglion extend backward to form ventral nerve cord ventrally. Cerebral ganglion sent out about 11 radial branch nerves to connect with lateral nerve cord, then elongate to the body side. Two-sided cerebral ganglions are connected by densely no branching transverse nerves ( Fig. 20 ). It is difficult to define the boundary between cerebral ganglion and ventral nerve cord. Ventral nerve cord lies in both sides of the body with a diameter of about 20–50 μ m ventrally. In the anterior end, they join with two-sided cerebral ganglion respectively and extend along the body to the posterior end connecting by arc-shaped nerves. The point where ventral nerve cord sent out branch nerves shows obvious bulge, which contains nerve cells inside. The branch nerves emitted by the right and left ventral nerves connect with lateral nerve cord transversally ( Fig. 19 ). Lateral nerve cord locates in both sides of the body and parallel ventral nerve cord. They come from the first pair of branch nerves and extend backward along the body side to the terminal end of ventral nerve cord ( Figs 19–20 ). In the central part of the body, there are 1–2 incomplete longitudinal nerves between lateral nerve cord and ventral nerve cord ( Fig. 21 ).

Phylogenetic analysis. The phylogenetic trees generated using maximum-parsimony ( MP) and maximum-likelihood ( ML) analyses were shown in Fig. 23 and Fig. 24 , respectively. Both results suggested that the COI sequences from Girardia sinensis sp. 1–5 formed a clade with 100% bootstrap probabilities. In addition, these sequences also formed a clade with 100% bootstrap probabilities with the sequence of Girardia tigrina . It should be noted that Dugesia japonica and Dugesia ryukyuensis formed a 100% probabilities clade, suggesting that the genus Dugesia is likely to be a sister group of the genus Bipalium , Dugesia , Girardia and Novibipalium from the family Geoplanoidea .

Figures

Figs 1–7. Girardia sinensis Chen & Wang, sp. nov. 1–4. Mature. 1–2. Mature individuals, dorsal view. 3. Mature individuals, ventral view. 4. Pseudopodium. 5. New egg. 6. Egg clusters and the incubative individual. 7. Immature individual, dorsal view. Scale bars: 1–3 =1mm; 4–7=500μm.

Figs 8–14. Girardia sinensis Chen & Wang, sp. nov. 8–10. Body anterior part close to ventral side. 9. Ovaries. 10. Testes. 11–14. Sagittal section copulatory apparatus. Scale bars: 8, 11–14=100 μm; 9–10=50μm.

Figs 15–17. Girardia sinensis Chen & Wang, sp. nov., longitudinal section of copulatory apparatus. Scale bars =100μm.

Fig. 18. Girardia sinensis Chen & Wang, sp. nov., sagittal reconstruction of copulatory apparatus. Scale bar=100 μm.

Figs 19–21. Girardia sinensis Chen & Wang, sp. nov., AChE+ nerve structure. 19. Mature individuals, ventral view. 20. Head. 21. Body, middle part. Scale bars: 19=200μm; 20–21=100 μm.

Fig. 23. Phylogenetic tree generated based on Maximum-parsimony (MP) analysis of partial sequence of COI gene. Dendrocoelum lacteum (Planarioidea) was chosen as outgroup.

Fig. 24. Phylogenetic tree generated based on maximum-likelihood (ML) methods with Tamura-Nei model analysis of partial sequence of COI gene. Dendrocoelum lacteum (Planarioidea) was chosen as outgroup.