Two new species of brackish-water Macrostomum (Platyhelminthes, Macrostomida) from southern China Author Fang, Chu-Yu Author Wang, Lei Author Zhang, Yu Author Wang, An-Tai text Zootaxa 2016 4170 2 298 310 journal article 10.11646/zootaxa.4170.2.4 dd8bd107-12fe-4ff7-900d-b0633408d394 1175-5326 263628 666CFB09-4980-448A-9571-1DA0C34DBB00 Macrostomum baoanensis Wang & Fang , n. sp. ( Figs. 7–10 ) Material examined. Observations were made on several live and preserved specimens. Holotype (PLA–Ma0081): permanent slides of stylet in polyvinyl-lactophenol. Paratype (PLA–Ma0082–87): six serially-sectioned specimens. The sampling locality was Baoan District , Shenzhen City , Guangdong Province ( 22°41′35″N , 113°45′52″E ) ( Fig. 1 ). Samples were taken from floating Spirogyra in a brackish-water artificial fishpond near littoral zone in July , 2015. All specimens are deposited in IZCAS . Etymology. The name of this species is derived from the name of Baoan District, Shenzhen City, Guangdong Province , China . Description. The body is dorsoventrally flattened and transparent. The length and width of the body is 994 ± 65.5 µm (n=5) and 208 ± 32.3 µm (n=5), respectively ( Fig. 7 B). Live squash-mounted specimens are 1290 ± 103.9 µm (n=3) in length and 240 ± 68.4 µm (n=3) in width ( Fig. 7 A). Anterior and posterior ends are bluntly rounded. Cilia are 4–7 µm in length and distributed densely over the body surface. Rigid cilia are 10–15 µm in length and are sparsely distributed ( Fig. 9 A). Rhabdites are 12–14 µm in length and distributed along the dorsal body surface. A pair of black-colored eyes (6.2 ± 1.3 µm in diameter, 33 ± 2.4 µm in eyes distance), are located at anterior 17% position of the body. ( Fig. 7 B). Similar to other species of Macrostomum , the pharynx is located posterior to the eyes, while the pharyngeal gland cell bodies are resided further back laterally on the ventral side of the animal. ( Figs. 7 B, 9C). A pair of elongated oval-shape testes is located one at each side of the anterior 50% position of the intestine. Mature sperm are 110–130 µm in length. The lengths of feeler, body and shaft of the sperm are 38–45 µm, 23–27 µm, and 43–57 µm (n=3), respectively. The sperm has no brush at the posterior end, but has a pair of bristles in the body part ( Figs. 8 D, 10F). The false vesicula seminalis, vesicula seminalis, vesicula granulorum are arranged in a left- to right-hand sequence in the posterior part of the body ( Figs. 5A , C, 6B). The spherically-shaped vesicula seminalis is surrounded by a muscular wall that is 6–8 µm in thickness in squashed samples. The penis stylet is “J”- shaped and connects to the posterior end of the vesicula granulorum, and points towards the male gonopore at its distal end. The curved-line (marked as ‘a’) and straight-line (marked as ‘b’) distances between the base and distal end of the penis stylet are 171 ± 45.8 µm (n=5) and 140 ± 38.2 µm (n=5), respectively. The penis stylet is slightly broader at the base end, and gradually narrower towards distal end. The diameters of basal, middle and distal parts of penis stylet are 25 ± 4.3 µm (n=5), 13 ± 2.9 µm (n=5) and 6.2 ± 0.7 µm (n=5), respectively. The penis stylet bends 90° leftwards at 75% of its length. The inner wall of the arc-shaped distal end of the penis stylet is somewhat similar to that of M. tuba (von Graff 1882 ) . In the new species, the external wall of distal end is thickened and bents outwards into a hook-shape ( Figs. 8 A–C, 10D–E). For the female reproductive system, a pair of ovaries is located one at each side of the posterior region of the intestine. Their size varies according to the state of sexual maturity. In living specimens, the ovaries usually extend forwards to meet the testes, but they are usually separated in histological sections, possibly due to shrinkage of the specimen ( Figs. 7 B, 9A). The female antrum, which is involved in egg laying, also varies in size according to the state of sexual maturity. Its epithelium is thickened, but the cellular valve is not found from the histological sections ( Figs. 9 D–E). The female gonopore, which is the opening of female antrum, is surrounded by numerous cement glands ( Fig. 9 D). FIGURE 7. Macrostomum baoanensis Wang & Fang , n. sp. : (A) squeezed living animal, ventral view; (B) whole body photographed in life, ventral view; (C) copulatory apparatus of a squeezed animal; (D) copulatory apparatus of a living animal. FIGURE 8. Macrostomum baoanensis Wang & Fang , n. sp. : (A–C) penis stylet; (D) mature sperm. FIGURE 9. Macrostomum baoanensis Wang & Fang , n. sp. : (A–B) horizontal whole body section; (C–E) longitudinal whole body section. Remarks. There are 7 species similar to Macrostomum baoanensis n. sp. identified in this study (see Tab. 2 ). These species are common in having J-shaped penis stylets. However, these species can be distinguished by other morphological details of the penis stylet. These differences can be categorized into length of penis stylet, diameter of basal and distal opening, features of distal opening, and bending angle and position of penis stylet ( Tab. 2 ). Among these 7 species, the distal opening of penis stylet is in groove shaped in Macrostomum amurense , truncated shaped in M. burti , gutter shaped in M. clavistylum , and slightly bevel shaped in M. curvistylum and M. japonicum . The basal region of stylet in M. curvistylum is characterized by its crenulated shape. In M. extraculum , the lower margin of their penis stylets has a conical enlargement, while in M. sinensis , the lower margin is foliar shaped. In addition, the stylet distal openings of these 7 species are non-thickening. In contrast, the stylet distal opening of M. baoanensis n. sp. is thickened and the lower margin is in hook-shaped, which is distinct compared to the other 7 similar species. The length (the straight-line ‘b’ distances between the base and distal end) of the penis stylet of M. burti (45– 53 µm), M. curvistylum (68 µm) and M. extraculum (68–72 µm) is shorter than that of M. baoanensis n. sp. (140 ± 38.2 µm). In M. amurense (150 µm), M. clavistylum (150 µm) and M. japonicum (170 µm), although the lengths are similar to that of M. baoanensis n. sp. , the ratios of total length to basal diameter and basal diameter to distal diameter of penis stylet vary among these three species and M. baoanensis n. sp. (total length: basal diameter and basal diameter: distal diameter, 5.6 and 4.0 in M. baoanensis n. sp. , 4.4 and 8.5 in M. amurense , and 2.7 and 6.0 in M. clavistylum , 4.3 and 13.3 in M. japonicum ). Macrostomum sinensis is most similar to M. baoanensis n. sp. in the overall morphology of their penis stylet. However, these two species are distinct in penis stylet ( Tab. 2 ), as well as the overall body size and type of habitat. Although the ratio of total length to basal diameter of stylet in M. sinensis is similar to that of M. baoanensis n. sp. (5.3 vs. 5.6), the ratio of basal diameter to distal diameter of penis stylet differs a lot between these two species ( 7.1 in M. sinensis vs. 4.0 in M. baoanensis n. sp. ). In M. sinensis , the penis stylet bends 130° rightwards at 75% position while in M. baoanensis n. sp. , it bends 90° at 68% position. Other differences between these two species include body size ( 1.7 mm in M. sinensis vs. 990 µm in M. baoanensis n. sp. ) and living environment (freshwater in M. sinensis vs. brackish-water in M. baoanensis n. sp. ). TABLE 2. Comparison of the penis stylets among Macrostomum baoanensis n. sp. and other 7 similar species within the genus Macrostomum .

Length of stylet (µm)	Diameter of basal /distal opening (µm)	Features of stylet distal opening	Bending angle and position of stylet	Habitat	Locality	Reference
M. baoanensis 140 ± 38.2 n. sp.	25 ± 4.3/ 6.2 ± 0.7	Hook-shaped, with thickened wall	90° at 68% position	Brackish	China	This study
M. amurense 150	30–38/4	Groove-shaped Non-thickening	90° at 84% position	Freshwater	Russia	Beklemische v 1950
M. burti 45–53	9–10/2	Truncated shape Non-thickening	90° at 67% position	Brackish marine	Canada	Ax & Armonies 1987
M. clavistylum 150	50–60/8.5–10	Beak-shaped Non-thickening	>90° at 76% position	Freshwater	Russia	Beklemische v 1951
M. curvistylum 68	15–18/2.5–3.5	Beveled Non-thickening	>90° at 72% position	Freshwater	USA	Ferguson 1939
M. extraculum 68–72	18–22/3	Lower margin with a conical enlargement	>120° at 40% position, ≥60° at 70% position	Brackish	Alaska, USA	Ax & Armonies 1990
M. japonicum 170	40/3	Beveled Non-thickening	90° at 75% position	Freshwater	Japan	Okugawa 1930
M. sinensis 220–280 Average 245	40–52/6–7	Foliar-shape lower margin Non-thickening	≥120° at 75% position	Freshwater	China	Wang 2005

Discussion. To date, approximately 200 species of Macrostomum have been recorded worldwide (http:// macrostomorpha.info/). Around 50% of the species were found in freshwater, while 30% from brackish-water and marine environments. The taxonomic study of Macrostomum in China started relatively late. To our knowledge, all of the 10 Macrostomum species previously discovered in this region were from fresh-water environment, while brackish-water and marine species has never been recorded from China . In this work, the two new Macrostomum species, namely M. zhujiangensis n. sp. and M. baoanensis n. sp. , were described from brackish-water environments in Zhujiang River Estuary, representing the first descriptions of brackish-water Macrostomum species in southern China . The identification and classification of these species were based mainly on the distinct features of their penis stylet and for M. zhujiangensis n. sp. also on 18S rDNA phylogenetic analysis. According to the analysis of sperm and stylet morphology conducted by Schärer et al . (2011) , both new species should be categorized into Macrostomum clade 2 that are characterized by having bristle sperm and non-needle-like penis stylet. These features are evolutionary correlated with reciprocal copulation, which is considered to be less evolutionary stable and has greater diversification. These results indicated the importance of increasing taxon sampling of Macrostomum in this relatively seldom studied area should contribute a better understanding of their phylogeny. Since the sampling localities are only a small part of the Pearl River Delta characterized by a wide variety of freshwater and marine habitats, more systematic sampling should result in the discovery of more undescribed species.