Polydora lingshuiensis sp. nov. Materials. All polydorin species were sampled from one pearl oyster farm (18 �� 24 ��� 28.048 ���N, 109 �� 58 ��� 55.730 ���E) in Xincun Bay, Lingshui County, Hainan Province, China. Holytype, NSB 20140001, from burrows in the shells of P. martensi, 11 September 2013, coll. L.T. Ye; 8 paratypes, NSB 20140002, from mudtubes on the surface of pearl oyster cages, 12 October 2012, coll. L.T. Ye; 5 paratypes, NSB 20140003, from burrows in the shells of P.penguin, 14 October 2012, coll. L.T. Ye. FIRURE 1. Live worms of Polydora lingshuiensis sp. n. (A) Worms (arrows) wriggled out of the mudtubes detached from the surface of pearl oyster cages; (B) Large number of burrows (arrows) excavated by polydorins distributed in the inner surface of a cultured P. martensi; (C) Palps and posterior chaetigers protruding from self-excavated burrows near the rim of cultured P. martensi, with the whitish pygidium (arrow) of the worm exposed; (D) Polydorin curled up in its calcareous habitat, showing the U shape of the burrow; (E) Entire live worm pulled out from its burrow habitat; (F) Magnification of five anterior chaetigers of (E), showing the extension of the caruncle (arrow) to the middle or posterior part of the third chaetigers of the worm. Scale bar: A���B, 20 mm; C���F, 1 mm. FIRURE 2. SEM images of Polydora lingshuiensis. (A) Anterior end, dorsal view, showing the digitiform nuchal antenna(arrow) and the caruncle extending to the end of chaetiger 3; (B) Lateral view of neuropodia, showing the hooded hooks (arrow); and (C) Dorsal view of pygidium and the pygidium flaring disc with one dorsal notch. Diagnosis. A moderate-sized Polydora species inhabiting burrows in the shells of pearl oysters and mudtubes on the surface pearl oyster cages. Body pigmentation absent. Prostomium anteriorly round or weakly incised. Eyes present or absent. Caruncle extending to the middle of chaetiger 3. Median antenna present on the caruncle. Specialized chaetae absent from posterior notopodia. Chaetiger 5 with both dorsal superior capillaries and posterioventral capillaries; major spines falcate, with one concavity at subterminal ends, alternating with foliaceous companion chaetae. Branchiae from chaetiger 7. Pygidium disc-like with dorsal notch. Description of Holotype. A complete 160 -chaetiger individual measuring 48 mm long and 1 mm wide, and palps 3 mm long. Body tan to greenish-brown in life. Prostomium anteriorly round, tapering as caruncle to the posterior margin of chaetiger 3 (Figs. 1 F and 2 A). Two pairs of eyes present, anterior pair wider and larger than posterior pair, trapezoid arrangement (Fig. 4 A). Median antenna digitiform, located between the bases of palps (Fig. 2 A). FIRURE 3. Light micrographs and SEM images of major spines in chaetiger 5 of Polydora lingshuiensis sp. n. collected from Lingshui, China. (A, B) Lateral view of major spines alternating with companion chaeta, major spine falcate, companion chaeta (arrows at Fig. A) foliaceous, and subterminal ends of spines having one concavity (arrowheads at Fig. B); (C) Apical view of major spines, showing four superior notochaetae (arrow) and six neurochaetae (arrowhead) in chaetiger 5; and (D) Apical view of major spines, showing subterminal concavity in major spines (arrowhead). Scale bar: 50 um. Chaetiger 1 reduced in size, with digitiform notopodial lamellae and auricular neuropodial lamellae. Winged capillary neurochaetae present, notochaetae absent. Winged capillary notochaetae of Chaetigers 2 to 4 and 6, as well as subsequent chaetigers arranged in three rows, chaetae of hind row longer and thinner. Notochaetae fewer towards the posterior end. Specialized chaetae absent from posterior notopodia. Winged capillary neurochaetae present in Chaetigers 2 to 4 and 6, but replaced by hooded hooks from chaetigers 7, to end of body without accompanying capillaries. Bidentate hooded hooks with distinct manubrium and constriction on shaft (Figs. 2 B and 4 D), exhibit a reduced angle between apical tooth and main fang, up to 10 in a series. Chaetiger 5 expanded, almost twice as large as preceding and succeeding chaetigers; 5-8 major spines arranged in a slightly oblique row, alternating with foliaceous companion chaetae (Fig. 3 A and B), tuft of four dorsal superior geniculate capillaries and posterioventral fascicle of five winged neurochaetae present(Fig. 3 C). Major spines falcate, without conspicuous flange, with concavity at subterminal ends (Figs. 2 B and D and 4 B). Branchiae from chaetiger 7 to the end of the fragment, strap-like, free from notopodia. Pygidium white in life, wider than the last chaetigers as a flared cup with dorsal notch (Figs. 2 C and 4 C). FIRURE 4. Polydora lingshuiensis sp. nov. (A) Anterior end, dorsal view; (B) Major spines and companion chaetae of chaetiger 5; (C) Posterior end, dorsal view; and (D) Neuropodial bidentate hooded hook. Scale bars: A���C, 0.5 mm; D, 0.05 mm. Variability. Mid-sized specimens measuring up to 24 mm long and 1.3 mm wide at chaetiger 5, with up to 78 chaetigers. The average length of shell-boring worms was 33 mm (22-48), but the average length of mudtubedwelling worms was 17 mm (14-24). Eyes were present or absent, and if present, there were four. Body pigmentation was absent. Black pigmentations were sometimes scattered along margin of palp edge. Prostomium was anteriorly round or weakly incised. Caruncle extended either to the posterior border of chaetiger 2 or to the middle of chaetiger 3. Pygidium was usually disc-like, but sometimes cup-shaped, with dorsal notch. Habitat. A large number of P. lingshuiensis inhabited mudtubes on the surface of pearl oysters and their cages, and others were extracted from U-shaped burrows in the inner surface of the shells of P. martensi and P. penguin. P. lingshuiensis was commonly found co-occurring with Polydora haswelli Blake and Kudenov, 1978 in calcareous burrows of P. martensi and P. penguin. Reproduction. Egg strings were found inside some large burrows of P. lingshuiensis, and not found inside the worm mudtubes. The egg string, which was cylindrical in shape, was full of thousands of eggs, and no individual egg capsules were formed within the egg string. Egg strings were attached to the inner wall of the burrows by several thin filaments along their length. Etymology. The specific name lingshuiensis refers to Lingshui County, Hainan Province, China, which is the type locality of the new species. Molecular analyses. The 18 S rDNA sequences of P. lingshuiensis showed 99.9 % to 100 % sequence similarity to one another (Fig. 5). Polydora uncinata collected from Japan and Australia had the most similar sequences to P. lingshuiensis, showing 98.7 % sequence similarity. The 18 S data set consisted of 1660 nucleotide positions, with 164 parsimony informative, 161 uninformative, and 1335 constant. The 50 % majority rule consensus tree generated from Bayesian analysis indicated that Polydora species formed three well-supported clades, namely, P. haswelli / Polydora websteri (HW), Polydora brevipalpa / Polydora onagawaensis / Polydora calcarea (BOC), and Polydora aura / P. uncinata (AU) complex species (Fig. 5). All P. lingshuiensis samples grouped together, forming a sister clade to P. uncinata, and fell within the AU clade (Fig. 5). Genetic distances between polydorin species according to mitochondrial 16 S rDNA sequence are shown in Table 1. The 16 S data set consisted of 320 nucleotide positions, with 82 parsimony informative, 6 uninformative, and 232 constant.We found that the intraspecific genetic distance of P. lingshuiensis was only 0.01 (Table 1). Comparing interspecific genetic distance with other species, P. lingshuiensis had the smallest interspecific genetic distance (d = 0.11) with Polydora haswelli and Polydora triglanda, and the greatest (d = 0.16) interspecific genetic distance with D. cardalia (Table 1). The intraspecific genetic distance ranged from 0.00 to 0.01, except for P. triglanda, the intraspecific genetic distance of which surprisingly reached as high as 0.06. Nevertheless, the interspecific genetic distance ranged from 0.06 to 0.17. In most cases, the interspecific genetic distances based on 16 S sequence were evidently greater than the intraspecific genetic distances. FIRURE 5. Fifty percent major-rule consensus tree obtained from Bayesian analysis on the basis of 18 S rDNA sequences of annelids, rooted at oligochaeta, Tubifex tubifex. Numbers beside the nodes represent the Bayesian posterior probabilities (the posterior probabilities translated into percentages). Species sequences submitted in the present study are shown in bold. Unknown information is labeled as NK. The 28 S rDNA sequences of P. lingshuiensis were approximately 3400 bp in length, and three sequences of worms extracted from burrows (accession nos. KF 562244, KF 562245, and KF 562247) showed 100 % sequence identity to two sequences of worms extracted from mudtubes (accession nos. KF 562243 and KF 562246). P. lingshuiensis showed 97.7 % (3252 nt/ 3328 nt) sequence identity to P. haswelli (accession no. KF 562248), which was collected from the same host species and location as P. lingshuiensis. Remarks. P. lingshuiensis collected from burrows had almost the same morphological characteristics as those from mudtubes, except that the sizes of the former were relatively larger than those of the latter. Comparing with those Polydora species which had median antenna on the caruncle, P. lingshuiensis most closely resembled P.bioccipitalis, P.aura, P.latispinosa, P.cornuta, P.triglanda, and P.uncinata (Table 2). However, P. lingshuiensis could be readily differentiated from P.bioccipitalis, which had 2 median antenna and 2 lateral teeth on major spines, the caruncle extending to beyond chaetiger 5. P.a ur a and P.latispinosa differed from P. lingshuiensis in that they had lateral flange on major spines, and needle-like modified spines in posterior notopodia. P.cornuta differed from P. lingshuiensis in that it had a lateral tooth on major spines. P.triglanda and P.uncinata also easily distinguished from P. lingshuiensis because both of them had distinctive black bars in the palps. P. lingshuiensis morphologically resembled P.alloporis because both of them had subdistal cancavity on major spines. However, P.alloporis had distinctive black bars in the palps, and there was no median antenna on caruncle. P.triglanda, P.c o r n ut a, P. villosa, P. vicina, and P.onagawaensis were common Polydora species reported in Chinese waters (Radashevsky & Hsieh, 2000; Zhou et al. 2010; Sato-Okoshi et al. 2013). P. lingshuiensis could be differentiated from them based on morphological characteristics (see table 2). We first misidentified P. lingshuiensis as P. onagawaensis because of the high morphological similarity between these species. For example, they had similar shape of prostomium and pygidium, both with superior notochaetae and neurochaetae on chaetiger 5, without modified spines in posterior notopodia. Furthermore, similar to P. onagawaensis, some individuals of P. lingshuiensis showed some variability in palp pigmentation and length of caruncle extension. However, P. lingshuiensis had subdistal concavity on major spines, one median antenna on the caruncle, and no pigmentation all over the body. Furthermore, the distinction of egg capsule type also indicated the existence of two different species. All eggs of P. lingshuiensis were gathered together in one hollow cylinder, whereas P. onagawaensis formed egg strings, which included several egg capsules joining one another. Additionally, we could easily distinguish Polydora species by comparing their molecular characteristics. For example, the sequence identity between P. aura and P. lingshuiensis was 98.5 % (1746 nt/ 1771 nt), the resolution of which was sufficiently high to distinguish different polydorin species because we found that the intraspecific sequence identity of P. lingshuiensis reached as high as 99.9 % to 100 % (Fig. 5). P. haswelli also showed as high as 99.9 % to 100 % intraspecific sequence identity despite being collected from China and Japan, respectively (Fig. 5) (Sato-Okoshi & Abe 2013). TABLE 2. Some morphorlogical characteristics of the Polydora species most closely resembling Polydora lingshuiensis sp. and other Polydora species reported in Chinese waters. a Zhou et. (2010) reported that the palps of P.vicina were missing, so we treated palp pigmentation characteristics of P.vicina as NM(not mention). References: (1) Present study; (2) Blake (1978); (3) Radashevsky and Hsieh (2000); (4) Sato-okoshi (1998); (5) Blake (1987); (6) Light (1970); (7) Teramoto et al. (2013); (8) Zhou et al. (2010). Species Palp Prostomium Median Caruncle Superior Neuro- Major spines Modified Pygidium Habitat Reference pigmentation (anterior edge) antenna (maximal length) notochaetae chaetae on on chaetiger 5 spines in on chaetiger 5 posterior chaetiger 5 notopodia lingshuie-nsis absent entire or digitiform end of chaetiger 3 present present subdistal absent disc-like burrows, 1 weakly incised cancavity mud-tubes bioccipitalis absent incised present beyond chaetiger 5 absent absent 2 teeth and 1 absent disc-like burrows 2, 3 flange aura absent entire or present end of chaetiger 2-4 absent present lateral flange needle-like flaring disc burrows 3, 4 weakly incised latispino-sa absent incised triangular end of chaetiger 2 absent present lateral flange needle-like flaring disc burrows 2 or sheath cornuta absent incised present end of chaetiger 3 absent present lateral tooth absent disc-like mud-tubes 5 triglanda black bars incised present end of chaetiger 3 present present lateral flange absent cup-shaped burrows, 3 mud-tubes uncinata black bars weakly incised present middle or end of present present lateral tooth recurved flaring disc burrows 4 chaetiger 3 hook alloporis black bars entire absent middle or end of present present subdistal absent disc-like burrows 6 chaetiger 4 cancavity onagaw-aensis absent weakly incised absent middle or end of present present lateral tooth absent disc-like burrows 7 chaetiger 4 or flange villosa absent incised absent end of chaetiger 3 present present lateral tooth separate cuff-like coral-borer 3 on 1 side and needles shelf on the other vicina NM a incised absent middle of chaetiger absent absent lateral flange absent cup-shaped mud-tubes 8 3, Published as part of Ye, Lingtong, Tang, Bin, Wu, Kaichang, Su, Youlu, Wang, Ruixuan, Yu, Ziniu & Wang, Jiangyong, 2015, Mudworm Polydora lingshuiensis sp. n is a new species that inhabits both shell burrows and mudtubes, pp. 88-100 in Zootaxa 3986 (1) on pages 90-97, DOI: 10.11646/zootaxa.3986.1.4, http://zenodo.org/record/235791, {"references":["Blake, J. 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