Poecilobdella nanjingensis Yang, 1996

Nakano, Takafumi & Lai, Yi-Te, 2016, First Record of Poecilobdella nanjingensis (Hirudinida: Arhynchobdellida: Hirudinidae) from Taiwan and its Molecular Phylogenetic Position within the Family, Species Diversity 21, pp. 127-134 : 129-133

Poecilobdella nanjingensis Yang, 1996 ( Figs 1–4 )

Poecilobdella nanjingensis Yang, 1996: 128 , 129, fig. 80; Yang

2014: 128, fig. 4-5.

Material examined. Two specimens collected from Neidong Forest Trail , Wulai District, New Taipei City, Taiwan, by Takafumi Nakano on 3 February 2013: KUZ Z1742 (elev. 565 m, 24.8300°N, 121.5320°E) and KUZ Z1743 (elev. 569 m, 24.8297°N, 121.5324°E), dissected . Two specimens collected from Rueiyan River Major Wildlife Habitat , Renai Township, Nantou County, Taiwan: KUZ Z1744 (elev. 2238 m, 24.1151°N, 121.1969°E) collected by Yu-Chang Yang on 19 January 2009, and KUZ Z1745 (elev. 2200 m, 24.1056°N, 121.1836°E), dissected, collected by Huei-Ping Shen on 2 July 2009 .

Description. Body firm, muscular, with constant width posteriorly, BL 12.9–49.4 mm, BW 2.8–8.7 mm ( Fig. 1 ). Caudal sucker ventral, elliptical, CL 2.1–5.5 mm, CW 2.0– 5.9 mm; ventral surface with several radial rays ( Figs 1B , 2D ).

Somite I completely merged with prostomium ( Fig. 2A ). Somites II and III uniannulate ( Fig. 2A ). Somites IV and V biannulate, (a1+a2)=a3; somite V forming posterior margin of oral sucker ( Fig. 2A, B ). Somite VI dorsally triannulate/ventrally biannulate, a1=a2=a3/(a1+a2)> a3 ( Fig. 2A, B ). Somite VII triannulate, a1=a2=a3, or a1=a2 <a3 (with dorsally obvious furrow, b5=b6) ( Fig. 2A, B ). Somite VIII 4-annulate, a1=a2=b5=b6, or a1 (with dorsally obvious furrow, b1=b2)> a2=b5> b6 ( Fig. 2A, B ). Somites IX–XXIII 5-annulate, b1=b2=a2=b5=b6 ( Fig. 2A, C–E ). Somite XXIV 4-annulate, b1=b2=a2=a3 ( Fig. 2C, D ). Somite XXV triannulate, a1=a2=a3 or a1> a2 <a3 ( Fig. 2C ); a3 being last complete annulus ventrally. Somite XXVI biannulate, (a1+a2) (with obvious furrow, a1=a2)> a3 ( Fig. 2C ). Somite XXVII uniannulate ( Fig. 2C ). Anus at posterior margin of XXVII.

X b5 and XIII a2, respectively, being first and last annuli of clitellum.

Male gonopore in XI b5/b6 ( Fig. 2E ). Female gonopore in XII b5/b6 ( Fig. 2E ). Gonopores separated by 5 annuli.

Eyes in 5 pairs, in parabolic arc; 1st pair on II, 2nd pair on III, 3rd pair on IV (a1+a2), 4th pair on V (a1+a2), and 5th pair on VI a2. Sensillae developed, 1 row on every annulus. Somital papillae undeveloped.

Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of a1 of VIII, and at posterior margin of b2 of each somite in IX–XXIV ( Fig. 2B, D, E ).

One median longitudinal furrow on ventral surface of oral sucker ( Fig. 2B ). Three jaws in oral cavity, 1 dorsal and 2 ventrolateral; each jaw with uncountable barely detectable salivary papillae; monostichodont, median jaw bearing 85 diminutive teeth (KUZ Z1743 only), right jaw bearing 82 (KUZ Z1745) or 88 (KUZ Z1743) teeth, and left jaw bearing 80 (KUZ Z1745) or 81 (KUZ Z1743) teeth. Pharynx reaching to IX a2/b5. Crop reaching to XIX a2/b5, giving rise to 10 pairs of crop caeca: 1st pair in XI b1 and b2; 2nd pair in XI a2–b6; 3rd pair in XII a2–b6; 4th pair in XIII b2–b5 or a2–b6; 5th pair in XIV a2–b6 or XIV b2–XV b1; 6th pair in XV a2–b6 or XV b2–XVI b1; 7th pair in XVI a2–b6 or XVI b2–XVII b1; 8th pair in XVII b2–b6 or XVII b2–XVIII b2; 9th pair in XVIII a2–b6 or XVIII b2–XIX b1; and 10th pair (post-crop caeca) in XIX b2–XXIV a3. Intestine reaching to XXIII b1. Rectum simple tubular.

Testisacs in 9 pairs ( Fig. 3A ): 1st pair in XIII b5–XIV b1; 2nd pair in XIV b5–XV b1; 3rd pair in XV b5–XVI b1, or right sac in XV b5 and b6 (KUZ Z1743); 4th pair in XVI b5–XVII b1; 5th pair in XVII a2–b6 or XVII b5–XVIII b1; 6th pair in XVIII b5–XIX b1, or left sac in XVIII a2– b6 (KUZ Z1743); 7th pair in XIX b5–XX b1, or left sac in XIX a2–b2 (KUZ Z1743); 8th pair in XX b5–XXI b1 (KUZ Z1745), or right sac in XX b5 and b6, left sac in XX a2–b6 (KUZ Z1743); 9th pair in XXI a2–b6. Paired epididymides developed, globular ( Fig. 3A–C ); right epididymis in XII b1 and b2 (KUZ Z1745) or XII b1–b5 (KUZ Z1743) and left epididymis in XII b1–a2 (KUZ Z1745) or XI b6–XII a2 (KUZ Z1743); from each epididymal mass, thick sperm duct reaching to right or left ejaculatory bulb, respectively. Ejaculatory bulbs developed, ovoidal ( Fig. 3A–C ); right bulb in XI b5 and b6 (KUZ Z1743) or XI b6–XII b2 (KUZ Z1745) and left bulb in XI b5 and b6 (KUZ Z1743) or XI b5–XII b1 (KUZ Z1745). Ejaculatory ducts narrow, running inward toward male atrium in somite XI b5–b6 ( Fig. 3B, C ); right duct crossing ventrally beneath nerve cord. Male atrium covered with prostate gland and continuous with penis sheath ( Fig. 3A–C ). Penis sheath hook-like, reaching to XII b2–XIII b1, then turning anteriorly to male gonopore ( Fig. 3B, C ).

Paired ovisacs globular, in XII b6 and XIII b1 ( Fig. 3A, D, E ). Oviducts short ( Fig. 3D, E ); right oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XII b6–XII/XIII. Common oviduct thick, short, descending to female vagina in XII b6–XIII b1 ( Fig. 3D, E ). Female vagina covered with prostate gland, continuous to vaginal duct ( Fig. 3A, D, E ); vaginal duct hook-like, slightly folded, reaching to XIII a2–XIII/XIV, then turning anteriorly to female gonopore ( Fig. 3D, E ); vaginal caecum absent.

Colouration. In life, dorsal surface greenish yellow with 7 discontinuous black longitudinal stripes, each stripe present in b2–b5 and absent in b1 and b6 of each midbody somite ( Figs 1A , 4 ): median stripe darker than lateral stripes, with weaker striping in cephalic region (somites I–X) and darker striping from somites XI to XXV; 4 lateral inner stripes, lighter than 2 lateral outer stripes; irregular black markings present in b1 and b6 of each mid-body somite; in cephalic region, irregular markings and lateral inner stripes forming chainlike pattern; ventral surface paler than dorsal surface. Colour faded in preservative, but longitudinal stripes and markings still present on dorsal surface ( Fig. 1A ).

Distribution. In addition to the Taiwanese localities, this species has been found at two locations in continental China ( Fig. 5 ): the Nanjing University campus, Nanjing, Jiangsu (type locality) ( Yang 1996) and Tianmu Mountain, Zhejiang ( Yang 2014).

Natural history. The Neidong individuals were found curled up under rocks in moist mountainous habitats ( Fig. 4B ). Thus, they are indeed terrestrial as was mentioned by Yang (2014). A mature leech, KUZ Z1745, with an obvious clitellum was collected on 2 July; therefore, P. nanjingensis is thought to enter its reproductive season before early July in Taiwan.

Phylogenetic position. The obtained BI ( Fig. 6 ) tree had a topology identical to that of the ML tree (ln L =−17190.50; not shown). The Taiwanese P. nanjingensis belong to a well supported clade (BS=98%, PP=0.99) that consists of Goddardobdella Richardson, 1969 , Hirudinaria , Aliolimnatis Richardson, 1972 species , and Asiaticobdella fenestrata (Moore, 1939) . In the ML analyses, the monophyly of the Taiwanese P. nanjingensis and Aliolimnatis + As. fenestrata clade was supported (BS=89%); however, this relationship was not recovered in the BI analysis (PP=0.85). The two Taiwanese individuals formed a fully supported clade (BS=100%, PP=1.0).

Remarks. Chinese P. nanjingensis possess over 100 teeth on each jaw with salivary papillae arranged in four longitudinal stripes ( Yang 1996, 2014). The present specimens possess jaws without developed salivary papillae, and they have a lower number of teeth (~80) on each jaw compared to Chinese individuals. The Taiwanese specimens were identified as P. nanjingensis based on the presence of the following characteristics: dorsal surface with seven dark, discontinuous stripes; somital annulation excluding uniannulate somite XXVII (biannulate in Chinese leeches); male gonopore in XI b5/b6 and female gonopore in XII b5/b6; crop caeca in 10 pairs, 10th pair being post-crop; globular epididymides in posterior part of XI to anterior part of XII; atrium continuous with penis sheath; penis sheath hooklike, reaching to body area between posterior part of XII and anterior part of XIII, and then turning to male gonopore; vaginal duct hook-like, reaching to XIII b2, then turning to female gonopore.

Yang (1996) stated that this species has a vaginal caecum. However, according to the original description and figure of P. nanjingensis , its common oviduct clearly descends to the terminus of its female vagina. Therefore, the vagina does not consist of a vaginal ‘caecum,’ but forms a simple ‘duct’-like structure. The Taiwanese P. nanjingensis also has a vaginal duct only. This morphological characteristic sheds light on its generic position, because Poecilobdella has been differentiated by a developed vaginal caecum in the female median structure. It is thus highly possible that P. nanjingensis should be placed under another hirudinid genus. To deter- mine the precise generic position of P. nanjingensis , further taxonomic studies on the nominal genera of Hirudinidae should be conducted.

The molecular phylogenies showed that P. nanjingensis forms a monophyletic lineage with a clade of Aliolimnatis / Asiaticobdella , which are diagnosed by an acaecate female vagina ( Richardson 1969, 1972). In contrast, Goddardobdella and Hirudinaria , both of which have a vaginal caecum ( Richardson 1969; Sawyer 1986), forms a distinctive lineage. However, the present phylogenetic analyses did not cover all of the known hirudinid genera. A future phylogenetic analysis based on a larger number of the known hirudinid taxa will lead to a better understanding of whether the characteristics of the vagina reflect hirudinid phylogenetic relationships.

Figures

Fig. 1. Poecilobdella nanjingensis Yang, 1996, from Neidong, Wulai, KUZ Z1743. A, dorsal view; B, ventral view. Scale bar: 5mm.

Fig. 2. Poecilobdella nanjingensis Yang, 1996, from Neidong, Wulai, KUZ Z1743. A, dorsal view of somites I–XII; B, ventral view of somites I–VIII; C, dorsal view of somites XXIII–XXVII and caudal sucker; D, ventral view of somites XXIII and XXIV and caudal sucker; E, ventral view of somites XI and XII. Scale bars: 2mm (A); 1mm (B–E). Abbreviations: fg, female gonopore; mg, male gonopore; np, nephridiopore.

Fig. 3. Poecilobdella nanjingensis Yang, 1996, from Neidong, Wulai, KUZ Z1743. A, dorsal view of reproductive system including ventral nervous system; B, dorsal view of male median reproductive system with outline of prostate gland including positions of ganglia XI and XII; C, left lateral view of schematic drawing of male median reproductive system; D, dorsal view of female reproductive system with outline of prostate gland including position of ganglion XIII; E, lateral view of schematic drawing of female reproductive system. Scale bars: 2mm (A); 0.5mm (B, D). Abbreviations: at, atrium; eb, ejaculatory bulb; ep, epididymis; od, oviduct; ov, ovisac; pes, penis sheath; pg, prostate gland; ts, testisac; vd, vaginal duct; vg, vagina.

Fig. 4. Poecilobdella nanjingensis Yang, 1996, from Neidong, Wulai, KUZ Z1743. A, dorsal view of live animal; B, live animal found under a stone at the locality.Scale bar: 5mm (B).

Fig. 5. Map showing the localities where Poecilobdella nanjingensis Yang, 1996 has been recorded. Closed circles indicate localities reported herein, open circles those from previous studies: aYang (1996) and bYang (2014).

Fig. 6. Bayesian inference tree for 5519 bp alignment positions of nuclear 18S rRNA, 28S rRNA, mitochondrial cytochrome c oxidase subunit I, and 12S rRNA markers. Numbers on nodes indicate bootstrap values for maximum likelihood and Bayesian inference posterior probabilities.