A New Quadrannulate Species of Orobdella (Hirudinida: Arhynchobdellida: Orobdellidae) from Pingtung, Taiwan Nakano, Takafumi Lai, Yi-Te Species Diversity 2017 2017-11-25 22 143 150 Nakano & Lai, 2017 Nakano & Lai 2017 [996,1174,1109,1133] Clitellata Orobdellidae Orobdella GBIF Animalia Arhynchobdellida 1 144 Annelida species meisai sp. nov.   ( Figs 2–4)   Diagnosis.Body length of mature individual exceeding 6 cm. Somite IV uniannulate, somites VIII–XXV quadrannulate. Clitellum in somite XI b5 to somite XIII a2. Male gonopore in somite XI b5/b6, female gonopore slightly posterior to anterior margin of somite XIII a1, behind gastropore, gonopores separated by 5+1/4 annuli. Pharynx reaching to somite XV b5–b6. Gastropore conspicuous, slightly posterior to anterior margin of somite XIII a1. Gastroporal duct rudimentary tubular. Paired vasa deferentia thick. Epididymis lacking. Pre-atrial loop present. Atrial cornu lacking.   Material examined.  Holotype: KUZ Z1917, dissect- ed, collected from under a stone along the Jinshui Ying Old Trail, Fanliao, Pingtung County, Taiwan( 22.40730°N, 120.758568°E; elev.  1470 m) by Takafumi Nakano( TN) on  9 March 2017.  Paratypes( 12 specimenscollected from around the type locality): KUZ Z1908( 22.407°N, 120.756°E; elev.  1490 m) by Wei-Ren Liang on  26 August 2014; KUZ Z1909– Z1910( 22.407°N, 120.756°E; elev.  1490m) by Fu-Sheng Huang on  23 February 2014; KUZ Z1911– Z1912( 22.40824°N, 120.75264°E; elev.  1650 m), KUZ Z1914– Z1915( 22.40790°N, 120.75304°E; elev.  1620m) by Yi-Te Lai (YL) on  9 March 2017; KUZ Z1913( 22.40809°N, 120.75288°E; elev.  1620 m), KUZ Z1916( 22.40772°N, 120.75662°E; elev.  1450m) by TN on  9 March 2017; KUZ Z1918– Z1919( 22.407933°N, 120.756530°E) by YL on  22 January 2017; and KUZ Z1920( 22.409192°N, 120.754820°E) by Kao-Shr Shiang on  9 December 2016( KUZ Z1910, Z1914, and Z1915 were dissected).   Description.Body firm and muscular, elongate, with constant width in caudal direction, dorsoventrally compressed, BL 62.5 mm, BW 4.7mm( Fig. 2A, B). Caudal sucker ventral, slightly elliptic, CL 2.7 mm, CW 2.9 mm( Figs 2B, 3D). Somite I completely merged with prostomium ( Fig. 3A). Somites II (=peristomium), III and IV uniannulate ( Fig. 3A); somite II not separated from somite I. Somite V biannulate, (a1+a2)=a3; a3 forming posterior margin of oral sucker ( Fig. 3A, B). Somites VI and VII triannulate, a1=a2=a3 ( Fig. 3A, B). Somites VIII–XXV quadrannulate, a1=a2=b5=b6 ( Fig. 3A–E). Somite XXVI triannulate, a1>a2<a3; a3 being ventrally last complete annulus ( Fig. 3C, D). Somite XXVII uniannulate with slight dorsolateral furrow on respective sides ( Fig. 3C). Anus behind somite XXVII; post-anal annulus absent ( Fig. 3C). Somite X b5 and somite XIII a2, respectively, being first and last annuli of clitellum ( Fig. 3E). Male gonopore in somite XI b5/b6 ( Fig. 3E). Female gonopore slightly posterior to anterior margin of somite XIII a1, inconspicuous, located posterior to gastropore ( Fig. 3E, F). Gonopores separated by 5+1/4 annuli ( Fig. 3E). Anterior ganglionic mass in somite VI a2 and a3. Ganglion VII in a1 and a2. Ganglia VIII–XIX, of each somite, in a2 ( Fig. 4B). Ganglia XX–XXII, of each somite, in a1 and a2. Ganglion XXIII in a2. Ganglia XXIV–XXV, of each somite, in a1 and a2. Ganglion XXVI in somite XXV b6. Posterior ganglionic mass in somite XXVI a2 and a3. Eyes in 3 pairs, 1st pair dorsally on anterior margin of somite III, 2nd and 3rd pairs dorsolaterally on posterior margin of somite V (a1+a2) ( Fig. 3A). Papillae numerous, minute, hardly visible, on row on every annulus.  Fig. 3.  Orobdella meisai sp. nov., holotype, KUZ Z1917. A, dorsal view of somites I–VIII; B, ventral view of somites I–VIII; C, dorsal view of somites XXIV–XXVII and caudal sucker; D, ventral view of somites XXIV–XXVI and caudal sucker; E, ventral view of somites X–XIII; F ventral view of gastropore and female gonopore. Scale bars: 1mm (A, B); 2mm (C–E); 0.25 mm (F). Abbreviations: af, annular furrow; an, anus; cl, clitellum; fg, female gonopore; gp, gastropore; mg, male gonopore; np, nephridiopore. Nephridiopores in 17 pairs, each situated ventrally at posterior margin of a1 of each somite in somites VIII–XXIV ( Fig. 3B, D, E). Pharynx agnathous, euthylaematous, reaching to somite XV b5 ( Fig. 4A). Crop tubular, acecate, reaching to somite XXI a2. Gastropore conspicuous, ventral, slightly posterior to anterior margin of somite XIII a1 ( Fig. 3E, F). Gastroporal duct rudimentary tubular, reaching to somite XV a1/a2 ( Fig. 4A); its pore opening to crop hardly detectable. Intestine tubular, acecate, reaching to somite XXIV a1/a2. Rectum tubular, thin-walled, straight. Testisacs multiple ( Fig. 4B); on right side, in somite XVIII a2 to somite XXV a2, in total ~40 testisacs, 4 inXVIII, 5 inXIX, 6 inXX, 6 inXXI, 5 inXXII, 5 inXXIII, 6 inXXIV, 3 inXXV; on left side, in somite XVIII a2 to somite XXIV  Fig. 4.  Orobdella meisai sp. nov., holotype, KUZ Z1917. A, ventral view of gastroporal duct; B, dorsal view of reproductive system including ventral nervous system; C, dorsal view of male atrium including position of ganglion XI; D, dorsal view of female reproductive system including position of ganglion XIII. Scale bars: 1mm (A); 2 mm (B); 0.25mm (C); 0.5 mm (D). Abbreviations: at, atrium; cod, common oviduct; cp, crop; ed, ejaculatory duct; gd, gastroporal duct; od, oviduct; ov, ovisac; ph, pharynx; ts, testisac; vd, vas deference. b6, in total ~35 testisacs, 3 inXVIII, 4 inXIX, 7 inXX, 5 inXXI, 5 inXXII, 5 inXXIII, 6 inXXIV. Paired vasa deferentia thick ( Fig. 4B). Epididymis absent. Paired ejaculatory ducts in somite XI a2 to somite XVIII a1 ( Fig. 4B); coiled in position posterior to ovisacs; each duct crossing ventrally beneath each ovisac, then coiled in position anterior to ovisacs; each winding from respective junction with vas deferens, narrowing toward atrium with pre-atrial loop extending to middle of somite XI a2. Atrial cornu absent. Atrium small, globular, in somite XI b5 and b6 ( Fig. 4B, C). Penis sheath and penis absent. Paired ovisacs globular, in somite XIII a2 and b5 ( Fig. 4B, D). Oviducts thin-walled ( Fig. 4D); both oviducts converging in to common oviduct in somite XIII a1; dorsal surface of junction of both oviducts covered with ventral nerve cord. Common oviduct thin-walled, short, directly descending to female gonopore ( Fig. 4D).  Variation.Measurements (mean±1SD, followed by ranges in parentheses; n=13, including holotype): BL 48.9± 16.3 mm( 27.4–73.1 mm), BW 4.2± 1.5 mm( 2.1– 7.2 mm), CL 2.1± 0.68 mm(1.2–3.0 mm), CW 2.5± 0.79 mm( 1.4–4.2 mm). Somite XXVI a3 sometimes with slight dorsal furrow, or dorsolateral furrow on respective sides. Somite XXVII generally uniannulate without secondary furrow, or rarely biannulate. First pair of eyes rarely dorsally on posterior margin of somite II, or on somites II/III; small unpaired eye dorsally on left posterior margin of somite III (KUZ Z1912). Pharynx reaching to somite XV b5/b6–b6. Crop reaching to somite XXI a1–a2. Gastroporal duct reaching to somite XV a2–b5/b6. Intestine reaching to somite XXIII b5/ b6–somite XXIV a2/b5. Testisacs; right side, ~34–42 sacs in somite XVII b5–b6 to somite XXV a1–a2; left side, ~36–42 sacs in somite XVII b5–b6 to somite XXIV b6–somite XXV a2. Paired ejaculatory ducts in somite XI a2 to somite XVII a2/b5–b5. Paired ovisacs in somite XIII a2, or in somite XIII a2 and b5.  Fig. 5. Maximum likelihood tree for 5207 bp of nuclear 18S rRNA and histone H3 and mitochondrial COI, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeuand ND1 markers. Numbers on nodes represent bootstrap values for maximum likelihood and Bayesian posterior probabilities.  Coloration.In life, dorsal-to-lateral surface yellowish brown with irregular brownish markings without distinct pattern ( Fig. 2C); ventral surface brownish white with irregular dark markings, ventral median region rarely with markings. Color faded in preservative, but irregular markings on surface remain ( Fig. 2A, B).   Distribution.This species was collected only from its typelocality. The present locality is the southernmost habitat within the known range of  Orobdellaspecies.  Natural history.This species was found curled up under rocks in moist mountainous habitats, and is thus terrestrial. A mature individual bearing a slight clitellum (= holotype) was collected on 9 March 2017. This finding suggests that the reproductive season of this species begins in early-to-middle March.  Molecular analyses results.The ML tree (ln L= −28606.84; Fig. 5) for estimating the phylogenetic position of the new species had an identical topology to that of the BI tree (mean ln L=−28193.30; not shown).  Orobdella meisaibelonged to a well-supported clade (BS=100%, PP=1.0) containing the other eight species inhabiting the western part of Honshu to Taiwan. The clade was divided into two subclades: a well-supported lineage (BS=99%, PP=1.0) consisted of four species, i.e.,  O. esulcata Nakano, 2010,  O. naraharaetmagarumNakano, 2016,  O. kanaekoikeaeNakano, 2017and  O. brachyepididymisNakano, 2016, inhabiting the western part of Honshu, Shikoku and Kyushu islands of Japan; another lineage was not fully supported in the ML tree (BS=49%, PP=0.92), comprising three species, i.e.,  O. mononokeNakano, 2012,  O. shimadaeNakano, 2011and  O. dolichopharynxNakano, 2011, from the Ryukyu Islands, and the two Taiwanese  O. ketagalanand  O. meisai. The monophyly of  O. shimadae,  O. dolichopharynx,  O. ketagalanand  O. meisaiwas supported (BS=83%, PP=1.0). The two Ryukyu species,  O. shimadaeinhabiting Okinawajima island, and  O. dolichopharynxknown from Amamioshima island, formed a fully supported clade (BS=100%, PP=1.0). Monophyly of the Taiwanese  O. ketagalanand  O. meisaiwas also well-supported (BS=92%, PP=1.0).   TheND1 sequences ( LC314421and LC314426) obtained from the holotypeand paratypeof  O . meisaiexhibit unique genetic variations compared with those of the other known  Orobdellaleeches. The ND1 sequences of  O. meisailack 97th–111th and 249th–260th bp positions in those of the other congeners. Since those missing positions are counted from the first position of the ND1 sequences, the former deletion indicates that the 33rd–37th codons are missing from the  O. meisaiND1 sequences, and the latter deletion shows that the 83rd–87th codons are compacted into a single DNA triplet.   Etymology.The specific name is from a Japanese word meisai (=camouflage) referring to the dorsal coloration of the new species. The specific name is thus treated as indeclinable.   Remarks.  Orobdella meisaiclearly belongs to  Orobdella, because it possesses the generic diagnostic characteristics defined by Nakano (2016a): post-anal annulus absent; male gonopore in posterior part of somite XI; female gonopore in anterior part of somite XIII; gonopores separated by more than one full somite; pharynx agnathous euthylaematous; gastropore in anterior part of somite XIII; gastroporal duct lying on female organ; testisacs multiple; male atrium in posterior part of somite XI, without penis sheath or penis; ovisacs globular in somite XIII; female median reproductive system essentially lacking.  Table 2. Comparisons of morphological characters between  Orobdella meisai sp. nov.and ten quadrannulate congeneric species.    Species Body length Somite IV Somite XXV Annuli between gonopores Pharynx length Gastroporal duct Epididymides Atrial cornua    O. meisai middle 1 4 5+1/4 to posterior XV rudimentary tubular absent absent    O. brachyepididymis small 1 4 1/2+4[+(<1/2)] to anterior XIV tubular XX to XXI small, ovate    O. esulcata middle 1 4 2/3+4+1/3 to anterior to posterior XIV tubular, but bulbous at junction with gastropore XVI to XX developed, ovate    O. kanaekoikeae small 1 4 1/2+4+1/2 to posterior XIII to anterior XIV bulbous XIV to XVIII developed, ovate    O. kawakatsuorum middle 2 4 6 to middle to posterior XIV simple tubular XVI to XVII undeveloped    O. ketagalan middle 1 4 1/2+4+1/2 to posterior XIV simple tubular absent undeveloped    O. koikei small 1 3 1/2+4+1/2 to posterior XIII to anterior XIV bulbous XV to XX developed, ovate    O. masaakikuroiwai small 1 4 1/2+4+1/2 to anterior to middle XIV bulbous XVI to XVIII developed, ovate    O. naraharaetmagarum small 1 4 1/2+4+1/2 to posterior XIII bulbous XV to XX developed, ovate    O. tsushimensis middle 1 4 1/2+5 to posterior XIII to posterior XIV bulbous XVII to XIX developed, ovate    O. whitmani middle 1 or 2 4 1/2+4+1/2 to anterior to posterior of XIV bulbous XVI to XVIII developed, ovate According to taxonomic studies ( Nakano 2010, 2011b, 2012a; Nakano and Lai 2012; Nakano 2014; Nakano and Seo 2014; Nakano 2016a, b, 2017b), the new species is distinguishable from the ten quadrannulate species (  O. brachyepididymis,  O. esulcata,  O. kanaekoikeae,  O. kawakatsuorumRichardson, 1975,  O. ketagalan,  O. koikeiNakano, 2012,  O. masaakikuroiwaiNakano, 2014,  O. naraharaetmagarum,  O. tsushimensis, and  O. whitmaniOka, 1895) by the following combination of characters ( Table 2): body length, annulation of somites IV and XXV, number of annuli between gonopores, pharynx length, gastroporal duct, epididymidal lengths and atrial cornua.  Orobdella meisaican clearly differ from these known quadrannulate species in having 5+1/4 annuli between the gonopores, the pharynx reaching to the posterior part of somite XV, the rudimentary tubular gastroporal duct, and in lacking atrial cornua in the male median reproductive system. The new species is obviously distinguished from the six sexannulate species, i.e.,  O. dolichopharynx,  O. ijimaiOka, 1895,  O. mononoke,  O. okanoiNakano, 2016,  O. shimadaeand  O. yamaneaeNakano, 2016, and two octannulate species, i.e.,  O. nakahamaiNakano, 2016and  O. octonariaOka, 1895, by its mid-body somites that are quadrannulate. 2017-03-09 KUZ, TN Taiwan Takafumi Nakano 1470 22.4073 Jinshui Ying Old Trail 1 120.75857 Fanliao 1 144 Z1917 1 Pingtung holotype 2014-02-23 2017-03-09 2014-02-23 KUZ Taiwan 1490 2 145 22.409191 1 120.75482 1 144 Z1908, Z1909, Z1910, Z1911, Z1912, Z1914, Z1915, Z1913, Z1916, Z1918, Z1919, Z1920 12 Pingtung paratype LC314421 and LC314426 The 5 148 2 holotype