Orobdella masaakikuroiwai , Nakano, Takafumi, 2014
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Taxon classification Animalia Arhynchobdellida Orobdellidae
Orobdella masaakikuroiwai sp. n. Figs 2, 3, 4, 5
Body length of mature individual less than 4 cm. Somite IV uniannulate, somites VIII–XXV quadrannulate. Clitellum in XI b5 to XIII a2. Pharynx reaching to XIV. Gastropore conspicuous in middle of XIII a1. Gastroporal duct bulbous, winding at junction with gastropore. Male gonopore in middle of XI b6, female gonopore inconspicuous in middle of XIII a1, behind gastropore, gonopores separated by 1/2 + 4 + 1/2 annuli. Paired epididymides in XV/ XVI–XVI b5/b6 to XVII b5/b6-XVIII/XIX, occupying 7-10 annuli (i.e. one and a half to two and a half somites). Atrial cornua developed, ovate.
Type materials (see Fig. 1 for the locality number).
Holotype. KUZ Z694, holotype, dissected, collected from under a rock along a forest road at Mt. Mitsugaisan, Ina, Nagano Pref., Japan (35°47.72'N, 138°04.70'E; Alt. 875 m; locality number 4), by TN on 20 July 2012.
Paratypes. Four paratypes from the type locality by TN on 20 July 2012: KUZ Z690, Z691 (35°47.72'N, 138°04.69'E; Alt: 872 m), and KUZ Z692, Z693 (35°47.74'N, 138°04.69'E; Alt: 872 m). KUZ Z693, dissected.
Additional materials (see Fig. 1 and Table 1 for the locality numbers).
In total, 11 specimens examined. KUZ Z684-Z686 (three specimens), collected from under rocks in Akiruno (locality number 1), by TN: KUZ Z684, from along a mountain trail at Mt. Kariyoseyama (35°42.37'N, 139°12.03'E; Alt. 341 m) on 29 March 2010; KUZ Z685, from along Ohikagedori Road (35°43.33'N, 139°11.98'E; Alt. 230 m) on 30 March 2010; KUZ Z686, from along Bonborisen Forest Road (35°47.73'N, 139°11.01'E; Alt. 284 m) on 30 March 2010. KUZ Z687, Z688 (two specimens), from under rocks along a forest road in Namesawakeikoku Valley (locality number 2), by TN on 9 July 2011: KUZ Z687 (34°50.59'N, 138°54.69'E; Alt. 551 m); KUZ Z688 (34°50.50'N, 138°54.59'E; Alt. 576 m). KUZ Z689, from under fallen leaves along a forest road at Shirabisotoge Pass (35°26'N, 138°01'E; Alt. 1840 m; locality number 5), by Yoshiko Yamane on 14 October 2011. KUZ Z695, Z696 (two specimens), from under rocks in Shiojidaira Nature Park (locality no 7), by TN on 10 August 2012: KUZ Z695 (35°40.62'N, 137°50.48'E; Alt. 1304 m); KUZ Z696 (35°40.66'N, 137°50.48'E; Alt. 1315 m). KUZ Z697, Z698 (two specimens), from under rocks along a mountain stream in Ikuta (locality no 6), by TN on 10 August 2012: KUZ Z697 (35°33.67'N, 138°00.04'E; Alt. 1098 m); KUZ Z698 (35°33.68'N, 138°00.04'E; Alt. 1099 m). KUZ Z699, from under fallen leaves near Shibunoyu (36°02.1'N, 138°19.5'E; Alt. 1860 m; locality number 3), by Yume Imada on 6 October 2012. KUZ Z684, Z687, Z689, Z696, Z697 and Z699 (six specimens), dissected.
The specific name is a noun in the genitive case formed directly from the name of Mr Masaaki Kuroiwa, who generously accompanied the field survey in Nagano Prefecture.
Description of holotype.
Body firm and muscular, elongate, with constant width in caudal direction, dorsoventrally compressed, BL 34.0 mm, BW 3.42 mm (Fig. 2). Caudal sucker ventral, elliptic, CL 1.7 mm (minor axis), CW 1.9 mm (major axis) (Figs 2B, 3D).
Somite I completely merged with prostomium (Fig. 3A). Somites II–IV uniannulate, II not separated from I (Fig. 3A). Somite V biannulate, (a1 + a2) = a3; a3 forming posterior margin of oral sucker (Fig. 3A, B). Somites VI, VII triannulate, a1 = a2 = a3 (Fig. 3A, B). Somites VIII–XXV quadrannulate, a1 = a2 = b5 = b6 (Fig. 3 A–E); b5 of X and a2 of XIII respectively being first and last annuli of clitellum (Fig. 3E). Somite XXVI triannulate, with slight furrow in a3, a1 > a2 < a3 (b5 = b6); a3 being ventrally last complete annuls (Fig. 3C, D). Somite XXVII biannulate, with slight dorsal furrow in last annulus; anus behind it with no post-anal annulus (Fig. 3C).
Anterior ganglionic mass in VI a2 and a3. Ganglia VII–X, of each somite, in a2 (Fig. 4A). Ganglion XI in a2 and b5 (Fig. 4A). Ganglia XII–XVIII, of each somite, in a2 (Fig. 4A). Ganglia XIX, XX, of each somite, in a1 and a2. Ganglia XXI, XXII, of each somite, in a2. Ganglion XXIII in a1 and a2. Ganglion XXIV in a1. Ganglion XXV in XXIV b6 and XXV a1. Ganglion XXVI in b5 and b6 of XXV. Posterior ganglionic mass in a1-a3 of XXVI.
Eyes in three pairs, first pair dorsally on anterior margin of III, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig. 3A). Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of a1 of each somite in VIII–XXIV (Fig. 3B, E). Papillae numerous, minute, hardly visible, one row on every annulus.
Pharynx agnathous, euthylaematous, reaching to XIV a1/a2 (Fig. 3G). Crop tubular, reaching to XIX b5/b6 (Fig. 3G). Gastropore conspicuous, ventral in middle of XIII a1 (Fig. 3E, F). Gastroporal duct bulbous, slightly winding at junction with gastropore, joining with crop in XIV b5 (Fig. 3G). Intestine tubular, acecate, reaching to XXIV a1/a2. Rectum tubular, thin-walled, descending to anus.
Male gonopore in middle of XI b6 (Fig. 3E). Female gonopore in middle of XIII a1, inconspicuous, located posterior to gastropore (Fig. 3G). Gonopores separated by 1/2 + 4 + 1/2 annuli (Fig. 3E). Testisacs multiple, one or two on each side in each an nulus, in XVIII a2 to XXV a1 (Fig. 4A). Paired epididymides in XVI a2 to XVIII a1, occupying 8 annuli (Fig. 4A). Ejaculatory bulbs absent. Paired ejaculatory ducts in XI a2/b5 to XVI a2, coiled in position posterior to ovisacs; each duct crossing ventrally beneath each ovisac, then loosely curved in position anterior to ovisacs; each widening from respective junction with epididymis, narrowing at junction with atrial cornua, then turning sharply inward toward atrial cornua with pre-atrial loop reaching to anterior margin of XI b5 (Fig. 4A). Pair of muscular atrial cornua ovate, in XI b5 and b6 (Fig. 4 A–D). Atrium short, muscular, globular in XI b6 (Fig. 4 B–D). Penis sheath and penis absent. Paired ovisacs elongated globular, one each in XIII a2-b6 (Fig. 4A, E). Oviducts thin-walled, left oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII a1/a2 (Fig. 4A, E). Common oviduct thin-walled, short, directly descending to female gonopore (Fig. 4E).
BL 22.4 (KUZ Z686) -35.2 (KUZ Z684) mm, BW 2.3 (KUZ Z691) -3.5 (KUZ Z684) mm, CL 1.1 (KUZ Z686)-1.7 (KUZ Z693) mm, CW 1.1 (KUZ Z686)-2.1 (KUZ Z689) mm. Somites III, IV uniannulate, each with slight dorsal furrow (KUZ Z695). Somite XXVI variable; often dorsally quadrannulate, ventrally triannulate, rarely with slight ventral furrow in a3; KUZ Z699 with quadrannulate; KUZ Z698, Z691 with triannulate with slight dorsal furrow in a3; KUZ Z689 with triannulate. Somite XXVII biannulate, or uniannulate with slight dorsal furrow. Eyes in three pairs; KUZ Z699 with one eye dorsoleft on posterior margin of III. Pharynx reaching to XIII/ XIV–XIV a2/b5. Crop reaching to XIX b5/b6-XX a1. Gastropore occasionally slightly posterior to middle of XIII a1. Gastroporal duct joining with crop in XIV a1/a2-XIV b6; KUZ Z687 with thick, tubular duct. Intestine reaching to XXIII/ XXIV–XXV a2. Male gonopore rarely slightly anterior to middle of XI b6, or slightly posterior to middle of XI b6. Female gonopore occasionally slightly posterior to middle of XIII a1. Testisacs in XVII b6-XIX a1 to XXIV b5-XXV a2. Epididymides in XV/ XVI–XVI b5/b6 to XVII b5/b6-XVIII/XIX; occupying 7-10 annuli. Atrial cornua generally ovate; KUZ Z696 ellipsoid; KUZ Z687 fusiform. Pre-atrial loop absent, or reaching to middle of XI b5 (KUZ Z693, Z697). Ovisacs often in XIII a2-b6; KUZ Z687, Z699 in XIII a2, b5; KUZ Z696 right one in XIII a2-XIV a1/a2, left one in XIII a2-XIV a1. Right or left oviduct crossing ventrally beneath nerve cord; KUZ Z684, Z693 both oviducts converging into common oviduct in XIII a2.
In life, dorsal surface ochre (Fig. 5), whitish brown, or brown, ventral surface grayish white or yellowish white; individuals from Shizuoka Pref. (KUZ Z687, Z688), dorsal surface whitish yellow. Colour faded in preservative, rarely with one dorsal black line from VII a3-IX a2 to XIX b5-XXVI b6 (KUZ Z691, Z693, Z694, Z698).
Distribution (see Fig. 1 for the locality numbers).
This species was primarily collected from localities in Nagano Prefecture: the east-central part (locality number 3), and the southeastern part along the Inadani Basin (locality numbers 4-7). This species was also found in the western mountainous part of the Metropolitan Tokyo area (locality number 1), as well as in the Amagi Mountain Range in the central part of the Izu Peninsula, Shizuoka Prefecture (locality number 2). The locality data for this species suggested that Orobdella masaakikuroiwai sp. n. would be widely distributed in mountainous regions such as the southwestern part of the Kanto Region and the southeastern part of the Chubu Region, Honshu, Japan. The lowest elevation among the localities was 230 m above sea level (a.s.l.) (locality number 1), and the highest was ca. 1860 m a.s.l. (locality number 3).
This species was generally found curled up under rocks or fallen leaves in moist mountainous habitats (Fig. 5B). Soil was sometimes observed in the digestive tract during specimen dissection. This species is therefore considered an earthworm-feeder as are the other known Orobdella leeches.
Mature leeches with an obvious clitellum were collected on 20 July (KUZ Z690, Z691, Z693, Z694) and 10 August (KUZ Z697) at two sites in Nagano Prefecture (locality numbers 4 and 7, elevation ca. 875 m and 1098 m, respectively). These findings indicate that the reproductive season of this species may begin in mid-to-late July.
Although the leech specimens examined in this study were small (up to 35 mm), several individuals, including the holotype, were determined to be mature due to the possession of an obvious clitellum and developed testisacs. Specimen KUZ Z687 possessed a tubular gastroporal duct and fusiform atrial cornua. Immature leeches may have these characteristics, because the sperm ducts and testisacs of specimen KUZ Z687 are undeveloped and barely detectable.
The new species unambiguously belongs to the genus Orobdella as it has all the generic diagnostic characteristics (see Nakano et al. (2012) for the generic diagnosis): post-anal annulus absent; pharynx agnathous, euthylaematous; gastropore in XIII; gastroporal duct lying on female organ; gonopores separated by more than one full somite; testisacs multiple; male atrium in XI without penis sheath and penis; ovisacs globular in XIII; female median reproductive system essentially lacking.
According to previous taxonomic studies ( Nakano 2010, 2011a, 2012b, Nakano and Gongalsky 2014, Nakano and Lai 2012, Nakano and Seo 2012, 2014), Orobdella masaakikuroiwai sp. n. differs from the six other quadrannulate species (i.e., Orobdella esulcata Nakano, 2010, Orobdella kawakatsuorum , Orobdella ketagalan Nakano & Lai, 2012, Orobdella koikei , Orobdella tsushimensis Nakano, 2011a, and Orobdella whitmani Oka, 1895) by the following combination of characteristics (Table 3): body length less than 4 cm, IV uniannulate, gonopores separated by 1/2 + 4 + 1/2, XXV quadrannulate, gastroporal duct bulbous, epididymides in XVI to XVIII, atrial cornua ovate. Among the six above-listed quadrannulate species, only Orobdella whitmani is present in Honshu. Both Orobdella masaakikuroiwai sp. n. and Orobdella whitmani possess 1/2 + 4 + 1/2 annuli between the gonopores, a bulbous gastroporal duct, and epididymides in XVI–XVIII. Thus, it is difficult to distinguish these two species using these diagnostic features. However, Orobdella whitmani is a large species and grows up to ca. 10 cm ( Nakano 2010, Oka 1895). Therefore, Orobdella masaakikuroiwai sp. n. clearly differs from mature individuals of Orobdella whitmani in body length. However, distinguishing the new species from a small juvenile of Orobdella whitmani can be complex. Because immature individuals of Orobdella whitmani possess a tubular gastroporal duct (Nakano, unpublished observation) and mature individuals of Orobdella masaakikuroiwai sp. n. possess a bulbous gastroporal duct, the characteristics of the duct could be used to distinguish between the two. However, insofar as immature leeches of both species have a tubular gastroporal duct, this characteristic is not useful for discriminating between immature individuals of Orobdella masaakikuroiwai sp. n. and Orobdella whitmani . DNA data might be useful for identification, similar to the DNA barcoding of freshwater leeches (e.g. Oceguera-Figueroa et al. (2010)). In addition to DNA data, interbreeding experiments or karyological studies may be crucial for definitive clarification between Orobdella masaakikuroiwai sp. n. and Orobdella whitmani as is the case with the species of Hirudo Linnaeus, 1758 in Europe ( Petrauskiene et al. 2009, Utevsky et al. 2009).
The quadrannulate Orobdella masaakikuroiwai sp. n. is unequivocally distinguishable from the four species Orobdella dolichopharynx Nakano, 2011b, Orobdella ijimai Oka, 1895, Orobdella mononoke Nakano, 2012a and Orobdella shimadae Nakano, 2011b, due to their sexannulate mid-body somites, as well as Orobdella octonaria , which possesses octannulate mid-body somites.
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