Research Article
Print
Research Article
Four new species of the genus Orobdella from Shikoku and Awajishima island, Japan (Hirudinida, Arhynchobdellida, Orobdellidae)
expand article infoTakafumi Nakano
‡ Graduate School of Education, Hiroshima University, Higashihiroshima, Japan
Open Access

Abstract

Four new species of the genus Orobdella Oka, 1895 from the mountainous regions of Shikoku and Awajishima island, Japan are described. These new species consist of one quadrannulate, two sexannulate and one octannulate species. The quadrannulate Orobdella brachyepididymis sp. n. is a small species with a body length reaching only ca. 5 cm. The sexannulate Orobdella okanoi sp. n. was collected from Shikoku, and the other sexannulate species, Orobdella yamaneae sp. n., inhabits Awajishima island. The octannulate Orobdella nakahamai sp. n. is a large species with a body length greater than 20 cm and is only the second large octannulate species known within this genus. Phylogenetic analyses using nuclear 18S rRNA and histone H3, as well as mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu and NADH dehydrogenase subunit 1 markers, indicated that O. brachyepididymis is a sister species of the quadrannulate O. naraharaetmagarum Nakano, 2016, while the other three new species formed a clade closely related to O. masaakikuroiwai Nakano, 2014 and O. whitmani Oka, 1895. The ranges of the distant phylogenetic lineage groups of Orobdella overlap in Shikoku and adjacent islets.

Key Words

Hirudinea, Orobdella, new species, gastroporous, molecular phylogeny, Japan

Introduction

The genus Orobdella Oka, 1895 is a terrestrial macrophagous leech taxon that inhabits the Japanese Archipelago, Korean Peninsula and Taiwan (Nakano and Lai 2012, Nakano and Seo 2014, Sawyer 1986). The latest taxonomic study on this genus showed that Orobdella contains 13 species (Nakano 2016) split into three groups based on their mid-body somite annulation: eight species in the quadrannulate (four annuli per one somite) group and four species in the sexannulate (six annuli) group. The other species O. octonaria Oka, 1895 is an octannulate (eight annuli) taxon.

Additionally, these 13 species can be also split into another three types based on the body lengths of mature leeches (Nakano 2016): three quadrannulate species belong to the small-type (of which the body lengths of mature leeches are shorter than 5 cm); the other five quadrannulate and all sexannulate species are designated the middle-type (reaching ca. 10 cm); and the octannulate species is assigned to the large-type. The body length of the octannulate O. octonaria has been described as being greater than 20 cm (Nakano 2012c, Richardson 1971). Molecular phylogenetic analyses of the Orobdella species clearly showed that these morphological groups or forms do not reflect phylogenetic relationships (e.g. Nakano 2016): the sexannulate mid-body somite annulation and the small-type body length have evolved in parallel in the genus.

Additional Orobdella leeches were collected from Shikoku and Awajishima island, Japan. The specimens collected from Shikoku comprised three morphological units: a quadrannulate small-type, a sexannulate middle-type and an octannulate large-type. Meanwhile, Orobdella leeches on Awajishima island belonged to the sexannulate middle-type. Each of these four units is described here as a new species. In addition, the phylogenetic positions of these new species were estimated using nuclear 18S rRNA and histone H3, as well as mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu, and NADH dehydrogenase subunit 1 sequence data.

Materials and methods

Sampling and morphological examination

Leeches were collected from five localities in Shikoku, and one locality on Awajishima island, Japan (Fig. 1). When possible, elevation and geographical coordinates for localities were obtained using a Garmin eTrex® GPS unit.

Figure 1.

Map showing the collection localities of the specimens examined in this study. The closed diamond (2) denotes the locality of Orobdella brachyepididymis sp. n., closed squares (4–6) show the localities of Orobdella nakahamai sp. n., closed triangles (3, 5) designate the localities of Orobdella okanoi sp. n., and the closed circle (1) specifies the locality of Orobdella yamaneae sp. n. Symbols in red indicate the type locality of each of the new species.

Almost all of the specimens were relaxed by the gradual addition of absolute ethanol to freshwater. For DNA extraction, botryoidal tissue was removed from the posterior part of the body around the caudal sucker of every specimen, and then preserved in absolute ethanol. The remainder of the body was fixed in 10% formalin and preserved in 70% ethanol. Four measurements were taken: body length (BL) from the anterior margin of the oral sucker to the posterior margin of the caudal sucker, maximum body width (BW), caudal sucker length (CL) from the anterior to the posterior margin of the sucker and caudal sucker width (CW) from the right to the left margin of the sucker. Examination, dissection, and drawing of the specimens were conducted using a stereoscopic microscope with a drawing tube (Leica M125). Specimens used in this study have been deposited in the Zoological Collection of Kyoto University (KUZ).

The numbering convention is based on Moore (1927): body somites are denoted by Roman numerals, and the annuli in each somite are given alphanumeric designations. The following morphological abbreviations are used in the figures: ac—atrial cornu; af—annular furrow; an—anus; at—atrium; cl—clitellum; cod—common oviduct; cp—crop; ed—ejaculatory duct; ep—epididymis; fg—female gonopore; gd—gastroporal duct; gp—gastropore; mg—male gonopore; np—nephridiopore; od—oviduct; ov—ovisac; ph—pharynx; ts—testisac.

RCP and DNA sequencing

The extraction of genomic DNA from botryoidal tissues preserved in absolute ethanol followed Nakano (2012b). Primer sets for the PCR and cycle sequencing (CS) reactions used in this study were as follows: for 18S rRNA, A and L (PCR and CS), C and Y (PCR and CS), as well as O and B (PCR and CS) (Apakupakul et al. 1999); for histone H3 (H3), H3aF and H3bR (PCR and CS) (Colgan et al. 1998); for cytochrome c oxidase subunit I (COI), LCO1490 (PCR and CS) and HCO2198 (CS) (Folmer et al. 1994), and LCO-in (CS) and HCO-out (PCR and CS) (Nakano 2012b) or HCO-outout (PCR and CS) (Nakano 2012a); for tRNACys, tRNAMet, 12S rRNA, tRNAVal and 16S rRNA (tRNACys–16S), 12SA-out and 12SB-in (PCR and CS), and 12SA-in and 12SB-out (PCR and CS) (Nakano 2012b); for tRNALeu and NADH dehydrogenase subunit 1 (ND1) (tRNALeu–ND1), LND3000 and HND1932 (PCR and CS) (Light and Siddall 1999). The PCR reactions and DNA sequencing were performed using the modified method mentioned in Nakano (2012a). The PCR reactions were performed using a GeneAmp PCR System 2700 and 9700 (Applied Biosystems) as well as a T100 Thermal Cycler (Bio-Rad). The PCR mixtures were heated to 94°C for 5 min, followed by 35 cycles at 94°C (10 s each), 52°C for 18S and H3, 50°C for the anterior and posterior parts of tRNACys–16S, 48°C for COI or 42°C for tRNALeu–ND1 (20 s), and 72°C (1 min 12 s for COI, 24 s for H3 or 42 s for the other markers), and a final extension at 72°C for 6 min. The sequencing mixtures were heated 96°C for 2 min, followed by 40 cycles at 96°C (10 s each), 50°C (5 s each) and 60°C (42 s each). The obtained sequences were edited using DNA BASER (Heracle Biosoft S.R.L.). The DNA sequences listed in Table 1 were newly obtained in this study and were deposited with the International Nucleotide Sequence Database Collaboration (INSDC) through the DNA Data Bank of Japan (DDBJ). The GenSeq nomenclature proposed by Chakrabarty et al. (2013) was followed in Table 1 to show the reliability of the obtained DNA sequences.

Samples used for the phylogenetic analyses. The information on the vouchers is accompanied by the collection locality numbers for the new species of Orobdella described in this study (see Fig. 1) and the INSDC accession numbers. Sequences marked with am asterisk were obtained for the first time in the present study. Acronyms: KUZ, the Zoological Collection of Kyoto University; UNIMAS, the Universiti Malaysia Sarawak.

Species Voucher (locality number) INSDC # GenSeq Nomenclature
18S Histone H3 COI tRNACys–16S tRNALeu–ND1
Orobdella brachyepididymis sp. n. KUZ Z1673 Holotype (2) LC106319* LC106321* LC106320* LC106318* LC106322* genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella brachyepididymis sp. n. KUZ Z1674 Paratype (2) LC106324* LC106323* LC106325* genseq-2 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella nakahamai sp. n. KUZ Z1352 Paratype (5) LC106327* LC106326* LC106328* genseq-2 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella nakahamai sp. n. KUZ Z1672 Holotype (4) LC106330* LC106332* LC106331* LC106329* LC106333* genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella nakahamai sp. n. KUZ Z1680 (6) LC106335* LC106334* LC106336* genseq-3 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella okanoi sp. n. KUZ Z1491 (5) LC106338* LC106337* LC106339* genseq-3 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella okanoi sp. n. KUZ Z1671 Holotype (3) LC106341* LC106343* LC106342* LC106340* LC106344* genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella yamaneae sp. n. KUZ Z1358 Paratype (1) LC106346* LC106345* LC106347* genseq-2 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella yamaneae sp. n. KUZ Z1678 Holotype (1) LC106349* LC106351* LC106350* LC106348* LC106352* genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1
Orobdella dolichopharynx Nakano, 2011b KUZ Z120 Holotype AB663665 AB698876 AB679680 AB679681 AB828558
Orobdella esulacata Nakano, 2010 KUZ Z29 Holotype AB663655 AB698873 AB679664 AB679665 AB828555
Orobdella ijimai Oka, 1895 KUZ Z110 Topotype AB663659 AB698877 AB679672 AB679673 AB828559
Orobdella kawakatsuorum Richardson, 1975 KUZ Z167 Topotype AB663661 AB698878 AB679704 AB679705 AB828561
Orobdella ketagalan Nakano & Lai, 2012 KUZ Z208 Holotype AB704785 AB704786 AB704787 AB828582 AB828563
Orobdella koikei Nakano, 2012b KUZ Z156 Holotype AB698883 AB698882 AB679688 AB679689 AB828560
Orobdella masaakikuroiwai Nakano, 2014 KUZ Z694 Holotype AB938003 AB938013 AB938006 AB937997 AB938016
Orobdella mononoke Nakano, 2012a KUZ Z224 Holotype AB698868 AB698869 AB698866 AB698867 AB828564
Orobdella naraharaetmagarum Nakano, 2016 KUZ Z1652 Holotype LC087143 LC087145 LC087144 LC087142 LC087146
Orobdella octonaria Oka, 1895 KUZ Z181 Topotype AB698870 AB698871 AB679708 AB679709 AB828562
Orobdella shimadae Nakano, 2011b KUZ Z128 Holotype AB663663 AB698875 AB679676 AB679677 AB828557
Orobdella tsushimensis Nakano, 2011a KUZ Z134 Holotype AB663653 AB698872 AB679662 AB679663 AB828554
Orobdella whitmani Oka, 1895 KUZ Z45 Topotype AB663657 AB698874 AB679668 AB679669 AB828556
Erpobdella japonica Pawłowski, 1962 KUZ Z178 AB663648 AB698879 AB679654 AB679655 AB828542
Gastrostomobdella monticola Moore, 1929 UNIMAS/A3/BH01/10 AB663649 AB698880 AB679656 AB679657 AB828543
Mimobdella japonica Blanchard, 1897 KUZ Z179 AB663650 AB698881 AB679658 AB679659 AB828544
Odontobdella blanchardi (Oka, 1910a) KUZ Z180 AB663651 AB938012 AB938004 AB937995 AB938014

Molecular phylogenetic and genetic distance analyses

Eighty-five published sequences were obtained from the INSDC for use in molecular phylogenetic analyses (Table 1). In addition to 13 known Orobdella species, the following four erpobdelliform species were used as outgroup taxa: Erpobdella japonica Pawłowski, 1962 (Erpobdellidae), Gastrostomobdella monticola Moore, 1929 (Gastrostomobdellidae), Mimobdella japonica Blanchard, 1897, and Odontobdella blanchardi (Oka, 1910a) (both Salifidae).

The phylogenetic relationships of the newly identified Orobdella species within the genus was estimated based on 18S, H3, COI, tRNACys–16S and tRNALeu–ND1 sequences. The alignments of H3 and COI were trivial, as no indels were observed. 18S, tRNACys–16S, and tRNALeu–ND1 were aligned using MAFFT v. 7.266 L-INS-I (Katoh and Standley 2013). The lengths of the 18S, H3, COI, tRNACys–16S, and tRNALeu–ND1 sequences were 1,844, 328, 1,267, 1,135, and 635 bp, respectively. The concatenated sequences thus yielded 5,209 bp of aligned positions.

Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI). ML phylogenies were constructed using RAxML v. 8.1.5 (Stamatakis 2014) with the substitution model set as GTRCAT, immediately after nonparametric bootstrapping (Felsenstein 1985) conducted with 1,000 replicates. The best-fit partitioning scheme for the ML analyses was identified with the Akaike information criterion (Akaike 1974) using PartitionFinder v. 1.1.1 (Lanfear et al. 2012) with the “greedy” algorithm: 18S/the 1st and 2nd positions of H3/the 3rd position of H3/the 1st position of COI/the 2nd position of COI/the 3rd positions of COI and ND1/the 1st position of ND1/the 2nd position of ND1/12S/16S/tRNACys, tRNAMet, tRNAVal and tRNALeu.

BI and Bayesian posterior probabilities (BPPs) were estimated using MrBayes v. 3.2.5 (Ronquist et al. 2012). The best-fit partition scheme and models for each partition were selected based on the Bayesian information criterion (Schwarz 1978) using PartitionFinder with the “greedy” algorithm: for 18S and the 1st position of H3, K80+I; for the 2nd position of H3, JC69; for the 3rd position of H3, HKY85+G; for the 1st position of COI, GTR+G; for the 2nd positions of COI and ND1, HKY85+I+G; for the 3rd positions of COI and ND1 plus 16S, HKY85+I+G; for the 1st position of ND1, 12S, tRNACys, tRNAMet, tRNAVal and tRNALeu, GTR+I+G. Two independent runs of four Markov chains were conducted 12 million generations, and the tree was sampled every 100 generations. The parameter estimates and convergence were checked using Tracer v. 1.6.0 (Rambaut and Drummond 2009) and the first 40,001 trees were discarded based on the results.

Nodes with bootstrap support (BS) values higher than 70% were considered sufficiently resolved (Hillis and Bull 1993). Nodes with BPPs higher than 0.95 were considered statistically significant (Leaché and Reeder 2002).

Pairwise comparisons of uncorrected p-distances for nine COI sequences (1,267 bp) obtained from specimens of the new species in this study and sequences from Orobdella masaakikuroiwai Nakano, 2014 and Orobdella naraharaetmagarum Nakano, 2016 were calculated using MEGA6.06 (Tamura et al. 2013) in accordance with the results of the molecular phylogenetic analyses.

Results

Taxonomy
Family Orobdellidae Nakano et al., 2012

Orobdella Oka, 1895

Orobdella Oka, 1895: 278–280; Oka 1910b: 177; Soós 1966: 377, 381, 382; Richardson 1975: 42; Lukin 1976: 463, 464; Sawyer 1986: 680; Nakano 2010: 881; Nakano 2011b: 3.

Kumabdella Richardson 1971: 590, 591 (type species, Orobdella octonaria Oka, 1895 by original designation).

Type species

Orobdella whitmani Oka, 1895 by subsequent designation of Soós (1966).

Diagnosis

Body firm and muscular, elongate, with constant width in caudal direction, dorsoventrally compressed. Somite I completely merged with prostomium. Somite II uniannulate, not separated from I. Mid-body somite annulation variable, complete quadr-, sex- or octannulate. Post-anal annulus absent. Male gonopore in posterior part of XI. Female gonopore in anterior part of XIII. Pappilae numerous, minute, hardly visible, one row on every annulus. Pharynx agnathous, euthylaematous. Crop tubular, acaecate. Gastropore, when present, in anterior part of XIII. Gastroporal duct generally lying on female organ. Intestine tubular, acaecate. Rectum tubular, thin-walled, straight. Testisacs multiple. Ejaculatory bulbs absent. Male median reproductive system in posterior part of XI, without penis or penis sheath. Ovisacs globular. Oviducts thin-walled. Common oviduct thin-walled, short. Female median reproductive system essentially lacking.

Orobdella brachyepididymis sp. n.

Figs 2, 3, 4, 5

Type materials

Holotype: KUZ Z1673, dissected, collected from under a rock along a road (33.87151°N, 134.12016°E; Elev. ca. 990 m; locality #2, see Fig. 1) at Mt. Ichinomori, Mima, Tokushima Prefecture, Japan, by TN on 7 July 2015. Paratype: KUZ Z1674, dissected, collected from under a rock along a road (33.87119°N, 134.12233°E; Elev. ca. 1014 m) at the type locality, by TN on 7 July 2015.

Type locality

Japan, Tokushima Prefecture: Mima, Mt. Ichinomori (Shikoku).

Diagnosis

Body length of mature individual reaching to ca. 5 cm. Somite IV uniannulate, somites VIII–XXV quadrannulate. Clitellum in XI b5 to XIII a2. Male gonopore in middle of XI b6, female gonopore in anterior margin of or slightly anterior to middle of XIII a1, behind gastropore, gonopores separated by 1/2 + 4 [+ (< 1/2)] annuli. Pharynx reaching to XIV a1–a1/a2. Gastropore conspicuous, in anterior margin of or slightly anterior to middle of XIII a1. Gastroporal duct tubular, slightly bulbous at junction with gastropore. Paired epididymides in XX to XXI, occupying four annuli (one somite). Atrial cornua small ovate.

Description of holotype

BL 51.6 mm, BW 5.2 mm (Fig. 2). Caudal sucker ventral, elliptic, CL 3.0 mm, CW 3.4 mm (Figs 2B, 3D).

Figure 2.

Orobdella brachyepididymis sp. n., holotype, KUZ Z1673. A dorsal, B ventral views. Scale bar: 5 mm.

Figure 3.

Orobdella brachyepididymis sp. n., holotype, KUZ Z1673. A dorsal, B ventral views of somites I–VIII; C dorsal, D ventral views of somites XXIV–XXVII and caudal sucker; E ventral view of somites X–XIII; F ventral view of gastropore and female gonopore; G ventral view of gastroporal duct. Scale bars: A, B, G = 1 mm; C–E = 2 mm; F = 0.25 mm.

Somites III, IV uniannulate; IV with slight dorsal furrow (Fig. 3A). Somite V biannulate, (a1 + a2) = a3; a3 forming posterior margin of oral sucker (Fig. 3A, B). Somites VI and VII triannulate; VI, a1 < a2 > a3, a2 with slight dorsal furrow; VII, a1 = a2 = a3 (Fig. 3A, B). Somites VIII–XXV quadrannulate, a1 = a2 = b5 = b6 (Fig. 3A–E). Somite XXVI triannulate; dorsally a1 > a2 < a3, a3 with slight furrow; ventrally a1 > a2 = a3; a3 being ventrally last complete annulus (Fig. 3C, D). Somite XXVII uniannulate with slight dorso-lateral furrows (Fig. 3C). Anus behind somite XXVII (Fig. 3C).

X b5 and XIII a2, respectively, being first and last annuli of clitellum (Fig. 3E).

Male gonopore in middle of XI b6 (Fig. 3E). Female gonopore in anterior margin of XIII a1, inconspicuous, located posterior to gastropore (Fig. 3E, F). Gonopores separated by 1/2 + 4 annuli (Fig. 3E).

Anterior ganglionic mass in VI a2 and a3. Ganglion VII in a2. Ganglion VIII in a2 and b5. Ganglia IX and X, of each somite, in a2. Ganglion XI in a2 and b5 (Fig. 4A). Ganglia XII–XVI, of each somite, in a2 (Fig. 4A). Ganglia XVII–XX, of each somite, in a1 and a2 (Fig. 4A). Ganglia XXI (Fig. 4A) and XXII, of each somite, in a2. Ganglion XXIII in a2 and b5. Ganglia XXIV and XXV, of each somite, in a1 and a2. Ganglion XXVI in XXV b6 and XXVI a1. Posterior ganglionic mass in XXVI a2 and a3.

Figure 4.

Orobdella brachyepididymis sp. n., holotype, KUZ Z1673. A dorsal view of reproductive system including ventral nervous system; B dorsal (including positions of ganglia XI and XII), C lateral, D ventral views of male atrium; E dorsal view of female reproductive system including position of ganglion XIII. Scale bars: A = 3 mm; B–D = 0.5 mm; E = 0.25 mm.

Eyes in three pairs, first pair dorsally on posterior margin of II, 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, D, E).

Pharynx reaching to XIV a1 (Fig. 3G). Crop reaching to XIX b5/b6. Gastropore conspicuous, ventral, in anterior margin of XIII a1 (Fig. 3E, F). Gastroporal duct tubular, but slightly bulbous and winding at junction with gastropore, joining with crop in XIV a1 (Fig. 3G). Intestine reaching to XXIV/XXV.

Testisacs (Fig. 4A); on right side, in XXI b6 to XXV b5, in total approx. 27 testisacs, 2 in XXI, 7 in XXII, 6 in XXIII, 7 in XXIV, 5 in XXV; on left side, in XXI b6 to XXV b6, in total approx. 25 testisacs, 2 in XXI, 5 in XXII, 7 in XXIII, 5 in XXIV, 6 in XXV. Paired epididymides; right epididymis in XX b5/b6 to XXI b5/b6, occupying 4 annuli; left epididymis in XX b6 to XXI b5/b6, occupying 4 annuli (Fig. 4A). Paired ejaculatory ducts, thick; right duct in XI b5 to XX b5/b6; left duct in XI b5 to XX b6; loosely coiled in position posterior to ovisacs; each widening from respective junction with epididymis, narrowing at junction with atrial cornua, then turning gradually inward toward atrial cornua without pre-atrial loop (Fig. 4A–D). Pair of muscular atrial cornua small ovate, in XI b5 and b6 (Fig. 4A–D). Atrium short, muscular, globular in XI b5 and b6 (Fig. 4A–D).

Paired ovisacs in XIII a2 and b5 (Fig. 4A, E). Oviducts; right oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII a1 (Fig. 4A, E). Common oviduct directly descending to female gonopore (Fig. 4E).

Variation

BL 36.3 mm, BW 3.5 mm, CL 1.8 mm, CW 2.2 mm. Somite IV uniannulate Somite VI triannulate, a1 = a2 = a3. Male gonopore in middle of XI b6, female gonopore in slightly anterior to middle of XIII a1, gonopores thus separated by 1/2 + 4 + (< 1/2) annuli. Pharynx reaching to XIV a1/a2. Crop reaching to XX a1/a2. Gastropore in slightly anterior to middle of XIII a1. Gastroporal duct tubular, but slightly bulbous at junction with gastropore, joining with crop in XIV a2/b5. Intestine reaching to XXIV a1/b5. Testisacs undetectable. Paired epididymides; right epididymis in XX b5 to XXI a2/b5; left epididymis in XX a2/b5 to XXI a2; each occupying four annuli. Atrium in XI b6. Left oviduct crossing ventrally beneath nerve cord.

Colouration

In life, dorsal surface grayish (Fig. 5); ventral surface whitish red; clitellum, when obvious, whitish grayish pale ochre (Fig. 5). Color faded in preservative.

Figure 5.

Orobdella brachyepididymis sp. n., holotype, KUZ Z1673. Dorsal view of live animal. Scale bar: 5 mm.

Etymology

The specific name is a compound noun in apposition derived from the Greek words transliterated into Latin, brachys (short) and epididymis (epididymis), referring to the fact that the epididymides of this species occupy only four annuli.

Distribution

This species was found only from its type locality.

Natural history

This species was found curled up under rocks in moist mountainous habitats. As oligochaete worms were observed in the digestive tract of a dissected specimen, KUZ Z1674, this species is an earthworm-eater, as are the other known Orobdella species.

A mature leech, KUZ Z1673, was collected on 7 July. Therefore, Orobdella brachyepididymis is considered to enter its reproductive season before early July.

Remarks

The specimens were small (up to 52 mm), but the holotype was determined to be mature because it possessed an obvious clitellum and developed testisacs.

According to taxonomic studies (Nakano 2010, 2012b, 2014, 2016, Nakano and Gongalsky 2014, Nakano and Lai 2012, Nakano and Seo 2014), the new species is distinguished from the eight quadrannulate species (i.e. O. esulcata Nakano, 2010, O. kawakatsuorum Richardson, 1975, O. ketagalan Nakano & Lai, 2012, O. koikei Nakano, 2012b, O. masaakikuroiwai, O. naraharaetmagarum, O. tsushimensis Nakano, 2011a and O. whitmani) by the following combination of characteristics (Table 2): body length less than or reaching ca. 5 cm, IV uniannulate, XXV quadrannulate, gonopores separated by 1/2 + 4 [+ (< 1/2)] annuli, gastroporal duct tubular, epididymides in XX to XXI and atrial cornua small ovate.

Comparisons of morphological characters between Orobdella brachyepididymis sp. n. and eight quadrannulate congeneric species.

Character Body length Somite IV Somite XXV Gastroporal duct Annuli between gonopores Epididymides Atrial cornua
Orobdella brachyepididymis sp. n. less than or reaching to ca. 5 cm uniannulate quadrannulate tubular 1/2 + 4 [+ (< 1/2)] XX to XXI small, ovate
Orobdella esulcata reaching to ca. 10 cm uniannulate quadrannulate tubular, but bulbous at junction with gastropore 2/3 + 4 + 1/3 XVI to XX developed, ovate
Orobdella kawakatsuorum reaching to ca. 10 cm biannulate quadrannulate simple tubular 6 XVI to XVII undeveloped
Orobdella ketagalan reaching to ca. 10 cm uniannulate quadrannulate simple tubular 1/2 + 4 + 1/2 absent undeveloped
Orobdella koikei less than 4 cm uniannulate triannulate bulbous 1/2 + 4 + 1/2 XV to XX developed, ovate
Orobdella masaakikuroiwai less than 4 cm uniannulate quadrannulate bulbous 1/2 + 4 + 1/2 XVI to XVIII developed, ovate
Orobdella naraharaetmagarum less than 5 cm uniannulate quadrannulate bulbous 1/2 + 4 + 1/2 XV to XX developed, ellipsoid or ovate
Orobdella tsushimensis reaching to ca. 10 cm uniannulate quadrannulate bulbous 1/2 + 5 XVII to XIX developed, ovate
Orobdella whitmani reaching to ca. 10 cm uni- or biannulate quadrannulate bulbous 1/2 + 4 + 1/2 XVI to XVIII developed, ovate

Orobdella nakahamai sp. n.

Figs 6, 7, 8, 9

Type materials

Holotype: KUZ Z1672, dissected, collected from under rocks of a small mountain stream at Mt. Takanosuyama (33.788°N, 133.271°E; Elev. ca. 1500 m; locality #4), Ino, Kochi Prefecture, Japan, by Ryosuke Okano on 4 July 2015. Paratype: KUZ Z1352, dissected, collected from under rocks of a small mountain stream at Mt. Iwagurosan (33.7516°N, 133.1533°E; Elev. ca. 1600 m; locality #5), Kumakogen, Ehime Prefecture, Japan, by Naoyuki Nakahama on 10 August 2010. For locality numbers, see Fig. 1.

Additional material

KUZ Z1680, collected from Kaminodoi (32.942714°N, 132.841350°E; Elev. ca 140 m; locality #6, see Fig. 1), Shimanto, Kochi Prefecture, Japan, by Ryosuke Okano on 2 November 2015.

Type locality

Japan, Kochi Prefecture: Ino, Ishizuchi Mountains, Mt. Takanosuyama (Shikoku).

Diagnosis

Body length of mature individual greater than 15 cm. Somites IX–XXV octannulate. Clitellum in X c9 to XIII b4. Male gonopore in slightly posterior to middle of XI c11 or XI c10/c11, female gonopore in XIII b2/b3, behind gastropore, gonopores separated by 1/2 + 11 or 12 annuli. Pharynx reaching to XIV b1–b3/b4. Gastropore conspicuous, in XIII b2/b3. Gastroporal duct bulbous, slightly winding at junction with gastropore. Paired epididymides in XV to XVII, occupying 12 or 13 annuli (one and half somites). Atrial cornua ovate or ellipsoid.

Description of holotype

BL 237.7 mm, BW 11.3 mm (Fig. 6). Caudal sucker ventral, elliptic, CL 3.9 mm, CW 6.5 mm (Figs 6B, 7D).

Figure 6.

Orobdella nakahamai sp. n., holotype, KUZ Z1672. A dorsal, B ventral views. Scale bar: 2 cm.

Figure 7.

Orobdella nakahamai sp. n., holotype, KUZ Z1672. A dorsal, B ventral views of somites I–VIII; C dorsal, D ventral views of somites XXIV–XXVII and caudal sucker; E ventral view of somites X–XIII; F ventral view of gastropore and female gonopore. Scale bars: A–E = 5 mm; F = 1 mm.

Somites III–V biannulate; III and V, (a1 + a2) = a3; IV, (a1 + a2) > a3; V a3 forming posterior margin of oral sucker (Fig. 7A, B). Somite VI dorsally quinquannulate, b1 = b2 < a2 > b5 < b6; ventrally quadrannulate, a1 = a2 > b5 < b6 (Fig. 7A, B). Somite VII quiquannulate, a1 > b3 = b4 = b5 = b6, a1 with slight lateral furrow on each side (Fig. 7A, B). Somite VIII dorsally septannulate, a1 (with obvious secondary furrow, b1 = b2) > b3 = b4 = c9 = c10 = c11 = c12; ventrally octannulate, b1 = b2 = b3 = b4 = c9 = c10 = c11 = c12 (Fig. 7A, B). Somite IX octannulate, b1 = b2 = b3 < b4 = c9 = c10 = c11 = c12. Somites X–XXV octannulate, b1 = b2 = b3 = b4 = c9 = c10 = c11 = c12 (Fig. 7C–E). Somite XXVI octannulate, b1 = b2 = b3 = b4 = c9 = c10 = c11 > c12. Somite XXVII comprises three annuli, second and third annuli, respectively, with slight dorsal furrow; first annulus being ventrally last complete annulus (Fig. 7C, D). Anus behind somite XXVII (Fig. 7C).

X c9 and XIII b4, respectively, being first and last annuli of clitellum (Fig. 7E).

Male gonopore in slightly posterior to middle of XI c11 (Fig. 7E). Female gonopore in XIII b2/b3, inconspicuous, located posterior to gastropore (Fig. 7E, F). Gonopores separated by 1/2 + 11 annuli (Fig. 7E).

Anterior ganglionic mass in VI b5 and b6. Ganglion VII in a1. Ganglia VIII–X, of each somite, in b3 and b4. Ganglion XI in b4 (Fig. 8B). Ganglion XII in b3 and b4 (Fig. 8B). Ganglion XIII in b4 (Fig. 8B). Ganglia XIV–XVI, of each somite, in b3 and b4 (Fig. 8B). Ganglion XVII in b3 (Fig. 8B). Ganglion XVIII in b3 and b4. Ganglion XIX in b3. Ganglia XX and XXI, of each somite, in b3 and b4. Ganglia XXII–XXV, of each somite, in b3. Ganglion XXVI in b2. Posterior ganglionic mass in XXVI c10–c12.

Figure 8.

Orobdella nakahamai sp. n., holotype, KUZ Z1672. A ventral view of gastroporal duct; B dorsal view of reproductive system including ventral nervous system; C dorsal (including positions of ganglia XI and XII), D lateral, E ventral views of male atrium; F dorsal view of female reproductive system including position of ganglion XIII. Scale bars: A = 3 mm; B = 1 cm; C–F = 1 mm.

Eyes undetectable.

Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of b2 of each somite in VIII–XXIV (Fig. 7B, D, E).

Pharynx reaching to XIV b1 (Fig. 8A). Crop reaching to XXII c9. Gastropore conspicuous, ventral, in XIII b2/b3 (Fig. 7E, F). Gastroporal duct bulbous, slightly winding at junction with gastropore, joining with crop in XIII c12 (Fig. 8A). Intestine reaching to XXV/XXVI.

Testisacs (Fig. 8B); on right side, in XVII c9 to XXV c11, in total approx. 164 testisacs, 7 in XVII, 18 in XVIII, 24 in XIX, 21 in XX, 21 in XXI, 20 in XXII, 23 in XXIII, 16 in XXIV, 14 in XXV; on left side, in XVII b4 to XXV c12, in total approx. 160 testisacs, 6 in XVII, 15 in XVIII, 24 in XIX, 20 in XX, 22 in XXI, 18 in XXII, 18 in XXIII, 21 in XXIV, 16 in XXV. Paired epididymides in XV c9 to XVI/XVII, occupying 12 annuli (Fig. 8B). Paired ejaculatory ducts in XI c9 to XV c9; coiled in position posterior to ovisacs; each duct crossing ventrally beneath each ovisac, then running straight 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 without pre-atrial loop (Fig. 8B–E). Pair of muscular atrial cornua ovate, in XI c9–c11 (Fig. 8B–E). Atrium short, muscular, globular in XI c10 and c11 (Fig. 8B–E).

Paired ovisacs in XIII b3 and b4 (Fig. 8B, F). Oviducts; right oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII b3/b4 (Fig. 8B, F). Common oviduct directly descending to female gonopore (Fig. 8B, F).

Variations

BL 162.3–180.4 mm, BW 7.6–98 mm, CL 3.3–4.3 mm, CW 5.6 mm. Somite VI quadrannulate, a1 (dorsally b1 = b2) = a2 (dorsally b3 = b4) > b5 < b6, or dorsally quinquannulate, b1 = b2 < a2 > b5 < b6. Somite VII quinquannulate, a1 (dorsally b1 = b2 in KUZ Z1680) > b3 = b4 = b5 = b6. Somite VIII octannulate, b1 = b2 < b3 < b4 = c9 = c10 = c11 = c12, or ventrally septannulate, a1 (b1 = b2) > b2 = b3 = b4 = c9 = c10 = c11 = c12. Somite IX octannulate, b1 = b2 = b3 = b4 = c9 = c10 = c11 = c12. Somite XXVI dorsally octannulate, b1 = b2 = b3 = b4 > c9 = c10 = c11 = c12, ventrally septannulate, b1 = b2 = b3 = b4 > c9 = c10 < b6, or sexannulate, b1 = b2 = b3 = b4 = b5 < b6 (c11 = c12). Somite XXVII comprises two annuli, first annulus with slight three dorsal furrows, or comprises four annuli. Eyes in one pair, dorsally on posterior margin of II (KUZ Z1352). Pharynx reaching to XIV b3/b4. Crop reaching to XXII b1/b2. Gastroporal duct joining with crop in XIV b3. Intestine reaching to XXV b4. Male gonopore in XI c10/c11 (KUZ Z1680), thus gonopores separated by 12 annuli. Testisacs in XVII c10 to XXV c11; on right side, in total approx. 122 testisacs, 5 in XVII, 18 in XVIII, 17 in XIX, 18 in XX, 16 in XXI, 15 in XXII, 14 in XXIII, 10 in XXIV, 9 in XXV; on left side, in total approx. 126 testisacs, 4 in XVII, 16 in XVIII, 19 in XIX, 21 in XX, 17 in XXI, 13 in XXII, 13 in XXIII 11 in XXIV, 12 in XXV. Paired epididymides in XV c10/c11 to XVII b3/b4, occupying 13 annuli. Pair of muscular atrial cornua ellipsoid. Left oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII b3.

Colouration

In life, dorsal surface bluish gray (Fig. 9); ventral surface bluish white; clitellum, when obvious, paler than other body parts (Fig. 9). Color faded in preservative.

Figure 9.

Orobdella nakahamai sp. n., holotype, KUZ Z1672. Dorsal view of live animal. Scale bar: 1 cm.

Etymology

The specific name is a noun in the genitive case formed directly from the name of Mr Naoyuki Nakahama, who collected a specimen of this new species.

Distribution

This species was collected from the Ishizuchi Mountains and south-western part of Kochi Prefecture. The lowest elevation among the localities was ca. 140 m, and the highest was ca. 1600 m. The locality data for this species suggested that it is distributed in mountainous regions in the western part of Shikoku, Japan. Its distribution may not be restricted by habitat elevation.

Natural history

Mature leeches with an obvious clitellum, KUZ Z1352 and Z1672, were collected on 4 July and 10 August, respectively, in the Ishizuchi Mountains. Therefore, the reproductive season of O. nakahamai in the mountains begins before or during July, and then continues at least to early August.

Remarks

Orobdella nakahamai is only the second octannulate large-type species known within the genus. According to Nakano (2012c), this species can be distinguished from another octannulate species O. octonaria by the following combination of characteristics (Table 3): female gonopore in XIII b2/b3, gonopores separated by 1/2 + 11 or 12 annuli and epididymides in XV to XVII. Orobdella nakahamai is clearly distinguishable from the quadrannulate and sexannulate species of this genus because it possesses octannulate mid-body somites.

Comparisons of morphological characters between octannulate Orobdella nakahamai sp. n. and Orobdella octonaria.

Character Female gonopore Annuli between gonopores Epididymides
Orobdella nakahamai sp. n. XIII b2/b3 1/2 + 11 or 12 XV to XVII
Orobdella octonaria middle of XIII b2 1/2 + 10 + 1/2 XVII to XIX

Orobdella okanoi sp. n.

Figs 10, 11, 12, 13

Type material

Holotype: KUZ Z1671, dissected, collected from under a rock along a mountain trail at Mt. Kuishiyama (33.67636°N, 133.51556°E; Elev. ca. 910 m; locality #3, see Fig. 1), Kochi, Kochi Prefecture, Japan, by Yoshiko Yamane on 5 July 2015.

Additional material

KUZ Z1491, dissected, collected from in soil along a road at Mt. Iwagurosan (33.755278°N, 133.148333°E; Elev. ca 1510 m; locality #5, see Fig. 1), Saijyo, Ehime Prefecture, Japan, by Yoshiko Yamane on 22 August 2013.

Type locality

Japan, Kochi Prefecture: Kochi, Tosayamatakakawa, Mt. Kuishiyama (Shikoku).

Diagnosis

Dorsal surface reddish. Somite VII quinquannulate. Somite VIII–XXVI sexannulate. Male gonopore in slightly posterior to middle of XI c11/c12, female gonopore in middle of XIII b2, behind gastropore, gonopores separated by 8 + 1/2 annuli. Pharynx reaching to XIV b1–b2/a2. Gastropore conspicuous, in middle of XIII b2. Gastroporal duct bulbous, slightly winding at junction with gastropore. Paired epididymides in XV to XVII, occupying 8–11 annuli (one and half to almost two somites). Pre-atrial loop absent. Atrial cornua ellipsoid.

Description of holotype

BL 95.2 mm, BW 5.8 mm (Fig. 10). Caudal sucker ventral, elliptic, CL 2.3 mm, CW 3.7 mm (Figs 10B, 11D).

Figure 10.

Orobdella okanoi sp. n., holotype, KUZ Z1671. A dorsal, B ventral views. Scale bar: 1 cm.

Figure 11.

Orobdella okanoi sp. n., holotype, KUZ Z1671. A dorsal, B ventral views of somites I–VIII; C dorsal, D ventral views of somites XXIV–XXVII and caudal sucker; E ventral view of somites XI–XIII; F ventral view of gastropore and female gonopore; G ventral view of gastroporal duct. Scale bars: A–E = 2 mm; F = 0.25 mm; G = 1 mm.

Somite III uniannulate (Fig. 11A). Somites IV and V biannulate; IV, (a1 + a2) > a3 (Fig. 11A); V, (a1 + a2) = a3; V a3 forming posterior margin of oral sucker (Fig. 11A, B). Somite VI triannulate, a1 (dorsally b1 = b2) > a2 = a3 (Fig. 11A, B). Somite VII quinquannulate, b1 = b2 = a2 = b5 = b6 (Fig. 11A, B). Somites VIII–XXVI sexannulate, b1 = b2 = a2 = b5 = c11 = c12 (Fig. 11A–E). Somite XXVII comprises two annuli; first annulus being ventrally last complete annulus (Fig. 11C, D). Anus behind somite XXVII (Fig. 11C).

Male gonopore in XI c11/c12 (Fig. 11E). Female gonopore in slight posterior to middle of XIII b2, inconspicuous, located posterior to gastropore (Fig. 11E, F). Gonopores separated by 8 + 1/2 annuli (Fig. 11E).

Anterior ganglionic mass in VI a1 and VII b1. Ganglion VII in b2 and a2. Ganglion VIII in a2. Ganglion IX in a2 and b5. Ganglion X in a2. Ganglion XI in a2 and b5 (Fig. 12A). Ganglion XII in a2 (Fig. 12A). Ganglion XIII in a2 and b5 (Fig. 12A). Ganglia XIV and XV, of each somite, in a2 (Fig. 12A). Ganglion XVI in b2 and a2 (Fig. 12A). Ganglia XVII (Fig. 12A) and XVIII, of each somite, in a2. Ganglion XIX in b2 and a2. Ganglia XX–XXII, of each somite, in a2. Ganglia XXIII–XXV, of each somite, in b2 and a2. Ganglion XXVI in b1 and b2. Posterior ganglionic mass in XXVI b5–c12.

Figure 12.

Orobdella okanoi sp. n., holotype, KUZ Z1671. A dorsal view of reproductive system including ventral nervous system; B dorsal (including positions of ganglia XI and XII), C lateral, D ventral views of male atrium; E dorsal view of female reproductive system including position of ganglion XIII. Scale bars: A = 3 mm; B–E = 0.5 mm.

Eyes in three pairs, first pair dorsally on posterior margin of II, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig. 11A).

Nephridiopore in 17 pairs, one each situated ventrally at posterior margin of b2 of each somite in VIII–XXIV (Fig. 11B, D, E).

Pharynx reaching to XIV b1 (Fig. 11G). Crop reaching to XXII b1. Gastropore conspicuous, ventral, in slightly posterior to middle of XIII b2 (Fig. 11E, F). Gastroporal duct bulbous, slightly winding at junction with gastropore, joining with crop in XIV b1 (Fig. 11G). Intestine reaching to XXV b5/c11.

Testisacs (Fig. 12A); on right side, in XVII b5 to XXV c12, in total approx. 94 testisacs, 5 in XVII, 13 in XVIII, 12 in XIX, 13 in XX, 11 in XXI, 12 in XXII, 10 in XXIII, 10 in XXIV, 8 in XXV; on left side, in XVII c11 to XXIV c12, in total approx. 86 testisacs, 3 in XVII, 15 in XVIII, 11 in XIX, 13 in XX, 13 in XXI, 10 in XXII, 11 in XXIII, 10 in XXIV. Paired epididymides; right epididymis in XV c11/c12 to XVII b2, occupying 9 annuli; left epididymis in XV c11/c12 to XVII b1/b2, occupying 8 annuli (Fig. 12A). Paired ejaculatory ducts in XI b5 to XV c11/c12; slightly 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 without pre-atrial loop (Fig. 12A–D). Pair of muscular atrial cornua ellipsoid, in XI b5–c12 (Fig. 12A–D). Atrium short, muscular, globular in XI c11 and c12 (Fig. 12A–D).

Paired ovisacs; right ovisac in XIII a2 and b5; left ovisac in XIII a2–c11 (Fig. 12A, E). Oviducts; left oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII a2 (Fig. 12A, E). Common oviduct directly descending to female gonopore (Fig. 12E).

Variation

BL 143.2 mm, BW 7.6 mm, CL 3.1 mm, CW 4.0 mm. Somite III uniannulate with slight dorsal furrow. Somite XXVII comprises two annuli, each annulus with slight dorsal furrow. Male gonopore in posterior margin of XI c11. Female gonopore in middle of XIII b2. Eyes in three pairs, firs pair dorsally on anterior margin of III. Pharynx reaching to XIV b2/a2. Crop reaching to XXII a2. Gastropore in middle of XIII b2. Gastroporal duct joining with crop in XIV b2. Intestine reaching to XXIV c12. Testisacs; on right side, in XVII b2 to XXI b5, in total approx. 64 testisacs, 8 in XVII 13 in XVIII, 18 in XIX, 17 in XX, 8 in XXI; on left side, in XVII b2 to XXIV c11, in total approx. 99 testisacs, 7 in XVII, 10 in XVIII, 17 in XIX 17 in XX, 15 in XXI, 13 in XXII, 11 in XXIII, 9 in XXIV. Paired epididymides; right epididymis in XV a2 to XVI/XVII, occupying 10 annuli; left epididymis in XV a2 to XVII b1, occupying 11 annuli. Paired ejaculatory ducts curved in position anterior to ovisacs. Paired atrial cornua; right cornu in XI c12 and XII b1; left cornu in XI b5 and c11. Paired ovisacs in XIII a2 and b5.

Colouration

In life, dorsal surface red-purple (Fig. 13) or pinkish gray; ventral surface grayish red-purple or whitish red. Color faded in preservative; dark line present from VI a1 (b2) to XIV b5 in KUZ Z1491.

Figure 13.

Orobdella okanoi sp. n., holotype, KUZ Z1671. Dorsal view of live animal. Scale bar: 5 mm.

Etymology

The specific name is a noun in the genitive case formed directly from the name of Mr Ryosuke Okano, who collected valuable specimens of Orobdella leeches.

Distribution

The type locality of this species is located in the central region of Shikoku, Japan. In addition, this species was collected from the Ishizuchi Mountains. According to the collection localities, this species is considered to inhabit the central mountainous region of Shikoku.

Natural history

This species was found curled up under a rock or in soil in moist mountainous habitats. Oligochaete worms were found in the digestive tract of the holotype, and thus this species is an earthworm-eater. The reproductive season of O. okanoi remains unclear because no individuals of this species with a clitellum have been collected.

Remarks

According to taxonomic studies on sexannulate Orobdella species (Nakano 2011b, 2012a), Orobdella okanoi differs from the four known sexannulate species, O. dolichopharynx Nakano, 2011b, O. ijimai Oka, 1895, O. mononoke Nakano, 2012a and O. shimadae Nakano, 2011b, as well as the new sexannulate species described below in having the following characteristics (Table 4): dorsal surface reddish, somite VII quinquannulate, somite VIII sexannulate, 8 + 1/2 annuli between gonopores, pharynx reaching to XIV, gastroporal duct bulbous, epididymides in XV to XVII, pre-atrial loop absent and atrial cornua ellipsoid. Orobdella okanoi is clearly distinguished from quadrannulate and octannulate species in having sexannulate mid-body somites.

Comparisons of morphological characters between Orobdella okanoi sp. n., Orobdella yamaneae sp. n. and four sexannulate congeneric species.

Character Dorsal colour Somite VII Somite VIII Annuli between gonopores Pharynx Gastroporal duct Epididymides Pre-atrial loop Atrial cornua
Orobdella okanoi sp. n. reddish quinquannulate sexannulate 8 + 1/2 reaching to XIV bulbous XV to XVII absent ellipsoid
Orobdella yamaneae sp. n. purplish quinquannulate sexannulate 1/2 + 7 + 1/2 reaching to XIV bulbous XVI to XVIII extending to anterior of XI c9 ovate
Orobdella dolichopharynx yellowish green quadrannulate quinquannulate 8 reaching to XVI tubular, reaching to XVI absent extending to ganglion XI absent
Orobdella ijimai yellowish green quadrannulate sexannulate 1/2 + 7 + 1/2 reaching to XIV bulbous XVI to XIX absent ellipsoid
Orobdella mononoke anterior and posterior parts grayish purple, mid-body amber quadrannulate sexannulate 8 + 1/2 reaching to XIV tubular, but bulbous at junction with crop XV to XIX absent ovate
Orobdella shimadae yellowish green triannulate quinquannulate 9 reaching to XVI tubular, reaching to XV absent extending to ganglion XI absent

The right atrial cornu of one specimen, KUZ Z1491, is caudad. Because its right side testisacs only reach somite XXI, the right side of its male genital organ may be a result of abnormal development.

Orobdella yamaneae sp. n.

Figs 14, 15, 16, 17, 18

Type materials

Holotype: KUZ Z1678, dissected, collected from under fallen leaves along a forest road, “Yuzuruha Forest Road” (34.24741°N, 134.80791°E; Elev. ca. 195 m; locality #1, see Fig. 1), at Mt. Yuzuruhasan, Awajishima island, Japan, by Yoshiko Yamane on 8 July 2015. Paratypes: six specimens collected from the type locality; KUZ Z1358 (34.245889°N, 134.811861°E; Elev. ca. 210 m), and Z1359 (34.246000°N, 134.812000°E; Elev. ca. 210 m), from under a rock along the forest road, by TN on 17 June 2011; KUZ Z1675–Z1677 (34.24743°N, 134.80777°E; Elev. ca. 195 m), and Z1679 (34.24711°N, 134.80884°E; Elev. ca. 195 m), from under fallen leaves along a forest road, by Yoshiko Yamane on 8 July 2015; three specimens, KUZ Z1358, Z1676 and Z1679, dissected.

Additional material

KUZ Z1488, collected from under fallen leaves along the forest road (34.247222°N, 134.808611°E; Elev. ca. 215 m) at the type locality, by Yoshiko Yamane on 20 August 2013.

Type locality

Japan, Hyogo Prefecture: Minamiawaji, Mt. Yuzuruhasan (Awajishima island).

Diagnosis

Dorsal surface purplish. Somite VII quinquannulate. Somite VIII–XXVI sexannulate, b1 = b2 = a2 = c9 = c10 = b6. Male gonopore in middle of XI b6, female gonopore in slightly posterior to middle of XIII b2, behind gastropore, gonopores separated by 1/2 + 7 + 1/2 annuli. Pharynx reaching to XIV a2/c9–c9. Gastropore conspicuous, in slightly posterior to middle of XIII b2. Gastroporal duct bulbous, slightly winding at junction with gastropore. Paired epididymides in XVI to XVIII, occupying 8–11 annuli (one and half to almost two somites). Pre-atrial loop present. Atrial cornua ovate.

Description of holotype

BL 142.2 mm, BW 8.1 mm (Fig. 14). Caudal sucker ventral, elliptic, CL 3.0 mm, CW 5.1 mm (Figs 14B, 15D).

Figure 14.

Orobdella yamaneae sp. n., holotype, KUZ Z1678. A dorsal, B ventral views. Scale bar: 1 cm.

Figure 15.

Orobdella yamaneae sp. n., holotype, KUZ Z1678. A dorsal, B ventral views of somites I–VIII; C dorsal, D ventral views of somites XXIV–XXVII and caudal sucker; E ventral view of somites X–XIII; F ventral view of gastropore and female gonopore; G ventral view of gastroporal duct. Scale bars: A, B, G = 2 mm; C–E = 3 mm; F = 0.25 mm.

Somites III–V biannulate; III and IV, (a1 + a2) > a3; V, (a1 + a2) = a3; V a3 forming posterior margin of oral sucker (Fig. 15A, B). Somite VI triannulate, a1 (dorsally b1 = b2) > a2 > a3 (Fig. 15A, B). Somite VII quinquannulate, a1 = a2 = c9 = c10 = b6 (Fig. 15A, B). Somites VIII–XXV sexannulate, b1 = b2 = a2 = c9 = c10 = b6 (Fig. 15A–E). Somite XXVI sexannulate dorsally, b1 = b2 = a2 > c9 = c10 = b6, quinquannulate ventrally, b1 = b2 = a2 < b5 (c9 = c10) > b6 (Fig. 15C, D). Somite XXVII comprises two annuli, first annulus with slight dorsal furrow; first annulus being ventrally last complete annulus (Fig. 15C, D). Anus behind somite XXVII (Fig. 15C).

X c9 and XIII a2, respectively, being first and last annuli of clitellum (Fig. 15E).

Male gonopore in middle of XI b6 (Fig. 15E). Female gonopore in slightly posterior to middle of XIII b2, inconspicuous, located posterior to gastropore (Fig. 15E, F). Gonopores separated by 1/2 + 7 + 1/2 annuli (Fig. 15E).

Anterior ganglionic mass in VI a2 and a3. Ganglion VII in a2. Ganglia VIII and IX, of each somite, in b2 and a2. Ganglia X–XII, of each somite, in a2 (Fig. 16A). Ganglion XIII in a2 and c9 (Fig. 16A). Ganglia XIV–XVIII, of each somite, in a2 (Fig. 16A). Ganglion XIX in b2 and a2. Ganglia XX–XXIV, of each somite, in a2. Ganglion XXV in b2 and a2. Ganglion XXVI in b1 and b2. Posterior ganglionic mass in XXVI c9–b6.

Figure 16.

Orobdella yamaneae sp. n., holotype, KUZ Z1678. A dorsal view of reproductive system including ventral nervous system; B dorsal (including positions of ganglia XI and XII), C lateral, D ventral views of male atrium; E dorsal view of female reproductive system including position of ganglion XIII. Scale bars: A = 5 mm; B–D = 1 mm; E = 0.5 mm.

Eyes in three pairs, first pair dorsally on II/III, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig. 15A).

Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of b2 of each somite in VIII–XXIV (Fig. 15B, D, E).

Pharynx reaching to XIV a2/c9 (Fig. 15G). Crop reaching to XXI b6. Gastropore conspicuous, ventral, in slightly posterior to middle of XIII b2 (Fig. 15E, F). Gastroporal duct bulbous, slightly winding at junction with gastropore, joining with crop in XIV a2/c9 (Fig. 15G). Intestine reaching to XXIV c9/c10.

Testisacs in XVIII c9 to XXVI b1 (Fig. 16A): on right side, in total approx. 107 testisacs, 7 in XVIII, 13 in XIX, 16 in XX, 17 in XXI, 14 in XXII, 14 in XXIII, 14 in XXIV, 11 in XXV, 1 in XXVI; on left side, in total approx. 108 testisacs, 5 in XVIII, 12 in XIX, 19 in XX, 15 in XXI, 15 in XXII, 13 in XXIII, 14 in XXIV, 14 in XXV, 1 in XXVI. Paired epididymides; right epididymis in XVI c9 to XVIII c9, occupying 13 annuli; left epididymis in XVI c9/c10 to XVIII c9, occupying 12 annuli (Fig. 16A). Paired ejaculatory ducts; right duct in XI c9 to XVI c9; left duct in XI c9 to XVI c9/c10; coiled in position posterior to ovisacs; each duct crossing ventrally beneath each ovisac, then 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 atrial loop extending to anterior of XI c9 (Fig. 16A–D). Pair of muscular atrial cornua ovate, in XI c9–b6 (Fig. 16A–D). Atrium short, muscular, globular in XI c10 and b6 (Fig. 16A–D).

Paired ovisacs in XIII a2 and c9 (Fig. 16A, E). Oviducts; left oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII a2 (Fig. 16A, E). Common oviduct directly descending to female gonopore (Fig. 16A, E).

Variations

BL 62.4–97.6 mm, BW 4.0–6.5 mm, CL 1.6–2.8 mm, CW 2.5–3.6 mm. Somite III uniannulate with slight dorsal furrow, [(a1 + a2) > a3]. Somite IV uniannulate with slight dorsal furrow, [(a1 + a2) > a3] in KUZ Z1359 (Fig. 17A). Somite VII quadrannulate in KUZ Z1359, a1 = a2 < b5 (ventrally c9 = c10) > b6 (Fig. 17A, B). Somite VIII quinquannulate in KUZ Z1359, b1 = b2 = a2 < b5 (c9 = c10) > b6 (Fig. 17A, B). Somite XXVI generally sexannulate, b1 = b2 = a2 = c9 = c10 = b6, or b1 = b2 = a2 = c9 > c10 = b6; KUZ Z1359, quinquannulate, b1 = b2 = a2 < b5 > b6. Somite XXVII comprises 2–4 annuli. XXVI b6, or first annulus of XXVII being ventrally last complete annulus. Female gonopore rarely in posterior margin of XIII b2, gonopores thus rarely separated by 1/2 + 8 annuli. First pair of eyes generally dorsally on posterior margin of II. Pharynx reaching to XIV a2/c9–c9. Crop reaching to XXI c9–XXII c9/c10. Gastroporal duct often tubular, slightly bulbous at junction with gastropore, joining with crop in XIV b1/b2–b2/a2. Intestine reaching to XXIV/XXV–XXV a2/c9. Testisacs hardly detected. Paired epididymides: right epididymis in XVI b2–b2/a2 to XVII c9–XVIII b1, occupying 8–11 annuli; left epididymis in XIV b1/b2–XVI b2/a2 to XVII c9–XVIII b1, occupying 9–11 annuli. Paired ejaculatory ducts running straight, or nearly straight in position anterior to ovisacs; pre-atrial loop reaching to XI a2/c9–c9. Pair of muscular atrial cornua fusiform or ellipsoid, often in XI c10 and b6. Paired ovisacs generally in XIII a2 and c9; KUZ Z1358, in XIII c9. Right or left oviduct crossing ventrally beneath nerve cord.

Figure 17.

Orobdella yamaneae sp. n., paratype, KUZ Z1359. A dorsal, B ventral views of somites I–VIII. Scale bars: A, B = 1 mm.

Colouration

In life, dorsal surface grayish purple or red-purple (Fig. 18); ventral surface whitish blue or grayish purple; clitellum, when obvious, paler than other body parts (Fig. 18). Color faded in preservative.

Figure 18.

Orobdella yamaneae sp. n., holotype, KUZ Z1678. Dorsal view of live animal. Scale bar: 1 cm.

Etymology

The specific name is a noun in the genitive case formed directly from the name of Ms Yoshiko Yamane, who collected specimens of this new species.

Distribution

This species was collected only from its type locality.

Natural history

This species was found curled up under fallen leaves in moist mountainous habitats. A mature leech, KUZ Z1678, was collected on 8 July. Therefore, Orobdella yamaneae is considered to enter its reproductive season before early July.

Remarks

The mid-body somite annulation of the known sexannulate Orobdella species was described as being composed of b1, b2, a2, b5, c11 and c12 (Nakano 2011b, 2012a). However, the annulation of somites VII [a1 = a2 < b5 (ventrally c9 = c10) > b6] and VIII [b1 = b2 = a2 < b5 (c9 = c10) > b6] of one specimen, KUZ Z1359, clearly suggests that b5 is divided into c9 and c10, and b6 remains undivided in O. yamaneae. The sexannulation of this species is thus b1 = b2 = a2 = c9 = c10 = b6.

In addition to its unique sexannulation, O. yamaneae is distinguishable from the four known sexannulate species and O. okanoi by the following characteristics (Table 4): dorsal surface purplish, somite VII quinquannulate, somite VIII sexannulate, 1/2 + 7 + 1/2 annuli between gonopores, pharynx reaching to XIV, gastroporal duct bulbous, epididymides in XVI to XVIII, pre-atrial loop present and atrial cornua ovate. Orobdella yamaneae obviously differs from quadrannulate and octannulate species of this genus in its mid-body somite annulation.

Except for the holotype, all dissected individuals possess the following characteristics of the male genital organ: ejaculatory ducts in position anterior to ovisacs running straight and male atrial cornua ellipsoid or fusiform. However, they seem to be immature leeches because all of them have undeveloped and undetectable testisacs. Therefore, straight ejaculatory ducts and ellipsoid or fusiform testisacs are considered to be immature characteristics of O. yamaneae.

Molecular phylogenies and genetic distances

The obtained BI tree (Fig. 19) had an almost identical topology to that of the ML tree (ln L = −27703.79; not shown). In the ML phylogeny, Orobdella tsushimensis formed a monophyletic lineage with the clade containing O. dolichopharynx, O. esulcata, O. ketagalan, O. mononoke, O. naraharaetmagarum, O. shimadae and O. brachyepididymis (BS = 41%).

Figure 19.

Bayesian inference tree for 5,209 bp of nuclear 18S rRNA and histone H3 and mitochondrial COI, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu and ND1 markers. Numbers on nodes represent bootstrap values for maximum likelihood and Bayesian posterior probabilities. A species name of Orobdella in red indicates a quadrannulate species; green, a sexannulate; and blue, an octannulate species. Locality numbers are shown in Fig. 1.

Orobdella brachyepididymis formed a monophyletic clade with O. naraharaetmagarum (BS = 100%, BPP = 1.0). This clade is a sister lineage to O. esulcata (BS = 100%, BPP = 1.0). The monophyly of the two specimens identified as O. brachyepididymis was fully supported (BS = 100%, BPP = 1.0). The other three new species, O. nakahamai, O. okanoi and O. yamaneae formed a monophyletic clade (BS = 100%, BPP = 1.0). The monophyly of this clade and O. masaakikuroiwai was revealed, but this relationship was not strongly supported in the BI tree (BS = 87%, BPP = 0.90). Within the three new species, O. okanoi and O. yamaneae formed a monophyletic lineage (BS = 78%, BPP = 0.94). The monophyly of the specimens of the three new species was fully recovered (O. nakahamai, BS = 98%, BPP = 1.0; O. okanoi, and O. yamaneae, respectively, BS = 100%, BPP = 1.0).

The pairwise COI uncorrected p-distances within each of the new species were as follows: in O. brachyepididymis, 0.5%; O. nakahamai, 2.1–4.6% (mean = 3.7%); O. okanoi, 2.4%; and O. yamaneae, 3.9% (Table 5). The genetic divergence between O. brachyepididymis and O. naraharaetmagarum was 4.7%. The genetic distances among the three new species were as follows: O. nakahamai and O. okanoi, 5.6–6.2% (mean = 5.8%); O. nakahamai and O. yamaneae, 5.6–5.8% (mean = 5.7%); and O. okanoi and O. yamaneae, 5.8–6.0% (mean = 5.9%) (Table 5). Those between O. masaakikuroiwai and each of the three new species were as follows: O. nakahamai, 5.9–7.2% (mean = 6.8%); O. okanoi, 7.1–7.3% (mean = 7.2%); and O. yamaneae, 6.6–6.8% (mean = 6.7%).

Uncorrected p-distances for the 1267 bp for the COI sequences of specimens of Orobdella nakahamai sp. n., Orobdella okanoi sp. n. and Orobdella yamaneae sp. n., with associated collection locality numbers (see Fig. 1).

Species Specimen (locality #) 1 2 3 4 5 6
Orobdella nakahamai sp. n. 1: KUZ Z1352 (5)
2: KUZ Z1672 (4) 0.021
3: KUZ Z1680 (6) 0.043 0.046
Orobdella okanoi sp. n. 4: KUZ Z1491 (5) 0.058 0.056 0.056
5: KUZ Z1671 (3) 0.062 0.058 0.058 0.024
Orobdella yamaneae sp. n. 6: KUZ Z1358 (1) 0.056 0.057 0.058 0.059 0.059
7: KUZ Z1678 (1) 0.057 0.056 0.058 0.058 0.060 0.004

Discussion

According to the morphological characteristics of the four new species, each of them can be well defined and distinguished from each other and from the previously known species of Orobdella. However, the genetic divergences of the COI sequences showed small interspecific divergences. The genetic distance between Orobdella brachyepididymis and its sister species O. naraharaetmagarum (4.7%) was equivalent to the largest intraspecific COI divergence of the latter species indicated by Nakano (2016). Although no clear genetic distance gap exists between O. brachyepididymis and O. naraharaetmagarum, the new species is clearly distinguished from O. naraharaetmagarum by the following combination of morphological characters (Table 2): morphology of the gastroporal duct, number of annuli between gonopores, lengths of epididymides and morphology of the male atrial cornua. Clearly, O. brachyepididymis is a distinctive new species within the genus Orobdella.

Calculated interspecific COI distances among the three other new species, O. nakahamai, O. okanoi and O. yamaneae, each of which is well defined by morphological characteristics, were also small. These values and the obtained phylogenetic trees indicated that these three species are closely related to each other. Orobdella nakahamai is sympatric with O. okanoi on Mt. Iwagurosan in the Ishizuchi Mountains (locality #5, see Fig. 1). Moreover, specimens of each new species collected from Mt. Iwagurosan belonged to its respective clade. The present genetic analyses clearly support that O. nakahamai and O. okanoi are well-defined species. The COI divergences between O. yamaneae and O. okanoi (mean = 5.9%), and those between O. yamaneae and O. nakahamai (mean = 5.7%), were almost equal to those between O. nakahamai and O. okanoi (mean = 5.8%). Therefore, these distance values confirmed the distinctiveness of O. yamaneae.

According to the obtained molecular phylogenies, mid-body somite annulation and body size of mature leeches within Orobdella clearly evolved in parallel. Nakano (2012b) stated that sexannulate mid-body somite annulation had evolved in parallel within this genus. The present finding of the second octannulate species, O. nakahamai, and its phylogenetic position indicate that octannulate mid-body somite annulation also evolved in parallel within Orobdella. Note that both of the octannulate species formed a clade with the sexannulate species. These phylogenetic relationships shed light on a possible evolutionary correlation between sexannulation and octannulation in Orobdella leeches. Richardson (1971) erected the genus Kumabdella Richardson, 1971, which has been considered subjective junior synonym of Orobdella, only for O. octonaria, based principally on its octannulate mid-body somite annulation. In the redescription of O. octonaria, Nakano (2012c) briefly reviewed the taxonomic history of Kumabdella and concluded that Kumabdella should retain its status as a junior synonym of Orobdella. The presence of the second octannulate species within this genus, and the fact that these two octannulate species are phylogenetically distant, fully support the present taxonomic treatment for the genus Kumabdella.

The sexannulation of O. yamaneae and the fact that this species is genetically quite close to O. okanoi imply the possibility that the sexannulation of O. okanoi also follows b1 = b2 = a2 = c9 = c10 = b6, and not the b1 = b2 = a2 = b5 = c11 = c12 observed in the other known sexannulate species of Orobdella. The annulation pattern of O. yamaneae suggests that annular formulae can vary among Orobdella species possessing the same annulation. The quadrannulate somite of Orobdella obviously consists of a1, a2, b5 and b6, according to the positions of paired nephridiopores and the ventral ganglion in each somite. However, the annulation pattern of sexannulate and octannulate Orobdella species should be determined based on several specimens, including immature individuals.

Nakano (2014, 2016) stated that the small-type body length also evolved in parallel within Orobdella. In addition to small size, the present phylogenies indicated that the large-type body length evolved in parallel within this genus. The close relation of the small- and large-types to mid-body somite annulation in Orobdella leeches is highly possible. According to current knowledge on Orobdella species, the small-type only occurs in the quadrannulate species, i.e. O. brachyepididymis, O. koikei, O. masaakikuroiwai and O. naraharaetmagarum, while the large-type species consist of octannulate species, O. nakahamai and O. octonaria. Future phylogenetic studies, including an ancestral state reconstruction, will elucidate the character state evolution of Orobdella leeches.

The present phylogenetic tree showed that 17 Orobdella species consist of four main lineages: a Hokkaido lineage containing two species, O. kawakatsuorum and O. koikei; an Orobdella tsushimensis lineage; a western lineage comprising four species inhabiting the Ryukyu Islands and Taiwan, along with O. esulcata distributed in Kyushu, O. naraharaetmagarum in the Chugoku district, western Honshu, as well as the new species O. brachyepididymis from Shikoku; and an eastern lineage comprising four species known from the eastern to central parts of Honshu, along with the other three new species, O. nakahamai and O. okanoi collected from Shikoku and O. yamaneae from Awajishima island. Therefore, the range of the western lineage group overlaps that of the eastern lineage group in Shikoku and adjacent islets. Because species belonging to both lineage groups are distributed in Shikoku, the species diversity of Orobdella in this region may be quite high compared to other regions. The new Orobdella species inhabiting Shikoku and Awajishima island would offer a suitable opportunity to reveal speciation events, as well as species coexistence mechanisms in the genus Orobdella.

Acknowledgements

The author is grateful to Naoyuki Nakahama (Kyoto University; KU), Ryosuke Okano (Ehime University), and Yoshiko Yamane (KU) for providing specimens of the new species, to Professor Hidetoshi Nagamasu (The Kyoto UniversityMuseum) for his helpful advice on a specific name of the new species. The author expresses his sincere thanks to Dr Yi-Te Lai (National Taiwan University) and Dr Michael Ohl (Museum für Naturkunde) for their constructive comments on this manuscript. A part of this study was financially supported by Grants for Biodiversity and Evolutionary Research of Global COE (A06) and for Excellent Graduate Schools, both from MEXT, Japan, to KU, and JSPS Grants-in-Aid for JSPS Fellows (#15J00720) and Young Scientists (B) (#26840127) to the author. The open access publication of this manuscript was supported by the Museum für Naturkunde.

References

  • Akaike H (1974) A new look at the statistical model identification. IEEE Transactions on Automatic Control 19(6): 716–723. doi: 10.1109/TAC.1974.1100705
  • Apakupakul K, Siddall ME, Burreson EM (1999) Higher level relationships of leeches (Annelida: Clitellata: Euhirudinea) based on morphology and gene sequences. Molecular Phylogenetics and Evolution 12(3): 350–359. doi: 10.1006/mpev.1999.0639
  • Blanchard R (1897) Hirudinées du Musée de Leyde. Notes from the Leyden Museum 19(1–2): 73–113.
  • Chakrabarty P, Warren M, Page L, Baldwin C (2013) GenSeq: An updated nomenclature and ranking for genetic sequences from type and non-type sources. ZooKeys 346: 29–41. doi: 10.3897/zookeys.346.5753
  • Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J, Cassis G, Gray MR (1998) Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology 46(5): 419–437. doi: 10.1071/ZO98048
  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4): 783–791. doi: 10.2307/2408678
  • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3(5): 294–299. doi:
  • Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42(2): 182–192. doi: 10.1093/sysbio/42.2.182
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780. doi: 10.1093/molbev/mst010
  • Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PartitionFinder: Combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29(6): 1695–1701. doi: 10.1093/molbev/mss020
  • Leaché AD, Reeder TW (2002) Molecular systematics of the eastern fence lizard (Sceloporus undulatus): a comparison of parsimony, likelihood, and Bayesian approaches. Systematic Biology 51(1): 44–68. doi: 10.1080/106351502753475871
  • Light JE, Siddall ME (1999) Phylogeny of the leech family Glossiphoniidae based on mitochondrial gene sequences and morphological data. The Journal of Parasitology 85(5): 815–823. doi: 10.2307/3285816
  • Lukin EI (1976) Fauna USSR. Leeches. Nauka, Leningrad, 484 pp.
  • Moore JP (1927) The segmentation (metamerism and annulation) of the Hirudinea. In: Harding WA, Moore JP. The Fauna of British India, including Ceylon and Burma. Hirudinea. Taylor & Francis, London, 1–12.
  • Moore JP (1929) Leeches from Borneo with descriptions of new species. Proceedings of the Academy of Natural Sciences of Philadelphia 81: 267–295.
  • Nakano T (2010) A new species of the genus Orobdella (Hirudinida: Arhynchobdellida: Gastrostomobdellidae) from Kumamoto, Japan, and a redescription of O. whitmani with the designation of the lectotype. Zoological Science 27(11): 880–887. doi: 10.2108/zsj.27.880
  • Nakano T (2011a) A new species of Orobdella (Hirudinida: Arhynchobdellida: Gastrostomobdellidae) from Tsushima Island, Japan. Species Diversity 16(1–2): 39–47.
  • Nakano T (2011b) Redescription of Orobdella ijimai (Hirudinida: Arhynchobdellida: Gastrostomobdellidae), and two new species of Orobdella from the Ryukyu Archipelago, Japan. Zootaxa 2998: 1–15.
  • Nakano T (2012a) A new sexannulate species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Yakushima Island, Japan. ZooKeys 181: 79–93. doi: 10.3897/zookeys.181.2932
  • Nakano T (2012b) A new species of Orobdella (Hirudinida, Arhynchobdellida, Gastrostomobdellidae) and redescription of O. kawakatsuorum from Hokkaido, Japan with the phylogenetic position of the new species. ZooKeys 169: 9–30. doi: 10.3897/zookeys.169.2425
  • Nakano T (2012c) Redescription of Orobdella octonaria (Hirudinida: Arhynchobdellida: Orobdellidae) with designation of a lectotype. Species Diversity 17(2): 227–233. doi: 10.12782/sd.17.2.227
  • Nakano T (2014) A new quadrannulate species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from central Honshu, Japan. ZooKeys 445: 57–76. doi: 10.3897/zookeys.445.7999
  • Nakano T (2016) A new quadrannulate species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from western Honshu, Japan. ZooKeys 553: 33–51. doi: 10.3897/zookeys.553.6723
  • Nakano T, Gongalsky KB (2014) First record of Orobdella kawakatsuorum (Hirudinida: Arhynchobdellida: Erpobdelliformes) from Kunashir Island, Kuril Islands. Biodiversity Data Journal 2: e1058. doi: 10.3897/BDJ.2.e1058
  • Nakano T, Lai Y-T (2012) A new species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Taipei, Taiwan. ZooKeys 207: 49–63. doi: 10.3897/zookeys.207.3334
  • Nakano T, Ramlah Z, Hikida T (2012) Phylogenetic position of gastrostomobdellid leeches (Hirudinida, Arhynchobdellida, Erpobdelliformes) and a new family for the genus Orobdella. Zoologica Scripta 41(2): 177–185. doi: 10.1111/j.1463-6409.2011.00506.x
  • Nakano T, Seo H-Y (2014) First record of Orobdella tsushimensis (Hirudinida: Arhynchobdellida: Gastrostomobdellidae) from the Korean Peninsula and molecular phylogenetic relationships of the specimens. Animal Systematics, Evolution and Diversity 30(2): 87–94. doi: 10.5635/ASED.2014.30.2.087
  • Oka A (1895) On some new Japanese land leeches. (Orobdella nov. gen.). The Journal of the College of Science, Imperial University, Japan 8(2): 275–306.
  • Oka A (1910a) Key to Japanese leeches. Dobutsugaku Zasshi 22(256): 56–64.
  • Oka A (1910b) Synopsis der japanischen Hirudineen, mit Diagnosen der neuen Species. Annotationes Zoologicae Japonenses 7(3): 165–183.
  • Pawłowski LK (1962) O występowaniu pijawki Erpobdella octoculata (L.) w Japonii. Zeszyty Naukowe Uniwersytetu Łódzkiego Seria II Nauki Matematyczno-przyrodnicze 12: 127–136.
  • Richardson LR (1971) Gastrostomobdellidae f. nov. and a new genus for the gastroporous Orobdella octonaria Oka, 1895, of Japan (Hirudinoidea: Arhynchobdellae). Bulletin of the National Science Museum (Tokyo) 14(4): 585–602.
  • Richardson LR (1975) A new species of terricolous leeches in Japan (Gastrostomobdellidae, Orobdella). Bulletin of the National Science Museum Series A (Zoology) 1(1): 39–56.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. doi: 10.1093/sysbio/sys029
  • Sawyer RT (1986) Leech Biology and Behaviour. Clarendon Press, Oxford, 1065 pp.
  • Schwarz G (1978) Estimating the dimension of a model. The Annals of Statistics 6(2): 461–464. doi: 10.1214/aos/1176344136
  • Soós Á (1966) Identification key to the leech (Hirudinoidea) genera of the world, with a catalogue of the species. III. Family: Erpobdellidae. Acta Zoologica Academiae Scientiarum Hungaricae 12(3–4): 371–407.
  • Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312–1313. doi: 10.1093/bioinformatics/btu033
  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30(12): 2725–2729. doi: 10.1093/molbev/mst197