First Record of Orobdella tsushimensis (Hirudinida: Arhynchobdellida: Gastrostomobdellidae) from the Korean Peninsula and Molecular Phylogenetic Relationships of the Specimens
First Record of Orobdella tsushimensis (Hirudinida: Arhynchobdellida: Gastrostomobdellidae) from the Korean Peninsula and Molecular Phylogenetic Relationships of the Specimens
Animal Systematics, Evolution and Diversity. 2014. Apr, 30(2): 87-94
Copyright © 2014, The Korean Society of Systematic Zoology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : July 09, 2013
  • Accepted : March 18, 2014
  • Published : April 30, 2014
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About the Authors
Takafumi, Nakano
Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
Hong-Yul, Seo
National Institute of Biological Resources, Incheon 404-708, Korea

Specimens of the genus Orobdella Oka, 1895 from Korea, including various locations in the Korean Peninsula, were identified as Orobdella tsushimensis Nakano, 2011. Phylogenetic analyses using mitochondrial cytochrome oxidase subunit 1 (COI), ND1, tRNA Cys , tRNA Met , 12S rRNA, tRNA val , and 16S rRNA markers show that the newly collected specimens form a monophyletic group with the known O. tsushimensis specimens. The genetic distance of COI of these specimens was in the range 0.4-6.6%. These results confirm that the newly collected specimens belong to O. tsushimensis . This is the first record of the genus Orobdella from the Korean Peninsula.
The genus Orobdella Oka, 1895 is a terrestrial macrophagous leech taxon that is distributed in the Far East. At present 11 species of this genus have been described: 10 from Japan ( Oka, 1895 ; Richardson, 1975 ; Nakano, 2010 , 2011a , 2011b , 2012a , 2012b , 2012c ); and the other from Taiwan ( Nakano and Lai, 2012 ). Additionally, Orobdella whitmani Oka, 1895 has been reported from Primorsky Krai, Russia ( Gilyarov et al., 1969 ), but the precise taxonomic status of the Russian specimens still remains uncertain ( Nakano, 2012b ). Yun (1997) provided a Korean leech list, but Orobdella leeches were not included in this list. Recently, quadrannulate Orobdella tsushimensis Nakano, 2011 has been recorded from Gageodo Island, which is located in the Yellow Sea, southwest of the Korean Peninsula ( Nakano and Seo, 2012 ). However, Orobdella leeches have never been reported from the Korean Peninsula.
Recently, additional specimens of the quadrannulate Orobdella were collected from various places in Korea, including locations in the Korean Peninsula. This is the first record of the genus Orobdella from the peninsula. The identification and a description of the newly collected specimens are herein presented. In addition, their phylogenetic relationships are reconstructed using mitochondrial cytochrome oxidase subunit 1 (COI), ND1, tRNA Cys , tRNA Met , 12S rRNA, tRNA val , and 16S rRNA (tRNA Cys -16S) sequence data.
Leeches were collected from various places in Korea (Fig. 1) . Altitudes and coordinates for localities were obtained using a Garmin eTrex GPS unit.
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Map showing the collection localities of the specimens examined in this study (nos. 1-11), Tsushima Island, Japan, and the type locality of Orobdella tsushimensis Nakano, 2011.
For DNA extraction, botryoidal tissue was taken from the posterior part of the body around the caudal sucker of the specimens. All of the specimens were 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; caudal sucker width (CW) from the right margin to the left margin of the sucker. Examination, dissection, and drawings of the specimens were accomplished using a stereoscopic microscope equipped with a drawing tube (Leica M125; Leica, Wetzlar, Germany). 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 extraction of genomic DNA and DNA sequencing methods for COI and tRNA Cys -16S followed Nakano (2012b) . For obtaining tRNA Leu and ND1 (tRNA Leu -ND1) sequence data, the procedure above was modified as follows: the primer sets for tRNA Leu -ND1 were LND300 and HND1932 ( Light and Siddall, 1999 ) or HND1932-in (5′-AATGGAGCACGA TTAGTTTC-3′) (in this study); the PCR reaction mixture for tRNA Leu -ND1 was heated to 94℃ for 5 min, followed by 35 cycles of 94℃(10 s), 45℃(20 s), and 72℃(39 s) and a final extension at 72℃ for 6 min; the sequencing reaction mixture for tRNA Leu -ND1 was incubated at 96℃ for 2 min, followed by 40 cycles of 96℃ (10 s), 50℃ (5 s), and 60℃ (39 s). Obtained sequences have been deposited in GenBank. Sequences used for molecular phylogenetic analyses are listed in Table 1 ; 16 sequences of COI, 3 those of tRNA Leu - ND1 and 15 those of tRNA Cys -16S, which were published in the previous studies ( Nakano, 2012a , 2012b ; Nakano and Lai, 2012 ; Nakano and Seo, 2012 ; Nakano and Gongalsky, 2014 ), were obtained from GenBank.
Samples used for the phylogenetic analyses, with the information on voucher and GenBank accession numbers
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Sources: aNakano and Lai (2012); bNakano (2012b); cNakano (2012a); dNakano and Seo (2012); eNakano and Gongalsky (2014). For the locality number, see Fig. 1. COI, cytochrome oxidase subunit 1; KOR, Korea; H, holotype; TWN, Taiwan; JPN, Japan; T, topotype; UNIMAS, The Universiti Malaysia Sarawak; MYS, Malaysia.
Sequences of mitochondrial COI and ND1(tRNA Leu region was removed from each sequence) were aligned by eye because there were no indels. Mitochondrial tRNA Cys -16S sequences were aligned using MAFFT L-INS-I ( Katoh et al., 2005 ) and then refined with GBLOCKS ( Castresana, 2000 ). The length of the aligned COI sequences was 1,266 bp, that of ND1 was 579 bp, and that of tRNA Cys -16S was 818 bp (288 characters were eliminated). The concatenated sequences thus yielded a total of 2,663 bp positions. Pairwise comparisons of Kimura-2 parameter (K2P) distance ( Kimura, 1980 ) of the COI sequences were calculated using MEGA5 ( Tamura et al., 2011 ).
Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) models. ML phylogenies were calculated using TREEFINDER v October 2008 ( Jobb et al., 2004 ) with the tool package PHYLOGEARS v 2.0 ( Tanabe, 2008 ), and then nonparametric bootstrapping ( Felsenstein, 1985 ) was conducted with 500 replicates. The best-fit models for each partition were selected based on the Akaike Information Criterion ( Akaike, 1974 ) by using KAKUSAN4 ( Tanabe, 2011 ): for the 1st position of COI, TN93 with gamma distribution (+G) and proportion of invariant sites (+I); for the 2nd position of COI, TVM+I; for the 3rd position of COI, HKY85+G; for the 1st position of ND1, GTR+G+I; for the 2nd position of ND1, J1+G; for the 3rd position of ND1, J2+G; and for tRNA Cys -16S, GTR+G was selected. BI and Bayesian posterior probabilities (BPPs) were estimated using MRBAYES v 3.2 ( Ronquist et al., 2012 ). The best-fit models for each partition were identified under the Bayesian information criterion ( Schwarz, 1978 ) and also by using KAKUSAN4: for the 1st position of COI, GTR+G+I; for the 2nd position of COI, F81+I; for the 3rd position of COI, HKY85+G; for the 1st position of ND1, GTR+G+I; for the 2nd position of ND1, HKY85+G; for the 3rd position of ND1, HKY85+G; and for tRNA Cys -16S, GTR+G was selected. Two independent runs of four Markov chains were conducted for 4 million generations, and the tree was sampled every 100 generations. The parameter estimates and convergence were checked using TRACER v 1.5 ( Rambaut and Drummond, 2009 ), and based on these results, the first 10,001 trees were discarded.
Nodes with bootstrap (BS) values higher than 70% were considered as sufficiently resolved ( Hillis and Bull, 1993 ). Nodes with BPPs higher than 95% were considered statistically significant ( Leaché and Reeder, 2002 ).
  • Order Arhynchobdellida Blanchard, 1894
  • Suborder ErpobdelliformesSawyer, 1986
  • 1*Family Gastrostomobdellidae Richardson, 1971
  • 2*GenusOrobdellaOka, 1895
3*Orobdella tsushimensis Nakano, 2011 (Figs. 2, 3)
Orobdella tsushimensis Nakano, 2011a : 41-46, figs. 2-4 (near the Orobashi Bridge at Kechi Dam, alt. 69 m, 34° 15′17′′N, 129° 17′17′′E, Tsushima, Tsushima Island, Nagasaki Prefecture, Japan, holotype, KUZ Z134); Nakano and Seo, 2012 : 237-239, figs. 2-4.
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Orobdella tsushimensis Nakano, 2011 from Seoul, KUZ Z616. A, Dorsal view; B, Ventral view. Scale bars: A, B=5 mm.
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Orobdella tsushimensis Nakano, 2011 from Seoul, KUZ Z616. A, Dorsal view of somites I-VIII; B, Ventral view of somites IVIII; C, Dorsal view of somites XXV-XXVII and caudal sucker; D, Ventral view of somites XXV-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; H, Dorsal view of epididymides and ventral nervous system; I, Dorsal view of male atrium showing position of ganglion XI. ac, atrial cornua; af, annular furrow; an, anus; at, atrium; cp, crop; ed, ejaculatory duct; ep, epididymis; fg, female gonopore; gd, gastroporal duct; gp, gastropore; mg, male gonopore; np, nephridiopore; ph, pharynx; ts, testisac; vd, vas deferens. Scale bars: A-E, G-I=1 mm, F=0.5 mm.
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The maximum likelihood (ML) tree of 2,663 bp of cytochrome oxidase subunit 1 (COI), ND1, tRNACys, tRNAMet, 12S rRNA, tRNAval, and 16S rRNA. The numbers associated with the nodes represent bootstrap values for ML (BS)/and Bayesian posterior probabilities (BPPs). BSs higher than 40% and/or BPPs higher than 95% are indicated. For the locality number, see Fig. 1 and Table 1.
Material examined (see Fig. 1 for the locality number). Korea: 3 specimens, Seoul: Nowon-gu, Mt. Suraksan, alt. 118 m, 37° 41′08′′N, 127° 04′07′′E (locality no. 1), 4 Jul 2012, Seo HY, KUZ Z615, Z616, dissected, and KUZ Z617; 2 specimens, Jongno-gu, Mt. Bukhansan, alt. 447 m, 37° 37′ 28′′N, 126° 57′41′′E (locality no. 2), 6 Jul 2012, Seo HY, KUZ Z621 and Z622, dissected; 2 specimens, Jeollanam-do: Sinan-gun, Gageodo Island, alt. 429 m, 34° 04′49′′N, 125° 96′27′′E, (locality no. 7), 10 Jul 2012, Seo HY, KUZ Z623 and Z624; 1 specimen, Wando-gun, Yeoseodo Island, alt. 46 m, 33° 59′03′′N, 126° 55′19′′E (locality no. 8), 20 Jul 2012, Seo HY, KUZ Z628, dissected; 1 specimen, Jejudo Island: Jeju-si, alt. 384 m, 33° 26′47′′N, 126° 33′49′′E (locality no. 9), 17 Jun 2012, Seo HY, KUZ Z614; 1 specimen, Seogwipo-si, alt. 183 m, 33° 18′05′′N, 126° 37′51′′E (locality no. 11), 27 Sep 2012, Seo HY, KUZ Z629; 1 specimen, Jeju-si, Mt. Hanlasan, alt. 1,601 m, 33° 21′56′′N, 126° 30′48′′E (locality no. 10), 2 Jul 2012, Kang SG, KUZ Z620, dissected; 1 specimen, Gwangju Metropolitan City: Dong-gu, Mt. Mudeungsan, alt. 533 m, 35° 07′11′′N, 126° 58′56′′E (locality no. 6), 1 Jul 2012, Seo HY, KUZ Z618, dissected; 1 specimen, Dong-gu, Mt. Mudeungsan, alt. 375 m, 35° 07′21′′N, 126° 58′34′′E (locality no. 6), 24 Jun 2012, Seo HY, KUZ Z619; 1 specimen, Incheon Metropolitan City: Gyeyang-gu, Mt. Gyeyangsan, alt. 63 m, 37° 33′33′′N, 126° 43′30′′E (locality no. 3), 11 Oct 2012, Seo HY, KUZ Z625, dissected; 1 specimen, Gangwon-do: Wonju-si, Mt. Chiaksan, alt. 790 m, 37° 18′07′′N, 128° 03′39′′E (locality no. 4), 5 Oct 2012, Seo HY, KUZ Z626, dissected; 1 specimen, Daejeon Metropolitan City: Seo-gu, Mt. Changtaesan, alt. 245 m, 36° 11′38′′N, 127° 20′19′′E (locality no. 5), 5 Oct 2012, Seo HY, KUZ Z627, dissected.
Description. Maximum BL 105.4 mm (KUZ Z622), BW 11.9mm(KUZ Z628), minimum BL 33.2 (KUZ Z626), BW 4.3mm(KUZ Z619). Caudal sucker ventral, ellipsoid, maximum CL 3.9 mm, CW 6.6 mm (KUZ Z622), minimum CL 1.6mm(KUZ Z624), CW 2.0mm(KUZ Z626).
Somites VIII-XXV quadrannulate, somite XXVI triannulate (Fig. 3A - E) . Anus behind somite XXVII with no postanal annulus (Fig. 3C) . Clitellum in XI b5 to XIII a2. Eyes often in 3 pairs, first pair dorsally in posterior margin of II, second and third pairs dorsolaterally in posterior margin of V (a1+a2) (Fig. 3A) . Nephridiopores in 17 pairs in VIII-XXIV (Fig. 3B , E) .
Pharynx reaching to XIII b5/b6-XIV b6 (Fig. 3G) ; each pharyngeal ridges reaching to crop straightly. Crop tubular, reaching to XIX b5/b6-XX a1/a2. Gastropore conspicuous in XIII a1/a2 (Fig. 3E , F) . Gastroporal duct developed, bulbous, or bottle-shaped, slightly winding at junction with gastropore; joining with crop in XIII b6-XIV b6 (Fig. 3G) .
Male gonopore in XI b6 (slightly anterior to middle of annulus), female gonopore inconspicuous in XIII a1/a2, behind gastropore, gonopores separated by 1/2+5 annuli (Fig. 3E , F) . Testisacs multiple, in XVIII b5-XX b6 to XXIV a1- XXVb6 (Fig. 3H) . Paired epididymides in XV b5-XVIII a1/a2 to XVIII b5-XX a1/a2 (Fig. 3H) . Ejaculatory ducts in XI b5 to XV b5-XVIII a1/a2; nearly straight in position anterior to ovisacs (Fig. 3I) ; coiled in position posterior to ovisacs (Fig. 3H) . Pair of muscular atrial cornua, ovate in XI b5 and b6 (Fig. 3I) or fusiform in XI b6. Atrium short, muscular, globular, in XI b5 and b6. One pair of ovisacs, thin-walled, globular, in XIII a2 and b5. Oviducts thin-walled, both oviducts converging into common oviduct in XIII a2.
Distribution. Known from mountainous regions of the Korean Peninsula and its peripheral islands including Tsushima Island, Japan ( Nakano, 2011a ; Nakano and Seo, 2012 ).
Phylogenetic relationships (see Fig. 1 for the locality number). The ML tree with ln L =−17134.56 (Fig. 4) was identical in topology to the obtained BI tree (not shown). Monophyly of the genus Orobdella was confirmed (BS=100%, BPP=100%). Although O. tsushimensis formed a monophyletic lineage with the clade that consists of ([ O. esulcata Nakano, 2010 + O. mononoke Nakano, 2012]+[ O. ketagalan Nakano and Lai, 2012 +{ O. dolichopharynx Nakano, 2011+ O. shimadae Nakano, 2011}]), the phylogenetic position of O. tsushimensis was not statistically resolved (BS=45%, BPP= 86%).
Monophyly of O. tsushimensis was confirmed (BS=100%, BPP=100%). Within O. tsushimensis , three lineages were recognized and defined. Lineage A contained only KUZ Z626 from Mt. Chiaksan, Gwangwon (locality no. 4). Lineage B consisted only of KUZ Z627 from Mt. Changtaesan, Daejeon (no. 5). Lineage C consisted of the other nine specimens. Lineages B and C formed a monophyletic lineage (BS=91%, BPP=99%). Within Lineage C, a monophyletic lineage (BS=100%, BPP=100%) of three specimens, KUZ Z616 (no. 1), Z622 (no. 2) and Z625 (no. 3) from around Seoul, KUZ Z620 from Jejudo Island (no. 10) and a monophyletic lineage (BS=100%, BPP=100%) of two specimens, KUZ Z133 (no. 12) and Z134 (no. 13), from Tsushima Island formed a monophyletic lineage (BS=82%, BPP=99%). KUZ Z215 (no. 7), Z618 (no. 6) and Z628 (no. 8) from the southwestern part of Korea, including Gageodo and Yeoseodo Islands, also formed a monophyletic lineage, but with low support values (BS=37%, BPP=58%).
Genetic divergence. The COI K2P distance within O. tsushimensis was between 0.4-6.6% (mean=5.0%).
Remarks. The present specimens newly collected from various places in Korea clearly belong to O. tsushimensis based on their possession of the following characteristics: somites VIII-XXV quadrannulate; gastropore conspicuous in XIII a1/a2; gastroporal duct developed, bulbous; male gonopore in middle of XI b6, female gonopore in XIII a1/a2, behind gastropore, gonopores separated by 1/2+5 annuli; paired epididymides in XV-XX, occupying more than 2 somites; atrial cornua developed, ovate or conical. One specimen, KUZ Z627, possesses tubular gastroporal duct and fusiform atrial cornua. However, these characteristics of this specimen seemed to be due to its immaturity, since this individual has undeveloped sperm ducts and undetectable testisacs. In Nakano and Seo (2012) , O. tsushimensis has been diagnosed as having epididymides in XVII-XIX (occupying approximately 2 somites). However, the present specimens have their epididymides in somites XV-XX(occupying 8-12 annuli, i.e., 2- 3 somites). Therefore, the diagnosis in Nakano and Seo (2012) should be amended to take this difference into account.
The molecular phylogenies obtained here showed that the specimens that were morphologically identified as O. tsushimensis form a well-supported monophyletic group. Additionally, the COI sequence divergence within these specimens was between 0.4-6.6% (mean=5.0%). For comparison, the intraspecific variation of COI sequences of O. koikei in Hokkaido, Japan was between 4.8-8.1% (mean=7.1%) ( Nakano, 2012b ). Thus, our molecular phylogenetic analyses and genetic distance data strongly support our morphological identification result that the Orobdella leeches newly collected in this study belong to O. tsushimensis .
The present molecular phylogenies showed that the specimen from Mt. Chiaksan (Lineage A; locality no. 4) first diverged from the others. Then, the specimen from Mt. Changtaesan (Lineage B; no. 5) diverged from the other leeches. It is noteworthy that the specimen from Jejudo Island (no. 10) and two specimens from Tsushima Island (nos. 12, 13) are monophyletic, which was statistically supported, with the three specimens from locations around Seoul (nos. 1-3), not with the specimens from adjacent localities (i.e., nos. 6-8). This geographic pattern of the phylogenetic relationships suggests a complex biogeographical history of O. tsushimensis . However, the present phylogenies failed to reconstruct fully robust phylogenetic relationships within Lineage C. Additional specimens of O. tsushimensis as well as suitable genetic markers are still needed to clarify the precise phylogenetic relationships and biogeographical history of this species.
Korean name: 왕산거머리과
Korean name: 왕산거머리속
Korean name: 왕산거머리
The authors are grateful to Professor Tsutomu Hikida (Kyoto University; KU) for his helpful comments and suggestions to improve this manuscript. We are also grateful to Dr. Seung-Gu Kang (National Institute of Biological Resources) for providing theOrobdellaspecimen from Jejudo Island, to three anonymous reviewers for their constructive comments on the manuscript, and to Dr. Elizabeth Nakajima (KU) for checking the English of this text. 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 Kyoto University, National Institute of Biological Resources, Ministry of Environment, the Republic of Korea, and a JSPS Research Fellowship for Young Scientists to TN.
Akaike H 1974 A new look at the statistical model identification IEEE Transactions on Automatic Control 19 716 - 723
Castresana J 2000 Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis Molecular Biology and Evolution 17 540 - 552
Felsenstein J 1985 Confidence limits on phylogenies: an approach using the bootstrap Evolution 39 783 - 791
Gilyarov MS , Lukin EI , Perel TS 1969 The first terrestrial leech- Orobdella whitmani Oka (Hirudinel, Herpobdellidae) in the fauna of the USSR: A Tertiary relict of forests of the southern Maritime Territory Doklady Akademii Nauk SSSR 188 235 - 237
Hillis DM , Bull JJ 1993 An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis Systematic Biology 42 182 - 192
Jobb G , von Haeseler A , Strimmer K 2004 TREEFINDER: a powerful graphical analysis environment for molecular phylogenetics BMC Evolutionary Biology 4 18 -
Katoh K , Kuma K , Toh H , Miyata T 2005 MAFFT version 5: improvement in accuracy of multiple sequence alignment Nucleic Acids Research 33 511 - 518
Kimura M 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences Journal of Molecular Evolution 16 111 - 120
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 44 - 68
Light JE , Siddall ME 1999 Phylogeny of the leech family Glossiphoniidae based on mitochondrial gene sequences and morphological data The Journal of Parasitology 85 815 - 823
Moore JP , Harding WA , Moore JP 1927 The segmentation (metamerism and annulation) of the Hirudinea; The Fauna of British India, including Ceylon and Burma. Hirudinea Taylor & Francis London 1 - 12
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 27 880 - 887
Nakano T 2011 A new species of Orobdella (Hirudinida: Arhynchobdellida: Gastrostomobdellidae) from Tsushima Island, Japan Species Diversity 16 39 - 47
Nakano T 2011 Redescription of Orobdella ijimai (Hirudinida: Arhynchobdellida: Gastrostomobdellidae), and two new species of Orobdella from the Ryukyu Archipelago, Japan Zootaxa 2998 1 - 15
Nakano T 2012 A new sexannulate species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Yakushima Island, Japan ZooKeys 181 79 - 93
Nakano T 2012 A new species of Orobdella (Hirudinida, Arhynchobdellida, Gastrostomobdellidae) and redescription of Orobdella kawakatsuorum from Hokkaido, Japan with the phylogenetic position of the new species ZooKeys 169 9 - 30
Nakano T 2012 Redescription of Orobdella octonaria (Hirudinida: Arhynchobdellida: Orobdellidae) with designation of a lectotype Species Diversity 17 227 - 233
Nakano T , Gongalsky KB 2014 First record of Orobdella kawakatsuorum (Hirudinida: Arhynchobdellida: Erpobdelliformes) from Kunashir Island, Kuril Islands Biodiversity Data Journal 2 e1058 -
Nakano T , Lai YT 2012 A new species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Taipei, Taiwan ZooKeys 207 49 - 63
Nakano T , Seo HY 2012 First record of Orobdella tsushimensis (Hirudinida: Arhynchobdellida: Orobdellidae) from Korea (Gageodo Island) and its molecular phylogenetic position within the genus Species Diversity 17 235 - 240
Oka A 1895 On some new Japanese land leeches (Orobdella nov. gen.) The Journal of the College of Science, Imperial University, Japan 8 275 - 306
Rambaut A , Drummond AJ 2009 Tracer v 1.5 [Internet]. Andrew Rambaut
Richardson LR 1975 A new species of terricolous leeches in Japan (Gastrostomobdellidae, Orobdella) Bulletin of the National Science Museum Series A (Zoology) 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 61 539 - 542
Sawyer RT 1986 Leech biology and behaviour Clarendon Press Oxford 1 - 1065
Schwarz G 1978 Estimating the dimension of a model The Annals of Statistics 6 461 - 464
Tamura K , Peterson D , Peterson N , Stecher G , Nei M , Kumar S 2011 MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods Molecular Biology and Evolution 28 2731 - 2739
Tanabe AS 2008 Phylogears v 2.0 [Internet]. Akifumi S. Tanabe
Tanabe AS 2011 Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data Molecular Ecology Resources 11 914 - 921
Yun IB 1997 Class Hirudinea;List of animals in Korea (excluding insects) Akademy Press Seoul 142 - 143