A new quadrannulate species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from central Honshu, Japan

Abstract A new quadrannulate species of Orobdella, Orobdella masaakikuroiwai sp. n., from the mountainous region of central Honshu, Japan is described. This is only the second small species known within this genus, with a body length of less than 4 cm for mature individuals. Phylogenetic analyses using nuclear 18S rDNA and histone H3 as well as mitochondrial COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, and ND1 markers showed that Orobdella masaakikuroiwai sp. n. is the sister species of the quadrannulate Orobdella whitmani Oka, 1895. Phylogenetic relationships within Orobdella masaakikuroiwai sp. n. conducted using mitochondrial markers reveled a distinction between eastern and western phylogroups.


Introduction
The genus Orobdella Oka, 1895 is an East Asian terrestrial macrophagous leech taxon assigned to the family Gastrostomobdellidae Richardson, 1971, along with the Southeast Asian terrestrial macrophagous genus Gastrostomobdella Moore, 1929(Richardson 1971. Gastrostomobdellidae was once classified within the suborder Hirudiniformes, which includes jawed blood-feeding taxa (Sawyer 1986). Recent molecular phyloge-netic studies revealed that Orobdella is part of the suborder Erpobdelliformes, which contains only predaceous leech taxa , Oceguera-Figueroa et al. 2011. The monotypic family Orobdellidae Nakano, Ramlah & Hikida, 2012 was established for Orobdella based on both morphological differences between Orobdella and Gastrostomobdella (gastroporal duct of Orobdella is tubular and positioned on top of the female organ while in Gastrostomobdella, this duct is columnar and vertical in position to the gastropore) and the results of an analysis by  which rejected the monophyly of these two taxa. Despite their failure to reconstruct the precise phylogenetic relationships of Orobdella and Gastrostomobdella within Erpobdelliformes, the classification by  is followed here.
Orobdella now consists of 11 nominal leech species from East Asia: nine species known from the Japanese Archipelago (Nakano 2010(Nakano , 2011b(Nakano , 2012a; one present in the Korean Peninsula and adjacent islands (Nakano 2011a, Nakano and Seo 2012; and the remainder in Taiwan (Nakano and Lai 2012). Species of Orobdella are usually large in size, with the body length of mature individuals reaching to around 10 cm (e.g. Nakano (2011b)). The largest species in this genus is the octannulate O. octonaria Oka, 1895 recorded from Honshu, Japan, with a body length often greater than 20 cm (Nakano 2012c, Oka 1895. In contrast, the smallest species is the quadrannulate O. koikei Nakano, 2012b found in Hokkaido, Japan with a body length of less than 4 cm, but which were considered to be mature due to the presence of developed testisacs. It is also noteworthy that the distribution of O. koikei in Hokkaido overlaps with that of the quadrannulate O. kawakatsuorum Richardson, 1975, which is present in Hokkaido as well as its adjacent islands and attains a body length of ca. 10 cm (Nakano 2012b, Nakano and Gongalsky 2014).
Several small Orobdella leeches were recently collected from east-central Honshu, Japan. Although the bodies of the specimens are up to 3.5 cm in length, some of them already possess an obvious clitellum and they are thus considered to be mature individuals. These leeches are described herein as a new species. The phylogenetic position of this new species was reconstructed using nuclear 18S and histone H3 (H3), and mitochondrial COI, tRNA Cys , tRNA Met , 12S, tRNA Val and 16S rDNA, and ND1 sequence data.

Sampling and morphological examination
Leeches were collected from seven localities in east-central Honshu, Japan (Fig. 1). These seven collection localities are numbered referring to the locality name listed in Table 1. When possible, altitudes above sea level and geographical coordinates for localities were obtained using a Garmin eTrex® GPS unit.
The specimens were relaxed by the gradual addition of absolute ethanol to fresh water. For DNA extraction, botryoidal tissue was taken from the posterior part of the body around the caudal sucker of every specimen, and then preserved in abso- Table 1. Collection localities in this study with the information on locality names.

Locality number
Locality name 1 Akiruno, Tokyo Metropolis, Japan 2 Namesawakeikoku Valley, Izu Shizuoka Prefecture, Japan 3 Shibunoyu, Kitayama, Chino, Nagano Prefecture, Japan 4 Mt. Mitsugaisan, Ina, Nagano Prefecture, Japan 5 Shirabisotoge Pass, Ida, Nagano Prefecture, Japan 6 Ikuta, Matsukawa, Nagano Prefecture, Japan 7 Shiojidaira Nature Park, Iizuna, Nagano Prefecture, Japan lute ethanol. The rest of the body was fixed in 10% formalin and then 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 margin to the left margin of the sucker. Examination, dissection, and drawing of the specimens were accomplished 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.

PCR 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, A and L (PCR and CS), C and Y (PCR and CS), and 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 COI, LCO 1490 (PCR and CS) and HCO 2198 (CS) (Folmer et al. 1994), and LCO-in (CS) and HCOout (PCR and CS) (Nakano 2012b); for tRNA Cys , tRNA Met , 12S, tRNA Val , and 16S (tRNA Cys -16S), 12SA-out (PCR and CS) and 12SB-in (CS), and 12SA-in (CS) and 12SB-out (PCR and CS) (Nakano 2012b); for tRNA Leu and ND1 (tRNA Leu -ND1), LND3000 and HND1932 (PCR and CS) (Light and Siddall 1999). The PCR reaction and DNA sequencing were performed using the modified methods outlined by Nakano (2012a). The 18S, H3 and ND1, and COI and tRNA Cys -16S reactions were respectively performed using a GeneAmp PCR System 2700 and a GeneAmp PCR System 9700 (Applied Biosystems). The PCR reaction mixtures were heated to 94 °C for 5 min, followed by 40 cycles at 94 °C (10 s each), 48 °C for 18S, H3, and tRNA Leu -ND1 or 45 °C for COI and tRNA Cys -16S (20 s), and 72 °C (48 s for 18S, H3 and tRNA Leu -ND1 or 1 min 12 s for COI and tRNA Cys -16S), and a final extension at 72 °C for 6 min. The sequencing mixtures were heated to 96 °C for 2 min, followed by 40 cycles at 96 °C (10 s each), 50 °C (5 s each), and 60 °C (48 s each). The obtained sequences were edited using DNA BASER (Heracle Biosoft S.R.L.). The DNA sequences listed in Table 2 were newly obtained in this study, and were deposited with the International Nucleotide Sequence Database Collaboration (INSDC).
The phylogenetic position of the new species within the genus Orobdella was estimated based on sequences of nuclear 18S and H3 and mitochondrial COI, tRNA Cys -16S, and ND1. Sequences of nuclear H3 and mitochondrial COI were aligned by eye because there were no indels. Nuclear 18S and mitochondrial tRNA Cys -16S and tRNA Leu -ND1 were aligned using MATTF L-INS-I (Katoh et al. 2005). Then, the tRNA Leu region was removed from each sequence of tRNA Leu -ND1. The length of the aligned 18S sequences was 1845 bp, that of H3 was 327 bp, that of COI was 1266 bp, that of tRNA Cys -16S was 1107 bp, and that of ND1 was 633 bp. The concatenated sequences thus yielded 5,124 bp positions. AB828544f Odontobdella blanchardi (Oka, 1910) KUZ 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 PHYLOGEARS v. 2.0 tool package (Tanabe 2008), followed by nonparametric bootstrapping (BS) (Felsenstein 1985) conducted with 1,000 replicates. The best-fit models for each partition were selected based on the Akaike Information Criterion (Akaike 1974) using KAKUSAN4 (Tanabe 2011): for 18S, TN93 with gamma distribution (+G) and proportion of invariant sites (+I); for the first, second, and third positions of H3, respectively, a homogenous (+H) TN93 model, JC69+H, and J2+G; for the first, second, and third positions of COI, respectively, TN93+G+I, TVM+I, and TIM+G; for tRNA Cys -16S, GTR+G; and for the first, second, and third positions of ND1, respectively, GTR+G+I, HYK85+G, and J2+G. 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 with the Bayesian information criterion (Schwarz 1978) using KAKUSAN4: for 18S, K80+G; for the first, second and third positions of H3, respectively, JC69+H, JC69+H, and HKY+G; for the first, second, and third positions of COI, respectively, GTR+G+I, F81+I, and HKY+G; for tRNA Cys -16S, GTR+G; and for the first, second, and third positions of ND1, respectively, GTR+G, HKY85+G, and HKY85+G. Two independent runs of four Markov chains were conducted for 10 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 the first 25,001 trees were discarded based on these results.
The phylogenetic relationships of the specimens of the new species were reconstructed based on sequences of mitochondrial regions. The alignment of the sequences as well as the reconstruction of the ML and BI phylogenies was accomplished followed the methods described above. The length of the aligned COI was 1266 bp, that of tRNA Cys -16S was 1056 bp, and that of ND1 was 579 bp. Thus, the concatenated sequences yielded 2,901 bp positions. The best-fit models for each partition selected for the ML phylogenies were as follows: for the first, second, and third positions of COI, respectively, TN93+G, TVM+H, and TN93+G; for tRNA Cys -16S, GTR+G; and for the first, second, and third positions of ND1, respectively, TN93+G, HKY85+H, and HYK85+G. The best-fit models identified for each partition for the BI analyses were as follows: the first, second, and third positions of COI, respectively, GTR+G, F81+H, and HKY85+G; for tRNA Cys -16S, GTR+G; and for the first, second, and third positions of ND1, respectively, GTR+G, F81+H, and HKY85+G. For BI and BPPs, two independent runs of four Markov chains were conducted for 6 million generations, and the tree was sampled every 100 generations. The first 15,001 trees were eliminated based on the results of the parameter estimates and convergence.
Nodes with BS values higher than 70% were considered sufficiently resolved (Hillis and Bull 1993). Nodes with BPPs higher than 95% were considered statistically significant (Leaché and Reeder 2002).
Type materials (see Fig. 1  Additional materials (see Fig. 1 and Table 1  Etymology. The specific name is a noun in the genitive case formed directly from the name of Mr Masaaki Kuroiwa, who generously accompanied the field survey in Nagano Prefecture. Description of holotype. Body firm and muscular, elongate, with constant width in caudal direction, dorsoventrally compressed, BL 34.0 mm, BW 3.42 mm (Fig. 2). Caudal sucker ventral, elliptic, CL 1.7 mm (minor axis), CW 1.9 mm (major axis) (Figs 2B, 3D).
Eyes in three pairs, first pair dorsally on anterior margin of III, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig. 3A). Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of a1 of each somite in VIII-XXIV (Fig. 3B, E). Papillae numerous, minute, hardly visible, one row on every annulus.
Coloration. In life, dorsal surface ochre (Fig. 5), whitish brown, or brown, ventral surface grayish white or yellowish white; individuals from Shizuoka Pref. (KUZ Z687, Z688), dorsal surface whitish yellow. Colour faded in preservative, rarely with one dorsal black line from VII a3-IX a2 to XIX b5-XXVI b6 (KUZ Z691, Z693, Z694, Z698). Distribution (see Fig. 1 for the locality numbers). This species was primarily collected from localities in Nagano Prefecture: the east-central part (locality number 3), and the southeastern part along the Inadani Basin (locality numbers 4-7). This species was also found in the western mountainous part of the Metropolitan Tokyo area (locality number 1), as well as in the Amagi Mountain Range in the central part of the Izu Peninsula, Shizuoka Prefecture (locality number 2). The locality data for this species suggested that O. masaakikuroiwai sp. n. would be widely distributed in mountainous regions such as the southwestern part of the Kanto Region and the southeastern part of the Chubu Region, Honshu, Japan. The lowest elevation among the localities was 230 m above sea level (a.s.l.) (locality number 1), and the highest was ca. 1860 m a.s.l. (locality number 3).
Natural history. This species was generally found curled up under rocks or fallen leaves in moist mountainous habitats (Fig. 5B). Soil was sometimes observed in the digestive tract during specimen dissection. This species is therefore considered an earthworm-feeder as are the other known Orobdella leeches.
Mature leeches with an obvious clitellum were collected on 20 July (KUZ Z690, Z691, Z693, Z694) and 10 August (KUZ Z697) at two sites in Nagano Prefecture (locality numbers 4 and 7, elevation ca. 875 m and 1098 m, respectively). These findings indicate that the reproductive season of this species may begin in mid-to-late July.
Remarks. Although the leech specimens examined in this study were small (up to 35 mm), several individuals, including the holotype, were determined to be mature due to the possession of an obvious clitellum and developed testisacs. Specimen KUZ Z687 possessed a tubular gastroporal duct and fusiform atrial cornua. Immature leeches may have these characteristics, because the sperm ducts and testisacs of specimen KUZ Z687 are undeveloped and barely detectable.
The new species unambiguously belongs to the genus Orobdella as it has all the generic diagnostic characteristics (see  for the generic diagnosis): post-anal annulus absent; pharynx agnathous, euthylaematous; gastropore in XIII; gastroporal duct lying on female organ; gonopores separated by more than one full somite; testisacs multiple; male atrium in XI without penis sheath and penis; ovisacs globular in XIII; female median reproductive system essentially lacking.

Molecular phylogenies and genetic distances
The ML tree (ln L = −23350.60) (Fig. 6) for estimating the phylogenetic position of the new species had an identical topology to the BI tree (not shown). The monophyly of the genus Orobdella was confirmed (BS = 99%, BPP = 100%) The genus was divided into two lineages (hereafter lineages A and B). Lineage A consisted of O. kawakatsuorum and O. koikei (BS = 99%, BPP = 100%). Monophyletic lineage B (BS = 97%, BPP = 100%) included the remaining 10 species (including the new species), and was divided into two sub-lineages (hereafter lineages B1 and B2). The monophyly of lineage B1, which consisted of six species, was not well supported by the ML analysis (BS = 50%, BPP = 99%). Lineage B2 included four species, but the monophyly of this lineage was also not well supported by the ML analysis (BS = 57%, BPP = 99%). The new species, O. masaakikuroiwai sp. n., was part of lineage B2, and was a sister taxon of O. whitmani within this lineage. However, this relationship was not fully supported by the ML analysis (BS = 57%, BPP = 99%).

Discussion
The current molecular phylogenies showed that the specimens morphologically identified as the new species form a monophyletic group with strong support values. In addition, the K2P genetic distance of the COI sequences detected within the specimens was 0.5-6.7% (mean = 4.4%). Nakano (2012b) stated that the COI K2P distance between the sister species of Orobdella, O. kawakatsuorum and O. koikei, was 8.1-9.9% (mean = 9.0%). Therefore, the present genetic analyses support the taxonomic designation of the specimens examined in this study as belonging to the new species, O. masaakikuroiwai sp. n. Orobdella masaakikuroiwai sp. n. was divided into two lineages (lineages 1 and 2) according to the molecular phylogenetic analyses. Lineage 1 consists of the individuals inhabiting the Kanto Region (KUZ Z684, locality number 1) and the Izu Peninsula (KUZ Z687, locality number 2). The Izu Peninsula is located on the Philippine Sea Plate and collided with Honshu island around 1 million years ago (Kitazato 1997). Therefore, O. masaakikuroiwai sp. n. likely migrated into the peninsula after this collision event. In addition to lineages 1 and 2 composed of specimens from the mountainous region of Nagano Prefecture, the individuals of O. masaakikuroiwai sp. n. were sub-divided into central (lineage 2''; locality numbers 3, 4, 7) and southern (lineage 2'; locality numbers 5, 6) phylogroups. The Ina Basin is located in the southern part of Nagano Prefecture along the Tenryu River. Mountain districts are present to the east (including locality numbers 4-6) and west (containing locality number 7) along this basin. The specimen from Shiojidaira is the closest to the holotype from Mt. Mitsugaisan even though the Ina Basin separates the mountainous regions. In addition, the COI divergence between the two specimens from Shiojidaira (KUZ Z696) and Mt. Mitsugaisan (KUZ Z694) was low (0.5%). This may indicate that O. masaakikuroiwai sp. n. leeches in this area have recently dispersed. The same low genetic distance (0.6%) was detected between the specimens collected from the southern part of Nagano Prefecture (KUZ Z689, locality number 5, and KUZ Z697, locality number 6).
Orobdella masaakikuroiwai sp. n. is the second known species in which the body length of a mature individual is less than 4 cm. Orobdella masaakikuroiwai sp. n. is syntopic with O. octonaria in the Izu Peninsula (locality number 2), and the distribution of this new species partly overlaps with that of the latter species (Nakano, unpublished data). In addition, both O. koikei and O. kawakatsuorum are present in Hokkaido (Nakano 2012b). Therefore, a difference in the body size of mature individuals may allow different species of Orobdella to coexist in the same region. The phylogeny indicates that the small size of mature leeches likely evolved in parallel within Orobdella. Orobdella whitmani is the sister species of O. masaakikuroiwai sp. n. and grows to ca. 10 cm. In addition, O. ijimai and O. octonaria are close congeners of O. masaakikuroiwai sp. n. and O. whitmani, and they grow to ca. 10 cm and ca. 20 cm, respectively. Therefore, the intermediate size of mature individuals may be a plesiomorphic characteristic of the clade consisting of these four species. However, several undescribed species of Orobdella are known including small-sized species (Nakano, unpublished observation). Further faunal and systematic studies will help to elucidate the evolutionary and biogeographical history of the predaceous genus Orobdella.