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

Abstract A new quadrannulate species of Orobdella Oka, 1895, Orobdella naraharaetmagarum sp. n., from the mountainous region of western Honshu, Japan is described. Orobdella naraharaetmagarum is a small species with a body length of less than 5 cm. 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 the present new species is the sister species of the quadrannulate Orobdella esulcata Nakano, 2010. Furthermore, mitochondrial DNA genealogy within Orobdella naraharaetmagarum demonstrated that this new species is divided into eastern and western lineages.


Introduction
The terrestrial macrophagous leech genus Orobdella Oka, 1895 contains 12 species that are distributed throughout the Japanese Archipelago, Korean Peninsula, and Taiwan (Nakano 2014, Nakano and Lai 2012, Nakano and Seo 2014. These 12 species are split into three groups based on their mid-body somite annulation: seven species in the quadrannulate (four annuli) group, four in the sexannulate (six annuli) group, and one octannulate (eight annuli) species.
Orobdella leeches had been considered large species, with body lengths reaching to 10 cm or greater (Sawyer 1986). In recent years, however, small mature leeches belonging to this genus have been discovered in Japan and described as new species: Orobdella koikei Nakano, 2012b from Hokkaido, and Orobdella masaakikuroiwai Nakano, 2014 from central Honshu. The bodies of mature individuals of these two species are shorter than 4 cm. Both species possess mid-body somites that are quadrannulate. Nakano (2014) suggested that differences in the body lengths of mature leeches might enable more than one species of Orobdella to coexist in the same region.
Additional small Orobdella leeches were collected from Chugoku District, western Honshu, Japan. The body lengths of the specimens were less than 5 cm. Nevertheless, a few individuals were regarded as mature leeches because they possessed an obvious clitellum. These specimens are described here as a new species. In addition, the phylogenetic position of this new species was estimated using nuclear 18S rRNA and histone H3, as well as mitochondrial cytochrome c oxidase subunit I, tRNA Cys , tRNA Met , 12S rRNA, tRNA Val , 16S rRNA, tRNA Leu , and NADH dehydrogenase subunit 1 sequence data.

Sampling and morphological examination
Leeches were collected from five localities in Chugoku district, western Honshu, Japan (Fig. 1). When possible, elevation and geographical coordinates for localities were obtained using a Garmin eTrex® GPS unit.
Almost all of the specimens were relaxed by the gradual addition of absolute ethanol (EtOH) 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 EtOH. The remainder of the body was fixed in 10% formalin and preserved in 70% EtOH. 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.

PCR and DNA sequencing
The extraction of genomic DNA from botryoidal tissues preserved in absolute EtOH 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), LCO 1490 and HCO 2198 (PCR and CS) (Folmer et al. 1994), and LCO-in (Nakano 2012b) and HCO-outout (Nakano 2012a) (PCR and CS), or LCO-inerpo2 (5'-GCTATTACAATATTACTTACAGATCG-3'; this study) and HCO-out (Nakano 2012b) (PCR and CS); for tRNA Cys , tRNA Met , 12S rRNA, tRNA Val and 16S rRNA (tRNA Cys -16S), 12SA-out and 12SB-in (PCR and CS), and 12SA-in and 12SBout (Nakano 2012b) or 12SB-outin (5'-AAAGGTACGAATATATTTAC-3'; this study) (PCR and CS); for tRNA Leu and NADH dehydrogenase subunit 1 (ND1) (tRNA Leu -  (5) indicates the type locality of the new species, Orobdella naraharaetmagarum sp. n., and closed circles (1-4) indicate additional localities. ND1), LDN3000 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 a GeneAmp PCR System 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, 60 °C, and 44 °C, respectively, for the anterior, and posterior parts of tRNA Cys -16S or 42 °C for COI and tRNA Leu -ND1 (20 s), and 72 °C (42 s each), 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 (48 s each). The obtained sequences were edited using DNA BAS-ER (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).
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 1 st and 2 nd positions of H3/the 3 rd position of H3/the 1 st position of COI/the 2 nd position of COI/the 3 rd positions of COI and ND1/the 1 st position of ND1/the 2 nd position of ND2/12S/16S/tRNA Cys , tRNA Met , tRNA Val and tRNA Leu .
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 1 st position of H3, K80+I; for the 2 nd position of H3, JC69; for the 3 rd position of H3, HKY85; for the  (Oka, 1910) KUZ Z180 AB663651 AB938012 AB938004 AB937995 AB938014 1 st position of COI, GTR+G; for the 2 nd positions of COI and ND1, HKY85+I; for the 3 rd positions of COI and ND1 plus 16S, HKY85+I+G; and for the 1 st position of ND1, and 12S, tRNA Cys , tRNA Met , tRNA Val and tRNA Leu , GTR+I+G. Two independent runs of four Markov chains were conducted for 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 30,001 trees were discarded based on these results.
The phylogenetic relationships within the available Orobdella materials were estimated based on sequences of COI, tRNA Cys -16S and tRNA Leu -ND1. tRNA Cys -16S and tRNA Leu -ND1 were aligned using MAFFT L-INS-i. The lengths of the COI, tRNA Cys -16S, and tRNA Leu -ND1 sequences were 1,267, 634, and 1,107 bp, respectively. The concatenated sequences yielded 3,008 bp of aligned positions. ML phylogenies were constructed in RAxML with the substitution model set as GTRCAT, immediately after nonparametric bootstrapping conducted with 1,000 replicates. The best-fit partitioning scheme was identified with the Akaike information criterion using PartitionFinder with the "greedy" algorithm: the 1 st position of COI/the 2 nd positions of COI and ND1/the 3 rd positions of COI and ND1/the 2 nd position of ND1/the 1 st position of ND1/tRNA-Cys , tRNA Met , tRNA Val , tRNA Leu /12S/16S. BI and BPPs were estimated using MrBayes. The best-fit partition scheme and models for each partition were selected based on the Bayesian information criterion using PartitionFinder with the "greedy" algorithm: for the 1 st positions of COI and ND1, GTR+I+G; for the 2 nd positions of COI and ND1, F81+I; for the 3 rd positions of COI and ND1 plus 16S, HKY+G; tRNA Cys , tRNA Met , 12S, tRNA Val and tRNA Leu , GTR+I+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 Traced, and the first 25,001 trees were discarded based on these results.
Nodes with bootstrap support (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).
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). Papillae numerous, minute, hardly visible, one row on every annulus.
Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of a1 of each somite in VIII-XXIV (Fig. 3B, D, E).
Distribution. This species was primarily collected in Hiroshima Prefecture in Chugoku District, Honshu, Japan, and in Tottori Prefecture. The lowest elevation among the localities was 470 m, and the highest was 1010 m. The locality data for this species suggested that it is distributed in mountainous regions in Chugoku District, Honshu, Japan.
Natural history. This species was generally found curled up under rocks or rotten trees in moist mountainous habitats (Fig. 5 C, D). Oligochaete worms were sometimes observed in the digestive tract during specimen dissection. Therefore, this species is an earthworm-eater, as are the other known Orobdella leeches.
A mature leech with an obvious clitellum was collected on 16 June. Moreover, the holotype, which appeared to have a clitellum (Fig. 5A), was collected on 25 April. These findings suggest that the reproductive season of the new species begins in May and then continues at least to mid-to-late June.
Remarks. The new species unambiguously belongs to Orobdella as it has all the generic diagnostic characteristics defined in : 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.
The specimens were small (up to 48 mm). However, one leech (KUZ Z1654) was determined to be mature because it possessed an obvious clitellum. The holotype, which had a body length of 40 mm, also possessed a slightly developed clitellum and developed testisacs. Two specimens (KUZ Z1360, Z1582) possessed a tubular gastroporal duct. This tubular gastroporal duct was thought to be observed in immature individuals because these two specimens had undeveloped, undetectable testisacs.

Discussion
The obtained molecular phylogenies showed that the present specimens formed a wellsupported clade. In addition, the mean value of the COI uncorrected p-distance among the individuals was 4.4%. This value indicated a clear gap between the present speci- mens and the closest congener, Orobdella esulcata. Therefore, all of the specimens examined can be considered to belong to the same species, Orobdella naraharaetmagarum. Although the precise phylogenetic position of O. tsushimensis from the Korean Peninsula and the adjacent islets could not be determined in the obtained phylogenies (see Fig. 6 naraharaetmagarum was a member of the last clade, and formed a monophyly with O. esulcata with strong support. Therefore, the range of the western lineage group covers the area from Chugoku District, at the western tip of Honshu, to Taiwan. As indicated in Figure 7, Orobdella naraharaetmagarum was divided into eastern (lineage 1; locality numbers 1-3) and western (lineage 2; locality numbers 4, 5) phylogroups. The COI uncorrected p-distances within lineage 1 were higher than those within lineage 2. The calculated genetic divergences between the three specimens collected from Mt. Kanmuriyama (locality number 5; KUZ Z1582, Z1652, and Z1654) and one individual, KUZ Z1360, from Kitahiroshima (locality number 4) was 1.1-1.4%. The geographic distance between these two collection localities is ca. 28 km. In comparison, the genetic distance between KUZ Z1380 collected on Mt. Azumayama (locality number 2) and KUZ Z1655 from Mt. Izaiyama (locality number 3) was greater than this value (3.1%), although these two localities are separated only by ca. 7 km. These phylogenetic relationships and genetic divergences implied that leeches belonging to lineage 2 dispersed more recently and rapidly than those of lineage 1. Such discordance between the COI genetic divergences and geographical distances was also seen in the small species O. masaakikuroiwai (Nakano 2014). Further molecular phylogenetic studies will help to reveal the biogeographical history of the Orobdella leeches.
The phylogenetic position of O. naraharaetmagarum also indicated that the small size of the mature leeches evolved in parallel within Orobdella, as mentioned in Nakano (2014). According to the obtained molecular phylogenies and the phylogenetic trees from studies (Nakano 2012b(Nakano , 2014, each of the three small species, O. koikei, O. masaakikuroiwai, and O. naraharaetmagarum may have diverged from a single large quadrannulate species. As with the other two small species (Nakano 2012b(Nakano , 2014, O. naraharaetmagarum is also distributed sympatrically with undescribed large quadrannulate species in Chugoku District (Nakano, unpublished data). Therefore, further systematic studies should be carried out to reveal the species diversity and evolutionary history of the genus Orobdella.