Four new species of the genus Orobdella from Shikoku and Awajishima island , Japan ( Hirudinida , Arhynchobdellida , Orobdellidae )

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.


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

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.
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).

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 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 12SB-out (PCR and CS) (Nakano 2012b); for tRNA Leu and NADH dehydrogenase subunit 1 (ND1) (tRNA Leu -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 tRNA Cys -16S, 48°C for COI or 42°C for tRNA Leu -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.
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/tRNA Cys , tRNA Met , tR-NA 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 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, tR-NA Cys , tRNA Met , tRNA Val and tRNA Leu , 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 andOrobdella naraharaetmagarum Nakano, 2016 were calculated using MEGA6.06(Tamura et al. 2013) in accordance with the results of the molecular phylogenetic analyses.
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).
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.
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).
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.
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).
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.
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.Oli-gochaete 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.
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.
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.
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).
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.
Except for the holotype, all dissected individuals possess the following characteristics of the male genital or-gan: 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.

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, midbody 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 (2014Nakano ( , 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.

Figure 1 .
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.

Figure 4 .
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.

Figure 8 .
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.

Figure 12 .
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.

Figure 16 .
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.

Figure 19 .
Figure 19.Bayesian inference tree for 5,209 bp of nuclear 18S rRNA and histone H3 and mitochondrial COI, tRNA Cys , tRNA Met , 12S rRNA, tRNA Val , 16S rRNA, tRNA Leu 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.

Table 5 .
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).Yoshiko Yamane (KU) for providing specimens of the new species, to Professor Hidetoshi Nagamasu (The Kyoto University Museum) 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.