A new sexannulate species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Yakushima Island, Japan

Abstract A new sexannulate species of the genus Orobdella Oka, 1895, Orobdella mononoke sp. n., is described on the basis of five specimens collected from Yakushima Island, Japan. Orobdella mononoke sp. n. differs from other sexannulate Orobdella species in its possessing the following combination of characters: dorsal surface bicolor in life, I–XIII, XXVII and caudal sucker grayish purple, XIV–XXVI amber, male gonopore at XI c11/c12, female gonopore at XIII b2, 8 + 1/2 between gonopores, tubular but bulbous at junction with crop gastroporal duct, epididymides in XV–XIX, and atrial cornua ovate. Phylogenetic analyses using nuclear 18S rDNA and histone H3, and mitochondrial COI, tRNACys, tRNAMet, 12S rDNA, tRNAVal and 16S rDNA markers show that Orobdella mononoke sp. n. is closely related to Orobdella esulcata Nakano, 2010 from Kyushu, Japan, and two species, Orobdella dolichopharynx Nakano, 2011 and Orobdella shimadae Nakano, 2011, from the Ryukyu Archipelago, Japan.

The nine Orobdella species are split into three groups based on their mid-body somite annulation (Nakano 2012: 1) the quadrannulate group containing five species; 2) the sexannulate containing three species; and 3) one octannulate species. Among these groups, the sexannulate Orobdella species consist of O. ijimai Oka, 1895 from Honshu, Japan, and two species, O. dolichopharynx O. shimadae Nakano, 2011, from the Ryukyu Archipelago, Japan. Recently, sexannulate Orobdella specimens were collected from Yakushima Island. These specimens are clearly distinguishable from the other three sexannulate species. Orobdella leeches from Yakushima Island are thus described as a new species herein. In addition, its phylogenetic position is estimated using nuclear 18S rDNA and histone H3, and mitochondrial COI and tRNA Cys , tRNA Met , 12S rDNA, tRNA Val and 16S rDNA (tRNA Cys -16S) sequence data.

Material and methods
Leeches were collected from Yakushima Island, Japan (Fig. 1), under rocks along mountain or forest trails. Altitude and coordinates for localities were obtained using a Garmin eTrex GPS unit.
Botryoidal tissue was taken from every specimen for DNA extraction, and the rest of the bodies were fixed in 10% formalin and preserved in 70% ethanol. Two measurements were made: body length (BL) from the anterior margin of the oral sucker to the posterior margin of the caudal sucker, and maximum body width (BW). Examination, dissection, and drawings of the specimens were accomplished under 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 annuli in each somite are given alphanumeric designations.

PCR and DNA sequencing
The extraction of genomic DNA followed Nakano (2012). Primer sets used in this study are listed in Table 1: for 18S, A and L, C and Y, and O and B (Apakupakul et al. 1999); for histone H3, H3aF and H3bR (Colgan et al. 1998); for COI, LCO1490 and HCO2198 (Folmer et al. 1994), and LCO-in (Nakano 2012) and HCO-outout; for tRNA Cys , tRNA Met , 12S, tRNA Val and 16S (tRNA Cys -16S), 12SA-out and 12SB-in, and 12SA-in and 12SB-out (Nakano 2012). All amplification reactions were performed using a GeneAmp PCR System 2700 (Applied Biosystems) or a MyCycler (Bi-Rad Laboratories) using an Ex Taq Polymerase Kit (Takara Bio Inc.). Only for primer set O and B of 18S, 10% DMSO was included in mixtures. Reaction mixtures were heated to 94°C for 5 min, followed by 35 cycles of 94°C (10 s), 42.5°C for 18S, COI and tRNA Cys -16S or 53°C for histone H3 (20 s), and 72°C (42 s for 18S, 21 s for histone H3, 1 min 13 s for COI, and 1 min for tRNA Cys -16S) and a final extension at 72°C for 6 min. The amplified DNA fragments were purified using polyethylene glycol (20% PEG 6000) precipitation. All samples were sequenced in both directions. Sequencing reactions were performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Each sequencing reaction mixture was incubated at 96°C for 2 min, followed by 40 cycles of 96°C (10 s), 50°C (5 s), and 60°C (42 s for 18S, 21 s for Histone H3, 45 s for COI, and 40 s for tRNA Cys -16S). The products were collected by ethanol precipitation and sequenced on an ABI 3130xl Genetic Analyzer (Applied Biosystems). The obtained sequences were edited using DNA BASER (Heracle Biosoft S.R.L.). In this study, the following DNA sequences were newly obtained and deposited in GenBank (  Oka, 1895; 2) histone H3 sequences from ten Orobdella species, Erpobdella japonica Pawłowski, 1962 (Erpobdellidae), Gastrostomobdella monticola Moore, 1929 (Gastrostomobdellidae) and Mimobdella japonica Blanchard, 1897 (Salifidae); 3) COI and tRNA Cys -16S sequences from the holotype (KUZ Z224) and two of the paratypes (KUZ Z221, 223) of the new species. Among the new species, DNA sequences of the holotype (KUZ Z224) were analyzed in the present study. The other DNA sequences were taken from GenBank (Table 2).

Phylogenetic analyses
Histone H3 and COI sequences were aligned by eye since there were no indels. Nuclear 18S and mitochondrial tRNA Cys -16S sequences were aligned using MAFFT X-INS-i (Hofacker et al. 2002;Katoh and Toh 2008;McCaskill 1990;Tabei et al. 2008) taking into account RNA secondary structure information, and then refined with GBLOCKS (Castresana 2000). The length of aligned sequences of 18S was 1787 bp, that of histone H3 was 327 bp, that of COI was 1266 bp, and that of tRNA Cys -16S was 787 bp. The concatenated sequences thus yielded a total of 4167 bp positions. Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI). 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 non-parametric bootstrapping (Felsenstein 1985) was conducted with 500 replicates. The best-fit models for each partition were selected using the Akaike Information Criterion (Akaike 1974) (Altekar et al. 2004;Huelsenbeck et al. 2001;Ronquist and Huelsenbeck 2003). The best-fit models for each partition were identified using the Bayesian Information Criterion (Schwarz 1978) also by using KAKUSAN4: for 18S, the Kimura 1980 model (K80)+I; for histone H3 1st and 2nd position, JC69; for histone H3 3rd position, the Hasegawa-Kishino-Yano model (HKY85)+G; for COI 1st position, GTR+I; for COI 2nd position, the Felsenstein 1981 model (F81)+I; for COI 3rd position, HKY85+G; and for tRNA Cys -16S, GTR+G. Two independent runs for four Markov chains were conducted for 7 million generations and the tree was sampled every 100 generations. Based on checking the parameter estimates and convergence using TRACER v 1.5 (Rambaut and Drummond 2009), the first 15,001 trees were discarded.
The nodes with bootstrap value (BS) higher than 70% were regarded as sufficiently resolved (Hillis and Bull 1993). Nodes with BPP higher than 95% were considered statistically significant (Leaché and Reeder 2002). Diagnosis. In life, dorsal surface of somites I-XIII, XXVII and caudal sucker grayish purple and of somites XIV-XXVI amber, ventral surface grayish white. Somite VI quadrannulate on dorsal, b1 = b2 < a2 = a3, and triannulate on venter, a1 = a2 = a3. Somite VII quadrannulate, somites VIII-XXV sexannulate, somite XXVI quinquannulate. Pharynx reaching to XIV. Gastropore conspicucous at XIII b2 (slightly anterior to middle of annulus). Gastroporal duct, winding at junction with gastropore, tubular but slightly bulbous at junction with crop. Male gonopore at XI c11/c12, female gonopore at XIII b2, behind gastropore, gonopores separated by 8 + 1/2 annuli. Paired epididymides in XV-XIX (approximately four somites Etymology. The specific name is from the Japanese animation movie title 'Mononokehime (Princess Mononoke)'. The type locality of this new species is the origin of an epic forest in that movie. The specific name is a Japanese word, not a Latin or latinized word.
Eyes three pairs, first pair dorsally on posterior margin of II (Fig. 4A), second pair dorsolaterally on middle of V (a1 + a2). Nephridiopores in 17 pairs, ventrally at posterior margin of a1 of each somite of VIII-XXIV (Fig. 4B, E). Papillae numerous, minute, hardly visible, one row on every annulus.
Orobdella mononoke sp. n. inhabits Yakushima Island, which is located in the northern part of the Ryukyu Archipelago (Fig. 1). In the Ryukyu Archipelago, two sexannulate Orobdella species have been described: 1) O. dolichopharynx from Amamioshima Island; and 2) O. shimadae from Okinawajima Island. These two species have the following characteristics in common: 1) long pharynx, reaching to somite XVI; 2) rudimentary gastroporal duct and absence of gastropore; 3) absence of epididymides; and 4) absence of male atrial cornua. Although Orobdella mononoke sp. n. is a sexannulate species, this species does not share such morphological characteristics. Orobdella mononoke sp. n. possesses 1) normal length pharynx for the genus Orobdella, 2) developed gastroporal duct and conspicuous gastropore, 3) epididymides in XV-XIX, 4) ovate atrial cornua. Molecular phylogenetic analyses in this study also could not show   (Toda et al. 2003). In the case of leeches, Haemadipsa japonica Whitman, 1886, which inhabits Honshu, Shikoku and Kyushu, Japan, is distributed in Yakushima Island (Itoh 2003). In islands of the Ryukyu Archipelago south of Yakushima Island, however, another species, Haemadipsa rjukjuana Oka, 1910, is distributed (Lai et al. 2011). A recent molecular phylogenetic study revealed that H. japonica and H. rjukjuana are not closely related species (Borda and Siddall 2011). These facts are collateral evidence that O. mononoke sp. n. is not very closely related to O. dolichopharynx and O. shimadae. Whether or not this is true, additional inventory surveys and molecular phylogenetic studies are needed to reveal the phylogenetic relationships within and the biogeographical history of the genus Orobdella.