First record of Orobdella kawakatsuorum (Hirudinida: Arhynchobdellida: Erpobdelliformes) from Kunashir Island, Kuril Islands

Abstract Specimens of the genus Orobdella Oka, 1895 from Kunashir Island, the Kuril Islands, are identified as Orobdella kawakatsuorum Richardson, 1975. Mitochondrial tRNALeu and ND1 data confirm the species identification of the Kunashir specimens. This is the first record of the genus Orobdella from the Kuril Islands.


Introduction
The genus Orobdella Oka, 1895 is a terrestrial macrophagous leech taxon that contains 11 described species from Japan, Korea, and Taiwan (Nakano 2012b, Nakano and Lai 2012, Nakano and Seo 2012. Orobdella whitmani Oka, 1895 was reported from Primorsky Krai in the continental part of the Russian Far East (Gilyarov and Perel 1971, Gilyarov et al. 1969, Gongalsky 2007, Lukin 1976. However, the identification of the Russian specimens is doubtful (Nakano 2012a). The northernmost distributional limits of this genus have been reported as Primorsky Krai of the Russian Far East, and the Shiretoko Peninsula of Hokkaido where two species, O. kawakatsuorum Richardson, 1975 andO. koikei Nakano, 2012, have been recorded (Nakano 2012a).
The Kuril Islands are a long archipelago off the coast of the Russian Far East located between the Kamchatka Peninsula and Hokkaido. Members of Orobdella have never been recorded from the Kuril Islands, although the southern part of the islands lies close to Hokkaido. Recently, several Orobdella specimens were collected on Kunashir Island by the second author during a survey of soil fauna on the island. Based on morphological examination of the specimens, the identification and brief description of the Kunashir Orobdella are herein presented. In addition, mitochondrial tRNA and ND1 sequence data of the specimens are presented as confirmation of species identity based on their morphological characteristics.

Materials and methods
Leeches were collected from Kunashir Island in the Kuril Islands (Fig. 1). The specimens were preserved in 95% ethanol in the field. In the laboratory, botryoidal tissue was taken from the posterior part of the body around the caudal sucker of each specimen for DNA extraction, and the rest of the bodies were re-fixed in 10% formalin for dissection and preserved in 70% ethanol. Examination, dissection, and drawings of the specimens were done under a stereoscopic microscope equipped with a drawing tube (Leica M125). Specimens used in this study have been deposited in the Zoological Collection of Kyoto University (KUZ).

Figure 1.
Map showing the Kuril Islands and adjacent areas. Filled circles indicate collection localities of the specimens examined in this study.
The numbering convention is based on the system adopted by Moore (1927): body somites are denoted by Roman numerals, and the annuli in each somite are given an alphanumeric designation.
Sequences of mitochondrial tRNA and ND1 (tRNA -ND1) were determined for 4 specimens of O. kawakatsuorum and O. koikei, and one specimen of O. whitmani Oka, 1895, in addition to the two specimens from Kunashir Island ( Table 1). The extraction of genomic DNA and DNA sequencing methods followed Nakano (2012a). For obtaining sequences of tRNA -ND1, the procedure was modified as follows: the primer set was LND300 and HND1932 (Light and Siddall 1999); the PCR reaction mixture was heated to 94°C for 5 min, followed by 40 cycles at 94°C (10 s), 55°C (20 s), and 72°C (39 s), and a final extension at 72°C for 6 min; the sequencing reaction mixture was incubated at 96°C for 2 min, followed by 40 cycles at 96°C (10 s), 50°C (5 s), and 60°C (39 s The length of the obtained tRNA -ND1 was 629 bp for O. whitmani (KUZ Z45) and 630 bp for the other specimens. These sequences were aligned using MAFFT FFT-NS-2 (Katoh et al. 2005). The length of the aligned sequences was 630 bp. A gap was inserted in the tRNA part of the sequence of O. whitmani. Genetic distances of the obtained sequences were calculated by Kimura 2-parameter (K2P) correction (Kimura 1980), and then a neighbor-joining tree was constructed with nonparametric bootstrapping based on 1000 replicates using MEGA 5 (Tamura et al. 2011

Distribution
Orobdella kawakatsuorum is distributed in Hokkaido, Japan, and its peripheral islands and inhabited in mountainous regions of these islands (Nakano 2012a). The present specimens have extended the known distributional range of the species north to include the southern tip of the Kuril Islands.

Genetic data
The obtained neighbor-joining tree (Fig. 4) showed that two Orobdella specimens from Kunashir Island (KUZ Z675, Z676) formed a monophyletic lineage with the individual of O. kawakatsuorum from Shiretoko, Hokkaido (KUZ Z152). No difference between the tRNA -ND1 sequences from the Kunashir specimens. The K2P distance was detected between these two specimens (KUZ Z675, Z676) and that from Shiretoko (KUZ Z152) was 0.5%.

Taxon discussion
Two specimens of Orobdella from Kunashir Island clearly belong to O. kawakatsuorum based on the following characteristics: male gonopore in the anterior margin of XI b6, female gonopore in the furrow of XIII a1/a2, 6 annuli between gonopores, and Leu Figure 4.
The neighbor-joining tree of 630 bp of tRNA and ND1. The numbers at the nodes represent the bootstrap values. Leu epididymides occupying 2 annuli. According to Nakano (2012a), O. kawakatsuorum grows to ca. 10 cm length. However, the body length of the Kunashir specimens is only ca. 3 cm. Since they have undeveloped male atria and undetectable testisacs, they were considered immature individuals. As noted in the Introduction, two quadrannulate species of Orobdella, O. kawakatsuorum and O. koikei, are distributed in Hokkaido. Orobdella koikei is the closest congener of O. kawakatsuorum according to the recent molecular phylogenetic study and the smallest species among the known species of Orobdella (Nakano 2012a). The body length of the known mature leeches of O. koikei is less than 4 cm. Therefore, based only on their body length, a possibility exists that the Kunashir specimens might be misidentified as O. koikei. However, O. kawakatsuorum, as well as the present specimens, are clearly distinguished from O. koikei in the characteristics mentioned above: the latter possesses 1/2 + 4 + 1/2 annuli between gonopores (male gonopore in the middle of XI b6, female gonopore in the middle of XIII a1), and the epididymides occupy 9-12 annuli. Our tRNA -ND1 data provided additional confirmation that the Orobdella leeches from Kunashir Island were identified correctly as O. kawakatsuorum.
Orobdella kawakatsuorum was collected from Rishirito Island (Nakano 2012a), which is located ca. 20 km away from Hokkaido (Fig. 1). Additionally, Kunashir Island lies offshore of Hokkaido. Thus, anticipating that Orobdella leeches might occur on the island was not difficult. Our findings suggest that Orobdella species may also be present in the southern part of the Kuril Islands, e.g., Iturup Island and Shikotan Island. Further faunal surveys should be conducted not only in the South Kurils, but also in the northern part of the Kuril Islands, as well as the Kamchatka Peninsula, to fully reveal the northern distributional limit of the genus Orobdella.