A new species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Taipei, Taiwan

Abstract A new quadrannulate species of Orobdella, Orobdella ketagalan sp. n., from Taipei, Taiwan, is described. This is the first record of Orobdella and the family Orobdellidae from Taiwan. This new species possesses small, paired sperm duct bulbs in the male reproductive system. In addition to these bulbs, the following combination of characters distinguishes this new species from other quadrannulate species: somite IV uniannulate, male gonopore at XI b6, female gonopore at XIII a1, 1/2 + 4 + 1/2 between gonopores, simple tubular gastroporal duct, lacking epididymides, and undeveloped atrial cornua. Phylogenetic analyses using nuclear 18S rDNA and histone H3 as well as mitochondrial COI, 12S rDNA, tRNAVal, and 16S rDNA markers showed that Orobdella ketagalan is related to the two Ryukyu Archipelago species Orobdella dolichopharynx Nakano, 2011 and Orobdella shimadae Nakano, 2011.


introduction
Species of the genus Orobdella Oka, 1895 are large annelids that feed on earthworms. They are usually 10-20 cm in length (except for O. koikei Nakano, 2012, approx. 5 cm) and they inhabit the banks of mountain streams in East Asia (Nakano 2012a, Oka 1895. The systematic position of the genus Orobdella has been contentious. Orobdella was initially included in the family Gastrostomobdellidae along with the Southeast Asian terrestrial macrophagous leech genus Gastrostomobdella Moore, 1929(Richardson 1971, Sawyer 1986. Although Sawyer (1986) placed Gastrostomobdellidae under Hirudiniformes, recent molecular phylogenetic studies reclassified the family under Erpobdelliformes (Nakano et al. 2012, Oceguera-Figueroa et al. 2011. Nakano et al. (2012) split Gastrostomobdellidae into two families based on phylogenetic analyses as well as morphological discontinuity, and the monotypic family Orobdellidae was erected for Orobdella.
Taxonomic and inventory studies on Orobdella have progressed recently, and this genus now includes ten species (Nakano 2010, 2011a, b, 2012a, b, in press, Oka 1895, Richardson 1975. Orobdella leeches exhibit various types of mid-body somite annulation; three types have been documented thus far: quadrannulate, sexannulate, and octannulate (Sawyer 1986). The quadrannulate mid-body somite is a plesiomorphy of this genus, and the sexannulate form is considered to have evolved in parallel (Nakano 2012a, b, Nakano et al. 2012. All of the known Orobdella species have been described based on specimens collected from Japan, and eight of the ten species have been reported only from Japanese islands (Sawyer 1986). Outside Japan, O. whitmani Oka, 1895, which is the type species of the genus, has been recorded from Primorsky Krai, Russia (Gilyarov et al. 1969). However, Nakano (2012a) noted that this specimen from Russia was misidentified as O. whitmani, and that it should be considered a new, undescribed species. In addition, O. tsushimensis Nakano, 2011 was recently collected from Gageodo Island, Korea (Nakano and Seo in press). In Taiwan, no studies have investigated the species diversity of terrestrial macrophagous leeches. Taiwanese leech species were catalogued by Lai and Chen (2010), but Orobdella leeches were not included. Recently, quadrannulate Orobdella specimens were collected from Taipei, Taiwan. These materials clearly differ from the other known quadrannulate Orobdella species. Therefore, in the present study, Orobdella leeches from Taipei are described as a new species. This is the first record of orobdellid leeches from Taiwan. In addition, their phylogenetic position is estimated using nuclear 18S and histone H3 (H3) and mitochondrial COI, 12S, tRNA Val , and 16S rDNA (12S-16S) sequence data.

Materials and methods
Leeches were collected from Taipei, Taiwan (Fig. 1). Botryoidal tissue was taken from specimens, which were fixed in ethanol, for DNA extraction. All of the specimens were preserved in 70% ethanol. Two measurements were taken: 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). The specimens have been deposited in the Zoological Collection of Kyoto University (KUZ).
We used the numbering convention of Moore (1927): body somites are denoted by Roman numerals, and annuli in each somite are given alphanumeric designations.
The extraction of genomic DNA followed (Nakano 2012a). The 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) were used; for H3, H3aF and H3bR (Colgan et al. 1998); for COI, LCO 1490 and HCO 2198 (Folmer et al. 1994), and LCO-in and HCO-out (Nakano 2012a); for 12S-16S, 12SA-in and 12SB-out (Nakano 2012a). The DNA sequencing methods for the above four markers followed Nakano (2012a). The following DNA sequences were newly obtained and deposited in GenBank (Table 2): 18S and H3 sequences from the holotype (KUZ Z208) of the new species, and COI and 12S-16S sequences from the holotype (KUZ Z208) and three paratypes (KUZ Z209-Z211) of the new species. The DNA sequences of the holotype (KUZ Z208) were analyzed in the following phylogenetic analyses. The other sequences were taken from GenBank (Table 2). For the outgroup, three Erpobdelliformes leeches were included in the phylogenetic analyses: Erpobdella japonica Pawłowski, 1962 (Erpobdellidae), Gastrostomobdella monticola Moore, 1929 (Gastrostomobdellidae), and Mimobdella japonica Blanchard, 1897 (Salifidae).

Gene
Primer name Reaction estimates and convergence were checked using Tracer v 1.5 (Rambaut and Drummond 2009), and based on the results the first 50,001 trees were discarded. Nodes with bootstrap (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). Diagnosis. Somite IV uniannulate, somites VIII-XXV quadrannulate. Pharynx reaching to posterior of XIV to anterior of XV. Gastropore conspicuous at XIII a1. Gas-

Taxonomy
troporal duct simple, tubular. Male gonopore at XI b6, female gonopore at XIII a1, gonopores separated by 1/2 + 4 + 1/2 annuli. Small paired sperm duct bulbs in XV. Epididymis absent. Atrial cornua, coniform, undeveloped. Etymology. The specific name is taken from the native Taiwanese tribe Ketagalan. The type locality of this new species is in an area settled by this aboriginal tribe. The specific name is a native word, not a Latin or Latinized word.
Distribution. Known from Yangmingshan National Park and adjacent areas in northern Taipei City, Taiwan (Fig. 1).

Discussion
The phylogenies obtained in this study are nearly identical to those obtained in other phylogenetic analyses of the genus Orobdella (Nakano 2012a, b, Nakano et al. 2012. The most ancestral clade of Orobdella (clade A in Fig. 5) is distributed in Hokkaido, Japan. The other species inhabit islands south of Hokkaido (clade B in Fig. 5) and are divided into three subclades (B1-3 in Fig. 5). In our analyses, however, the phylogenetic relationships of these subclades were not sufficiently resolved. Our phylogenetic trees clearly indicated that the quadrannulate mid-body somite annulation is a plesiomorphic character of Orobdella, and that sexannulate mid-body somites had evolved in parallel. This result was also mentioned in previous studies (Nakano 2012a, b, Nakano et al. 2012. Even in subclade B2, the sexannulate character was considered to have evolved in parallel. In this subclade, three sexannulate species from the Ryukyu Archipelago were included: O. mononoke is from Yakushima Island, which is located in the northern part of the Ryukyu Archipelago; O. dolichopharynx is from Amamioshima Island, which is located in the middle region of the Archipelago; and O. shimadae is from Okinawajima Island, which is also located in the middle region of the Archipelago, but south of Amamioshima Island. Our analyses showed that these three sexannulate species did not form a monophyletic clade. In contrast, two sexannulate species, O. dolichopharynx and O. shimadae, and the Taiwanese quadrannulate O. ketagalan formed a monophyletic clade. The other sexannulate species, O. mononoke, was not closely related to O. dolichopharynx and O. shimadae. This is in agreement with findings by (Nakano 2012b), who mentioned that O. mononoke was probably not very close to those two species. Our phylogenetic analyses supported his phylogenetic conclusion. According to the topologies of the ML and BI trees, O. mononoke is a sister taxon of a clade including O. ketagalan, O. dolichopharynx, and O. shimadae, but this phylogenetic position was not well resolved in either tree. To better understand the biogeographical history of Orobdella leeches, more robust trees for this genus based on either more DNA markers or specimens should be obtained.
Orobdella ketagalan possesses small, paired sperm duct bulbs in XV (Fig. 4A, B). Such small bulbs have never before been reported in Orobdella. Hence, small sperm duct bulbs could be considered an apomorphy of the Taiwanese O. ketagalan. Orobdella species generally possess eipididymides in their male reproductive systems (Nakano 2010(Nakano , 2011a(Nakano , b, 2012a (Nakano 2011b). These three species formed a monophyletic clade in our phylogenetic analyses (Fig. 5). Therefore, lacking epididymides could be considered a synapomorphy within O. ketagalan, O. dolichopharynx, and O. shimadae. Orobdella ketagalan also possesses a simple, tubular gastroporal duct, which is similar to that of O. kawakatsuorum (Nakano 2012a, Richardson 1975. This morphological similarity is clearly due to convergence, according to our phylogenetic analyses. This is the first record of the genus Orobdella from Taiwan. Moreover, we collected several other specimens that appear to be undescribed species of Orobdella (Nakano and Lai, unpublished observation). Further faunal and systematic studies will reveal the species diversity of Taiwanese Orobdella and further elucidate the biogeographical and evolutionary history of these macrophagous leeches.  Richardson, 1975