A new sexannulate species of Orobdella (Hirudinea, Arhynchobdellida, Orobdellidae) from Kii-Oshima Island, Japan

A large-type sexannulate leech species, Orobdella ibukifukuyamai sp. nov. , from Kii-Oshima Island, Japan, is described based on morphologic and molecular data. Phylogenetic analyses using nuclear 18S rRNA, 28S rRNA, histone H3, mitochondrial cytochrome c oxidase subunit I, tRNA Cys , tRNA Met , 12S rRNA, tRNA Val , 16S rRNA, tRNA Leu and NADH dehydrogenase subunit 1 markers showed that O. ibukifukuyamai formed a clade with the sexannulate O. okanoi Nakano, 2016 and O. yamaneae Nakano, 2016 and octannulate O. nakahamai Nakano, 2016, which are endemic to Shikoku island, Japan.


Introduction
Leeches that belong to the gastroporous genus Orobdella Oka, 1895 are indigenous to terrestrial habitats in Far East Asia (Sawyer 1986;Nakano 2017a), and are classified into 22 species (Nakano 2021). The species are grouped into three morphotypes by the mid-body somite annulation, i.e., 4-(quadr-), 6-(sex-), and 8-(oct-) annulate, and can also be categorized into three morphotypes by the body lengths of mature individuals, i.e., small (~5 cm), middle (~10 cm), and large (~20 cm) types (Nakano 2017a). All of the six sexannulate and two octannulate Orobdella leeches are denoted as the middle-or large-type species, and endemic only to the Japanese Archipelago, including the Ryukyu Islands (Nakano 2017a). Previous molecular phylogenetic analyses revealed that sexannulation and octannulation, respectively, have evolved in parallel within the genus Orobdella (Nakano 2016).
Unidentified large-type sexannulate individuals of Orobdella were collected from Kii-Oshima Island, which is located near the southern tip of the Kii Peninsula of Honshu island, Japan, and they are herein de-scribed as a new species. In addition, its phylogenetic position is investigated using nuclear and mitochondrial genetic markers.

Materials and methods
Samples and morphological examination Two leeches were collected from a small scree slope on Kii-Oshima Island, Japan. Specimens were relaxed by the gradual addition of absolute ethanol (EtOH) to fresh water, and then fixed in absolute EtOH. For DNA extraction, botryoidal tissue was removed from the posterior part around the caudal sucker of each 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 from the anterior margin of the oral sucker to the posterior margin of the caudal sucker, maximum body width, caudal sucker length from the anterior to the posterior margin of the sucker, and caudal sucker width from the right to the left margin of the sucker. Examination, dissection, and draw-ing of the specimens were conducted using a stereoscopic microscope with a drawing tube (M125, Leica Microsystems, Wetzlar, Germany). Specimens examined 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. The following abbreviations are using for morphological characters in the text and figures: ac atrial cornu; af annular furrow; an anus; at atrium; BL body length; BW body width; CL caudal sucker length; cod common oviduct; cp crop; CW caudal sucker width; ed ejaculatory duct; ep epididymis; fg female gonopore; gd gastroporal duct; gp gastropore; mg male gonopore; np nephridiopore; od oviduct; ov ovisac; ph pharynx; ts testisac.

Molecular analyses
The phylogenetic position of the new Orobdella species within the genus was investigated based on three nuclear and three mitochondrial markers, i.e., 18S rRNA, 28S rRNA, histone H3, cytochrome c oxidase subunit I (COI), tRNA Cys , tRNA Met , 12S rRNA, tRNA Val , and 16S rRNA (tRNA Cys -16S) and tRNA Leu and NADH dehydrogenase subunit 1 (tRNA Leu -ND1). Methods for the genomic DNA extraction, polymerase chain reactions and cycle sequencing reactions were elucidated in Lai 2016, 2017). In total, nine sequences were newly obtained and deposited with the International Nucleotide Sequence Databases (INSD) through the DNA Data Bank of Japan (Suppl. material 1: Table S1).
In addition to the newly obtained sequences, 181 sequences of 22 Orobdella species and eight erpobdelliform taxonomic units, which were selected as the outgroup, were obtained from the INSD in accordance with the previous studies (Nakano 2021), and were included in the present dataset (Suppl. material 1: Table  S1). The alignments of H3 and COI were trivial, as no indels were observed. The sequences of the other markers were aligned using MAFFT v. 7.471 L-INS-i (Katoh and Standley 2013). The lengths of the 18S, 28S, H3, COI, tRNA Cys -16S, and tRNA Leu -ND1 were 1850, 2823, 328, 1267, 1182, 640 bp, respectively. The concatenated sequences thus yielded 8090 bp of aligned positions.
Phylogenetic trees were reconstructed using maximum likelihood (ML) and Bayesian inference (BI). The best-fit partition scheme and models were identified based on the Bayesian information criterion using PartitionFinder v. 2.1.1 (Lanfear et al. 2017) with the 'greedy' algorithm (Lanfear et al. 2012). The selected partition scheme and models were as follows: for 18S and H3 1 st position, K80+I+G; for 28S, GTR+I+G; for H3 2 nd position, JC+I; for H3 3 rd position, GTR+G; for COI 1 st position, GTR+G; for 2 nd positions of COI and ND1, GTR+I+G for BI; for 3 rd positions of COI and ND1, KHY+G; for tR-NA Met and ND1 1 st position, GTR+I+G; for tRNA Cys , 12S, tRNA Val and tRNA Leu , GTR+I+G; and for 16S, GTR+G. The ML tree was calculated using IQ-Tree v. 2.1.3 (Minh et al. 2020) with non-parametric bootstrapping (BS) conducted with 1000 replicates. BI tree and Bayesian posterior probabilities (PP) were estimated using MrBayes v. 3.2.7a (Ronquist et al. 2012). Two independent runs for four Markov chains were conducted from 40 million generations, and the tree was sampled every 100 generations. The parameter estimates and convergence were checked using Tracer v. 1.7.1 (Rambaut et al. 2018), and then first 100 001 trees were discarded based on the results.
Pairwise comparisons of uncorrected p-distances for COI (1267 bp) sequences of the newly identified Orobdella and its close congeners were calculated using MEGAX (Kumar et al. 2018).
Coloration (based on the holotype KUZ Z4031 and paratype KUZ Z4032). In life, dorsal surface reddish brown (Fig. 1C); lateral surface ocher; ventral surface whitish yellow. Color faded in preservative; dark mid-dorsal line present from somite VII b6 to somite XXVII only in paratype.
Etymology. The specific name is dedicated to Mr Ibuki Fukuyama who collected the specimens of the new species.
Distribution. This species was collected only from the type locality.
Remarks. The morphological features (see Nakano 2016) as well as molecular phylogenetic position of O. ibukifukuyamai clearly elucidated that this new species belongs to the genus Orobdella. Only two specimens of the new species could be examined in this study, but nonetheless, their morphological characteristics and DNA sequences successfully ensured the distinct species status of O. ibukifukuyamai within Orobdella.