A new species of buffalo leech in the genus Hirudinaria Whitman, 1886 (Arhynchobdellida, Hirudinidae) from Thailand

Abstract Hirudinaria manillensis (Lesson, 1842), commonly known as the buffalo leech, shows a polymorphism of two ventral colorations. The green color morph has a plain green ventral surface and the red color morph has a brick-red ventral surface with two black submarginal stripes. Based on molecular and morphological evidence in the present study, these two color morphs were revealed as two different species. The red color morph fits well with the description of H. manillensis, while the green color morph showed some distinctions, and therefore is described herein as Hirudinaria thailandica Jeratthitikul & Panha, sp. nov. The new species can be distinguished from its congeners by the dark greenish or dark olive ventral surface and a round atrium with ventral insertion of ejaculatory ducts in the male reproductive organ. A phylogenetic tree based on concatenated data of COI and 28S genes supported the new species and further indicated it as a sister species to H. bpling Phillips, 2012.

introduction "Buffalo leech" is a common name of obligatory blood-feeding ectoparasitic leeches in the genus Hirudinaria Whitman, 1886 and Poecilobdella Blanchard, 1893. Both genera belong to the subfamily Hirudinariinae Sawyer, 1986, and are characterized by the presence of a large female vaginal caecum, but are distinguishable from each other by characteristics of the female reproductive system. Poecilobdella species have a well-developed vaginal stalk, whereas Hirudinaria species are considered to lack this structure (Sawyer 1986).
The genus Hirudinaria is widely distributed over tropical South and Southeast Asia (Moore 1938;Lai and Chen 2010), and includes three valid species: Hirudinaria javanica (Wahlberg, 1856), H. manillensis (Lesson, 1842), and the recently described H. bpling Phillips, 2012. Several authors have noted that H. manillensis showed a color polymorphism containing two color morphs: a green color morph with darkish green on the dorsal surface and paler green on the ventral surface, and a red color morph with dark reddish brown on the dorsal surface and paler brick-red with two black submarginal stripes on the ventral surface (Moore 1927;Sawyer et al. 1998). These two color morphs occurred sympatrically in some locations, with one color morph dominant over the other in each population (Sawyer et al. 1998).
In Thailand, the first scientific report of buffalo leeches was that of Baird (1869), which introduced a new name, Hirudo maculata Baird, 1869, based on materials collected from Siam (now Thailand). Dequal (1917) then reported the presence of buffalo leeches in Thailand under the name Limnatis maculosa (Grube, 1868), based on two specimens collected from Bangkok. Limnatis maculosa was originally described as Hirudo maculosa using specimens from Singapore (Grube 1868). Both H. maculata and H. maculosa were later synonymized under Hirudinaria manillensis by Moore (1927). Since that time, the systematics of buffalo leeches in Thailand have not received any attention. Nearly one hundred years later, Phillips (2012) studied freshwater leeches in the southern part of Thailand and described H. bpling based on specimens collected from Phang Nga Province. Recently, Tubtimon et al. (2014) studied freshwater leeches collected from northeastern Thailand by investigating their morphology, COI sequences and karyotypes. These authors suggested the possibility of a new species being present in their material based on the differentiation of chromosome numbers, although the genetic divergence for these leeches was relatively low.
In the present study, specimens of all valid species of the genus Hirudinaria, including the red and green color morphs of H. manillensis (Fig. 1), were acquired and examined for morphological and genetic characteristics. Based on molecular and morphological evidence, the two color-morphs of H. manillensis were revealed as two different species. The red color morph fits well with the description of H. manillensis, while the green color morph showed some differences. Therefore, it is described herein as a new species.

Specimen sampling
Hirudinaria leeches were collected from freshwater ponds, rice fields, and rivers in many locations in Thailand (Table 1). They were lured out of the substrate by creating gentle movements in the water. After appearing, leeches were collected by hand or with a dip net. The 2-Step Method was used for euthanasia, following AVMA Guidelines for the Euthanasia of Animals (AVMA 2013). First, animals were relaxed by the gradual addition of absolute ethanol (EtOH) to fresh water starting from approximately 5% (v/v) concentration until they became anesthetized. Then, they were moved to 70% (v/v) ethanol to complete the process. The leeches were then fixed and kept in 95% (v/v) ethanol for further external and internal morphological study. Vouchers were deposited in Mahidol University Museum of Natural History, Department of Biology, Faculty of Science, Mahidol University, Bangkok (MUMNH), and the Museum of Zoology, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok (CUMZ).
Morphological study and species identification of each specimen was based on Moore (1927), Lai and Chen (2010), Phillips (2012), and Tubtimon et al. (2014). Four measurements were taken: body length from the anterior-most point of the oral sucker to the posterior-most point of the caudal sucker (BL), maximum body width (BW), caudal sucker length (CL) and caudal sucker width (CW). Morphological examination and measurements were done under a stereo microscope (Zeiss, Stemi 305). Photographs were taken with a Nikon D5300 camera mounted with an AF-S VR Micro-Nikkor 105 mm f/2.8G IF-ED Macro Lens.

Molecular analysis
The total genomic DNA was extracted from the body-wall muscles to avoid contamination from any host DNA by using a NucleoSpin Tissue kit (MACHEREY-NAGEL), following the manufacturer's standard protocol. Fragments of mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear 28S ribosomal RNA large subunit gene (28S) were amplified and used to estimate molecular phylogeny. Primers used for polymerase chain reaction (PCR) and sequencing are presented in Table 2. For COI, two primer sets were used to amplify the 1266 bp sequence: 1) LCO1490hiru and HCO2198hiru and 2) LCOinHiru and HCO-out (Nakano 2012); two other  Nakano and Lai (2016) primer sets were used for the approximately 1200 bp sequence of the 28S gene: 1) 28F2-2 and 28R2 (Passamaneck et al. 2004) and 2) 28F2-3 and 28R3 (Jördens et al. 2004;Passamaneck et al. 2004). The PCR amplification was conducted in a final volume of 20 µl containing 1 µl of each primer (10 mM), 10 ng of the extracted genomic DNA, 10 µl of EmeraldAmp PCR Master Mix (TAKARA BIO INC.) and distilled water up to 20 µL total volume. Each PCR reaction was performed using a T100 thermal cycler (BIO-RAD). The thermal cycling was started at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 sec, 42-52 °C (depending on gene and primer set) for 60 sec, extension at 72 °C for 90 sec, then followed by a final 72 °C for 5 min. All PCR products were purified by PEG precipitation methods and then sent to Bio Basic Inc., Canada for bi-directional sequencing on an automated sequencer (ABI prism 3730XL). Nucleotide sequences were deposited in the GenBank database under GenBank submission numbers: MN882665-MN882698 (Table 1). Sequences were aligned and edited using ClustalW as implemented in MEGA7 (Kumar et al. 2016). The concatenated dataset of COI + 28S genes was used for phylogenetic tree reconstruction using maximum likelihood (ML) and Bayesian inference (BI) approaches, and with Poecilobdella nanjingensis Yang, 1996, Hirudo verbena Carena, 1820, and Aliolimnatis oligodonta (Johansson, 1913) as outgroups. The ML analysis was conducted using 1000 ML bootstrap replications and GTRGAMMA as the model for all gene partitions in the program RAxML v.8.2.10 (Stamatakis 2014). The BI analysis was performed in the program MrBayes 3.2.6 (Ronquist et al. 2012) with the Markov chain Monte Carlo analysis (MCMC) in two parallel runs and with four chains each. The best-fit evolution models based on the Akaike Information Criterion (AIC: Akaike 1974) as suggested by the program KAKUSAN4 (Tanabe 2011) were GTR+G for both COI and 28S. Markov chains were run using random starting tree for 10 million generations and tree sampling every 1000 th generation. The first 25% of obtained trees were discarded as burn-in. The remaining trees were used to estimate the consensus tree topology, bipartition posterior probability (bpp) and branch length. The effective sample size value sampled from the MCMC analysis was greater than 8000 for all parameters. Both Table 2. Sequences of primers used for PCR amplification and sequencing gene fragments in this study. Those marked with '*' were specifically modified for Hirudinaria species (F = Forward, R = Reverse).
Oral sucker, dorsal lip prominent, with several furrows. One median longitudinal furrow on ventral surface deep, extending from margin of lip to pharynx. Two deep furrows on each side, and one or two shallow furrows between deep furrows. Three jaws in oral cavity, 1 dorsal and 2 ventrolateral. Small salivary papillae circular, placed along denticular ridge. Large salivary papillae circular to ovate; irregularly placed laterally on each jaw; minimum of 30 visible on one side of right ventro-lateral jaw. Teeth Figure 3. Illustration of reproductive system of the holotype of Hirudinaria thailandica sp. nov. A dorsal view of male and female reproductive organs including positions of ganglia XII and XIII of the ventral nervous system. Lateral views of B male and C female reproductive organs. Scale bar: 2 mm. Abbreviations: atr, male atrium; cod, common oviduct; ej, ejaculatory bulb; ep, epididymis; fg, female gonopore; mg, male gonopore; ov, ovary; ps, penis sheath; ts, testisac. vas, vas deferens; vc, vaginal caecum; and vg, vagina. on median jaw 105-125 per jaw. Pharynx muscular and tubular reaching to somite IX b2/a2. Crop reaching to somite XIX b5. Crop caeca10 pairs: Intestine tubular, acaecate, extending from somite XIX b6 to somite XXIII. Rectum thin-walled, tubular, extending from somite XXIII to somite XXVII. CL 11.9 mm, 13.0 mm wide, round, with eight rays of sensillae on dorsal surface radiating out from center.
Male gonopore in somite XI b5/b6. Female gonopore in somite XII b5/b6. Gonopores separated by 5 annuli. Nerve cord passing along the left side of reproductive system. Male reproductive system in somites XI and XII. Atrium moderate-sized, bulbous, rather round. Penis sheath short, recurved anteriorly to atrium. Epididymes round, paired, lateral to atrium. Ejaculatory bulbs present, muscular, and thick. Ejaculatory ducts short, ventrally inserted into middle of atrium. Testisacs 11 pairs. Female reproductive system in somites XII and XIII.
Ovaries paired, small, droplet-shaped. Oviduct paired, narrow, short, join to form common oviduct. Common oviduct moderate in length, slightly bent distally, then descending to vagina. Albumin gland at point of intersection of two oviducts and common oviduct. Common oviduct opens into female bursa near the insertion point of vaginal caecum to female bursa. Vaginal caecum elongated, two times as long as wide at widest point. Vaginal caecum anteroventral end connected to female bursa.
Etymology. The specific name "thailandica" refers to the country in which specimens of the new species were collected.
Distribution. This species is known from several river basins in Thailand, including the Chao Phraya and the Middle Mekong river basins (Fig. 4B).

Molecular phylogenies and genetic divergence
Phylogenetic trees estimated by ML and BI gave equivalent topologies. Therefore, only a BI tree is shown in Fig. 4A. The monophyly of the genus Hirudinaria and of each analyzed Hirudinaria species was strongly supported (ML bootstrap values of 95-100% and a BI bpp of 0.99-1.0). Evolutionary relationships among Hirudinaria   dark greenish or dark olive ventral surface rather than a lighter green ventral surface; ejaculatory ducts that insert into the midventral part of the male atrium, rather than the anteroventral part of the male atrium; and in that the common oviduct opens into the female bursa near the insertion point between the vaginal caecum and the female bursa rather than directly opening into the dorsal portion of the vaginal caecum. Moreover, their distribution ranges are limited to different river basins (Fig. 4B).
Hirudinaria bpling is distributed in western coastal rivers that drain into the Andaman Sea and Peninsular Malaysia (Chong et al. 2014), while H. thailandica is restricted to the Chao Phraya and Mekong basins. The present new species can be clearly distinguished from the Singaporean Hirudo maculosa by its male ejaculatory ducts reaching to the midventral part of the male atrium, while the latter possesses ejaculatory ducts running toward the anteroventral part of the male atrium. In fact, morphological characteristics of H. maculosa resemble those of the recently described, Hirudinaria bpling, by Phillips (2012). However, DNA examination using fresh materials of Hirudo maculosa collected from the type locality is necessary before any further taxonomic conclusions can be made.
Genital characteristics of H. thailandica are most similar to that of H. manillensis. This is the reason that some authors included this species as a variant of H. manillensis (Moore 1927;Sawyer 1986;Tubtimon et al. 2014). However, their external morphologies are clearly distinct, especially in the coloration of the ventral surface. The new species possesses a dark greenish ventral surface, while H. manillensis has a brick-red ventral surface bordered by two additional black submarginal stripes (Fig. 1). The coloration and pattern of the ventral surface is one of the most helpful characters in identifying leech species, such as in the genera Hirudo Linnaeus (Trontelj and Utevsky 2012) and Hirudinaria.