First record of Limnatis paluda (Hirudinida, Arhynchobdellida, Praobdellidae) from Kazakhstan, with comments on genetic diversity of Limnatis leeches

Abstract Background Sawyer (1986) included three species in the nasal leech genus Limnatis Moquin-Tandon 1827: Limnatis nilotica (Savigny 1822), Limnatis bacescui Manoleli 1972 and Limnatis paluda (Tennent 1859). The first and last species have mainly been identified in Middle Eastern countries (e.g. Kinzelbach and Rückert 1985). The second species has been identified only in Romania Dobruja (Manoleli 1972). Although Limnatis leeches are well known species of endoparasitic leeches, Limnatis nilotica was recorded only once in Kazakhstan (Lukin 1976). New information Specimens of the genus Limnatis from Almaty Province, Kazakhstan are identified as Limnatis paluda. This is the first record of Limnatis paluda from Kazakhstan. Mitochondrial COI and 12S data demonstrated that the present specimens are genetically close to an Israeli specimen identified as Limnatis nilotica. In addition, molecular data suggest that some Limnatis specimens whose DNA sequences have been reported were misidentified. According to the observed phylogenetic relationships, the taxonomic status of the known Limnatis species should be revisited.

The type locality of L. nilotica is Egypt, and this species has been reported to occur mainly in Middle Eastern countries (Al-Safadi and El-Shimy 1993, Kinzelbach and Rückert 1985, Nesemann and Forster 1997, Rückert 1985. The second species, L. bacescui was only known from its type locality, Romanian Dobruja (Manoleli 1972). As L. nilotica, the last species L. paluda has mainly been identified in Middle Eastern countries, and is sometimes distributed sympatrically with the first species (Boye and Joshi 1994, Kinzelbach and Rückert 1985, Kuntz and Myers 1968, Rückert 1985. However, Limnatis paluda was first described from Ceylon (Sri Lanka) and was stated to be a cattle leech there (Tennent 1859). Its taxonomic status was revisited by Moore (1927a). Although Moore could not examine Ceylonese materials of L. paluda, many studies followed his classification on this species. Despite its impact as a parasite of humans and other large mammals, there have been very few distributional studies of Kazakhstan nasal leeches of Limnatis. To our knowledge, L. nilotica was identified from Bilikol Lake, Jambyl Province, southern Kazakhstan by Lukin (1976). In addition, this species was recently recorded in Kazakhstan along with neighboring countries, Azerbaijan and Uzbekistan (Kovalenko and Utevsky 2015). The second (TD) and last (KN) authors collected Limnatis leeches from Almaty Province, southeastern Kazakhstan, near the border with China. Based on morphological examination and molecular phylogenetic analyses, the taxonomic status of this leech was clarified. In addition, its genetic diversity based on mitochondrial DNA data is briefly discussed.

Materials and methods
Leeches were collected from the Suygaty Valley located at the left bank part of the Ily River Depression, Almaty Province, Kazakhstan (Fig. 1). The specimens were preserved in absolute ethanol (EtOH) in the field. For DNA extraction, botryoidal tissue was removed from the posterior part of the body around the caudal sucker of every 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 (BL) from the anterior margin of the oral sucker to the posterior margin of the caudal sucker, maximum body width (BW), caudal sucker length (CL) from the anterior to the posterior margin of the caudal sucker and caudal sucker width (CW) from the right margin to the left margin of the sucker. Examination, dissection of 2 specimens (KUZ Z702 and Z703; see below), and drawing of the specimens were conducted using 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 (1927b): body somites are denoted by Roman numerals and the annuli in each somite are given alphanumeric designations.

Habitat
During night time, the leeches examined in this study were found crawling in a small pond (Figs 9, 10a) fed from an artificial well on a small hill. The hill is situated in the clayey gravelly desert at the foot of the arid low mountains Ulken Boguty at 1270 a.s.l. (Fig. 10b). Although at one time local inhabitants grazed domesticated animals around the well, at present there are no dwellers in the area.

Genetic data
Neighbour-joining trees generated based on both the COI (Fig. 11) and 12S (Fig. 12) genes showed that the present Limnatis specimens from Kazakhstan form a monophyletic lineage with a sequence from L. nilotica collected in Israel (BS = 86% in COI, 98% in 12S). In the neighbour-joining tree based on COI sequences, this monophyletic lineage and one sequence obtained from the Afghan L. paluda formed a well-recovered clade (BS = 100%). In addition, the neighbour-joining trees revealed that sequences obtained from L. nilotica collected in Namibia and Croatia do not form a monophyletic group. Figure 11.
The neighbour-joining tree of available COI sequences. Numbers associated with nodes represent bootstrap values. Length of each sequence is listed in Table 1.

Figure 12.
The neighbour-joining tree of available 12S sequences. Numbers associated with nodes represent bootstrap values. Length of each sequence is listed in Table 1.
The COI uncorrected p-distance between the Kazakhstani L. paluda and the Israeli L. nilotica was 0.2% ( Table 2) based on 12S sequences consisting of 353 bp which showed that the sequences of the former are identical with that of the latter (Table 3). The COI uncorrected p-distance between Kazakhstani L. paluda and that from Afghanistan was 0.5%, and that between the Israeli L. nilotica and the Afghan L. paluda was 0.6%. The COI and 12S uncorrected p-distances between the Kazakhstani, Israeli, and Afghan Limnatis sequences and the remaining sequences of L. nilotica were 7.3-9.7% and 2.8%, respectively. The COI uncorrected p-distance between the Namibian L. cf. nilotica and the Croatian L. cf. nilotica was 11.9%, and that based on 12S was 3.9%.

Taxon discussion
The present 4 specimens of Limnatis clearly belong to Limnatis paluda sensu Moore (1927a) based on the following characteristics: VI a1 and a2 forming 1 annulus (a1 + a2) on venter; XXIV quadrannulate; sensillae small, undeveloped; each jaw bearing numerous salivary papillae; monostichodont teeth numbering 30-46 on each jaw; and body surface uniform brownish with lateral marginal stripes in orange. Tennent (1859) provided incomplete morphological characteristics of L. paluda, describing its colour as uniform brown without any bands but with reddish lateral margins, and not very numerous teeth. Although Moore (1927a) could not examine specimens of L. paluda from its type locality, he identified Limnatis leeches from Punjab and Baluchistan, which presently belong to Pakistan, as L. paluda based on their colouration as well as their limited number of teeth on each jaw.
Moore (1927a) stated that L. paluda could be clearly distinguished from Limnatis nilotica by its limited number (30-47) of teeth on each jaw (the latter species has numerous teeth on each jaw). He also mentioned that the numbers of teeth in L. paluda and L. nilotica never overlapped. Kinzelbach and Rückert (1985) also mentioned that the number of teeth in L. paluda was at most 45, but that of L. nilotica ranged from 45 to 60. However, Lukin (1962) and Lukin (1976) noted that L. nilotica possessed 30-50 teeth on each jaw. Based on the specimens collected in Azerbaijam, Kazakhstan and Uzbekistan, Kovalenko and Utevsky (2015) reported that L. nilotica bore 38-40 teeth on each jaw. In addition, L. bacescui possessed 50-54 teeth on each jaw (Manoleli 1972). Although Limnatis species are well known as nasal leeches, the taxonomic status of each species seems to be far from clarified. Each nominal species of the genus Limnatis including L. paluda should be revised based on specimens collected from the type locality.
As mentioned above, the taxonomic identities of Limnatis species have not been fully settled. According to the present neighbour-joining trees and p-distance data, however, the Israeli Limnatis leech, of which DNA sequences have been deposited with INSDC, should be assigned to Limnatis paluda as mentioned in (Phillips and Siddall 2009), because it forms a monophyly with the present Kazakhstani L. paluda specimens, and the p-distances of the COI and 12S sequences show extremely low genetic divergence (0.2% in COI and no differences in 12S). In addition, it is highly likely that L. cf. nilotica of Croatia and Bosnia and L. cf. nilotica of Namibia do not belong to the same species, because those specimens are paraphyletic consistently in the present phylogenetic trees, and the former is highly diverged from the latter (11.9% in COI and 3.9% in 12S). These uncorrected p-distance values are greater than those between L. paluda and the Namibian Limnatis species (7.3-7.4% in COI and 2.8% in 12S) as well as L. paluda and the Balkan Limnatis specimens (9.2-9.7% in COI and 2.8% in 12S).
It is noteworthy that the specimens of Limnatis paluda analysed in this study have low genetic divergences (0.2-0.5% in COI and 0% in 12S). The COI uncorrected pdistances between the present Kazakhstani specimens and the Israeli specimen are lower than that between the former and the Afghan specimen (0.5%) and that between the latter and the Afghan L. paluda (0.6%). The collection locality in Kazakhstan is ca. 4,000 km from Israel, and ca. 2,000 km from Afghanistan. In contrast, Israel is at most ca. 3,500 km from Afghanistan. Because few DNA sequences of L. paluda are available, it may be difficult to reveal its detailed genetic structure. However, the results of the mitochondrial genetic analyses at least shed light on the discordance between the COI genetic divergence between the Kazakhstani L. paluda and the Israeli specimen and the geographic distance between the collection localities. Trontelj and Utevsky (2012) revealed low genetic diversity in the European medicinal leeches, and suggested that medicinal leeches dispersed rapidly and widely via their host animals as these leeches are ectoparasitic species. Because Limnatis species are endoparasitic leeches that attach to the nasopharyngeal cavities of mammals, they likely achieve long-distance dispersal. Except when they parasitise the mammalian nasopharyngeal cavities, Limnatis species generally inhabit a freshwater environment. One of the routes of the Silk Road passed through the Ili River Depression (Baipakov 1998), near the collection locality of the present specimens. Therefore, Limnatis paluda have possibly dispersed by means of freshwater places along the trade route as stepping stones, and thus human activities may have influenced the distribution of L. paluda. In either case, further taxonomic studies of Limnatis species should be performed to clarify their taxonomic positions. In addition, future molecular studies should elucidate the biogeographical history of Limnatis paluda.