of and leeches of the genus Limnatis (Hirudinea, from the South Caucasus and Central Asia: phylogenetic relationships of Eurasian and African populations

New records of leeches of the genus Limnatis (Hirudinea, from the South Caucasus and Central Asia: phylogenetic relationships Eurasian African populations. Abstract New records of leeches of the genus Limnatis (Hirudinea, Praobdellidae) from the South Caucasus and Central Asia: phylogenetic relationships of Eurasian and African populations. Leeches of the genus Limnatis Moquin– Tandon, 1827 infest mucous membranes of various mammals, including humans and domestic ungulates. The type species of the genus L. nilotica (Savigny, 1822) was initially thought to occur throughout the Western Palaearctic, from North Africa to the Middle East and Central Asia. It was later found that L. paluda (Tennent, 1859) is a widespread Western Asian species. However, the South Caucasus and vast areas of Central Asia have not been explored sufficiently in terms of leeches of the genus Limnatis. We recorded L. paluda from Azerbaijan and Uzbekistan for the first time. We also carried out the first molecular characterisation of L. nilotica herein. We found a deep genetic differentiation (8 %) between the Western Asian L. paluda and North African (Moroccan) L. nilotica based on their COI sequences. This finding corroborates a previous morphology–based hypothesis on their separate species assignments . The low genetic diversity of L. paluda is explained by the recent colonisation of arid landscapes of Western Asia.


Introduction
The genus Limnatis Moquin-Tandon, 1827 comprises bloodsucking leeches that occur in the south-western Palaearctic. They infest mucous membranes of various organs such as the pharynx, nasopharynx, oesophagus, larynx, trachea, bronchial tubes and female genital organs in humans and domestic mammals, including horses, cattle, camels, deer, and dogs (Kaburaki, 1921;Moore, 1927;Almallah, 1968;Arenas et al., 1993;Boye and Joshi, 1994;Al-Ani and Al-Shareefi, 1995;Ağin et al., 2008;Bahmani et al., 2012Bahmani et al., , 2014Negm-Eldin et al., 2013;Rajaei et al., 2014;Raele et al., 2015). Moreover, leeches of the genus Limnatis can also be parasitic on amphibians (Lukin, 1976). The members of this genus are therefore important in terms of medicine, veterinary science, and parasitology. Taking global climate change into account, it is clear that they can pose a potential invasive threat by shifting their ranges, as has already happened with many species (Parmesan and Yohe, 2003).
According to the current view on the classification, the genus contains three species, Limnatis bacescui Manoleli, 1972, Limnatis nilotica (Savigny, 1822) and Limnatis paluda (Tennent, 1859) (Nakano et al., 2015). The type species of the genus, L. nilotica, was first described by the French zoologist Jules Cesar Savigny from Egypt under the name Bdella nilotica (Savigny, 1822). Later, in 1827, Moquin-Tandon renamed Savigny's Bdella nilotica to Limnatis nilotica in his seminal monograph (Moquin-Tandon, 1827). The second species is Limnatis bacescui Manoleli, 1972 from Romania (South-Eastern Europe), where it was described and is currently known only from its type locality (Manoleli, 1972). The third member of the genus, L. paluda, was described by Tennent (1859) as Haemopis paludum from Sri Lanka (= Ceylon). Its taxonomic status was revised by Moore (1927) so that the species was transferred to the genus Limnatis and its name was changed to L. paluda (Tennent, 1859).
Traditionally, praobdellid leeches, found both in North Africa and Western Asia, were identified as L. nilotica. Representatives of this species had been recorded for Kazakhstan and Central Asia (Lukin, 1976), and southern Iran (Grosser and Pešić, 2006). Subsequently, Siddall (2009) andNakano et al. (2015) found that the leeches of the genus Limnatis of Israel, Afghanistan and Kazakhstan should be assigned to L. paluda.
Despite the long history of previous studies, vast areas of Central Asia and the Caucasus have not been explored sufficiently in terms of their leeches of the genus Limnatis. There are no records based on molecular data concerning the species identity of those leeches in the South Caucasus and Central Asian countries except in Kazakhstan (Nakano et al., 2015), long known as regions of the Limnatis range (Lukin, 1976). Furthermore, North African leeches of the genus Limnatis have never been characterised based on their DNA sequences. The differentiation between the Western Asian L. paluda and the North African L. nilotica has relied on morphological and geographical considerations (Moore, 1938). For this reason we aimed to identify leeches of the genus Limnatis collected in Uzbekistan (Central Asia) and Azerbaijan (the South Caucasus) and to clarify taxonomic and phylogenetic relationships between North African and Western Asian leech populations of the genus Limnatis using both morphological and molecular characters.

Sample collection
Leeches were collected during field trips in Uzbekistan, Azerbaijan, and Morocco (table 1). These samples were anesthetized in 10 % ethanol, fixed, and preserved in 96 % ethanol for further examination using both morphological and molecular methods. The specimens are stored in the collection of invertebrate animals at the Department of Zoology and Animal Ecology, V. N. Karazin Kharkiv National University.

Morphological examination
Identification was carried out using a stereomicroscope Konus Crystal-45. Photo documentation was done using a USB HDCE-50B camera. We relied on Moore (1938) to find morphological features distinguishing L. nilotica and L. paluda.

DNA extraction, amplification and sequencing
Using molecular methods we analysed four specimens of L. paluda collected in Uzbekistan, one specimen from Azerbaijan and one from Morocco assigned to L. nilotica. A small piece of tissue from the posterior part of the body was taken for DNA extraction. Genomic DNA was isolated using a GeneElute Mammalian Genomic DNA Minprep Kits.
PCR products were cleaned using two enzymes, Exonuclease I and Shrimp alkaline phosphatase (SAP) (Fermentas, Thermo Fisher Scientific, USA). Exonuclease I (0.2 μl) and SAP (1 μl) were added to 10 μl of the PCR product. The mixture was then incubated for 45 min at 37 ºC followed by 15 min incubation at 80 ºC. The cleaned PCR product was then sequenced in both directions by Macrogen Inc. (the Netherlands) using the same primers as at the amplification stage. The chromatograms of sequences were processed in ChromasPro 1.32 (Technelysium Pty., Queensland, Australia). The length of the newly generated COI sequences was 650-661 bp.

Phylogenetic analysis
To reveal phylogenetic relationships of the Caucasian, Central Asian and North African leeches of the genus Limnatis, all available COI sequences of leeches assigned to that genus, sequences of Nearctic and Neotropic praobdellid leeches plus members of other families of Hirudiniformes and the erpobdellid leech Trocheta danastrica Stschegolew, 1938 were chosen for analysis and downloaded from GenBank (table 2).
The COI sequences were unambiguously aligned using MUSCLE algorithm in MEGA X. The final dataset contained a total of 1,302 positions. The alignment was checked for stop codons by translating it to amino acids using MEGA X (Kumar et al., 2018). Best-fit models of molecular evolution were determined for each codon position under the Bayesian information criterion using KAKUSAN4 (Tanabe, 2011): HKY85 with gamma distribution (+G) for the first codon position and GTR+G for the second and third positions. Phylogenetic relationships were assessed by Bayesian inference using MrBayes v3.2.7a (Ronquist and Huelsenbeck, 2003) as implemented in the CIPRES Science Gateway (Miller et al., 2010, accessible at www.phylo.org). Searches were performed in two parallel runs with eight chains each for ten million generations, sampled every 100th generation. After the first 25 % of the sampled trees were discarded, the final topologies were consented following the 50 % majority rule.
In addition, using MEGA X, we calculated the number of uncorrected base differences per site (based on p-distances) between species-level clades of the genus Limnatis.

Morphology
The specimens collected in Azerbaijan and Uzbekistan were identified as L. paluda based on their morphological characters. The leeches range from 12.6 to 95.4 mm in length. The specimen from Azerbaijan is 48.9 mm in length. The body is flattened, indistinctly separated into the trachelosome and the urosome. Some of the specimens have a well-defined clitellum. The oral sucker is confluent with the trachelosome. The caudal sucker is wide and constitutes 0.86 of the maximum width of the urosome. The oral sucker has a sulcus on its inner surface. The anus is inconspicuous. Mid-body segments are five-annulated. The gonopores are separated by five annuli. The leeches have five pairs of eyes that line up, creating a parabolic arc pattern. Papillae are weakly developed. Live leeches are greenish dorsally with no dark pattern. Lateral margins of the body are orange. The venter is bluish black ( fig. 1A, 1B). The Moroccan leeches, which were assigned to L. nilotica, are 7.7-13.0 mm in length. The posterior sucker constitutes 0.76 of the maximum body width. The dorsal coloration pattern includes six longitudinal rows of black dots and short lines. Pigmentation was largely bleached due to preservation in ethanol. The specimens do not have a well discernible sulcus. The gonopores are separated by five annuli (fig. 1C).

Phylogeny and genetic differentiation
Phylogenetic analyses show the family Praobdellidae is a well-supported monophyletic group with a posterior probability of 1.00. The genus Limnatis is also a wellsupported clade. All Middle Eastern, Caucasian and Central Asian samples joined a clade with a posterior probability of 0.78. This monophyletic group matches L. paluda. The phylogenetic structure of the clade is simple and shallow. The Namibian Limnatis sp. is sister to the clade of L. paluda with a posterior probability of 0.98. This group of Namibian and Western Asian leeches is sister to the clade consisting of the Moroccan L. nilotica and Croatian Limnatis sp. The latter clade is supported by a posterior probability of 0.62 ( fig. 2).
The number of base differences per site from averaging sequence pairs within the clade of L. paluda is as low as 0.0030 ± 0.0012. Uncorrected distances between the Western Asian L. paluda, Balkan Limnatis sp., North

Discussion
Morphological examination of the leeches of the genus Limnatis suggests the specimens collected in Azerbaijan and Uzbekistan should be assigned to the Middle Eastern and Central Asian L. paluda. This identification is based on the morphological features as follows: in contrast to the North African L. nilotica, Central Asian and South Caucasian leeches of the genus Limnatis are characterised by the monotonous green coloration with no dark dots or lines on the dorsum. The dorsal coloration pattern consisting of black stripes, lines and dots is characteristic of the North African L. nilotica (Moore, 1938). The Moroccan specimens have the typical coloration of L. nilotica. The phylogenetic analysis corroborated the morphological identification and revealed a deep differentiation between North African and Western Asian leeches of the genus Limnatis. Molecular characterisation of the North African Limnatis, which is currently assigned to L. nilotica in the strict sense, was performed herein for the first time. All Middle Eastern, Caucasian, Central Asian and Afghan leeches joined the clade of L. paluda ( fig. 2) corroborates independent species statuses of the North African and Western Asian populations, both of which had been assigned by some classical authors (Lukin, 1976) to L. nilotica. The Namibian Limnatis is sister to the L. paluda clade and belongs to an unidentified species, which may represent the littleknown taxonomical diversity of the genus Limnatis in Africa (Moore, 1938 Phillips and Siddall, 2009;Nakano et al., 2015), should be substantiated using fresh specimens and molecular data. Biogeographically, the occurrence of the same leech species in Sri Lanka and in Western Asia appears questionable.
Despite the vast range of L. paluda, the species was found to have low genetic diversity and a shallow phylogenetic structure. This may be explained by the recent colonisation of arid landscapes in Western Asia. The range expansion could be attributed to the parasitism of these leeches on their ungulate hosts that appear to be able to transmit their parasites over long distances (Nakano et al., 2015). The eastern medicinal leech Hirudo orientalis Utevsky and Trontelj, 2005 is another instance of the rapid colonization of that area, which caused comparable genetic consequences (Trontelj and Utevsky, 2012). Migrations of nomads and their livestock throughout vast territories of the Middle East, Caucasus and Central Asia or other human activities in that area (as was discussed in Nakano et al., 2015) could contribute to shaping the genetic structure of L. paluda. Obviously, more studies are needed to clarify the evolutionary history and to elaborate a robust classification of the leeches of the genus Limnatis.