A new species of Hirudo (Annelida: Hirudinidae): historical biogeography of Eurasian medicinal leeches
© The Author(s) 2016
Received: 11 December 2015
Accepted: 26 July 2016
Published: 23 August 2016
Species of Hirudo are used extensively for medicinal purposes, but are currently listed as endangered due to population declines from economic utilization and environmental pollution. In total, five species of Hirudo are currently described throughout Eurasia, with Turkey being one of the major exporters of medicinal leech, primarily H. verbana.
To define the distribution of Hirudo spp. within Turkey, we collected 18 individuals from six populations throughout the country. Morphological characters were scored after dorsal and ventral dissections, and Maximum Likelihood (ML) and Bayesian Inference (BI) analyses resolved phylogenetic relationships using mitochondrial cytochrome c oxidase subunit I (COI), 12S ribosomal RNA (rRNA), and nuclear 18S rRNA gene fragments. Our results identify a new species of medicinal leech, Hirudo sulukii n. sp, in Kara Lake of Adiyaman, Sülüklü Lake of Gaziantep and Segirkan wetland of Batman in Turkey. Phylogenetic divergence (e.g., 10–14 % at COI), its relatively small size, unique dorsal and ventral pigmentation patterns, and internal anatomy (e.g., small and pointed atrium, medium-sized epididymis, relatively long tubular and arc formed vagina) distinguish H. sulukii n. sp. from previously described Hirudo sp.
By ML and BI analyses, H. sulukii n. sp. forms a basal evolutionary branch of Eurasian medicinal leeches. Phylogeographic interpretations of the genus identify a European Hirudo “explosion” during the upper Miocene followed by geological events (e.g., Zanclean flood, mountain building) that likely contributed to range restrictions and regional speciation of extant members of the clade.
Hirudinid leeches are parasitic to a variety of vertebrates leading many to regard them with distaste, but their medicinal utility is well established. For centuries, Hirudo medicinalis and related species (e.g., H. verbana, H. troctina) were prescribed to treat virtually every human ailment from arthritis to yellow fever, most without efficacy. In 1830, during their peak usage, a Paris hospital employed more than five million medicinal leeches . Consequently, populations of H. medicinalis in Central Europe were depleted, and non-sustainable collecting led to their extinction in many areas. Pollution and habitat drainage further added to their decline, forcing Europe to import medicinal leeches from the Ottoman Empire (Anatolia), North Africa and Russia  to meet demand. By the late 1900’s, the advent of “modern” medicine drastically reduced clinical demand for leeches, allowing some threatened populations to rebound.
Leech therapy languished for most of the 20th century, considered “quackery” by mainstream medical practitioners , but the discovery of various bioactive compounds in leech saliva [27, 39], and recognition of the leech’s superior ability to relieve venous congestion (e.g., ), has led to renewed interest in clinical applications. Current fields of employment include reconstructive microsurgery, hypertension, and gangrene treatment . In light of 19th century threats to medicinal leech populations as demand increased, considerable conservation steps were implemented to ensure their continued availability. Pursuant to these efforts, much confusion resulted regarding the taxonomic status of different morphological forms [18, 28, 56, 65]. Phylogenetic analysis of nuclear and mitochondrial DNA sequences suggest that the genus Hirudo is monophyletic , and that species or morphological varieties can be readily identified by coloration patterns. Molecular studies have shown that European medicinal leeches, although usually marketed as H. medicinalis, comprise a complex of at least three species: H. orientalis, the commonly sold H. verbana and the relatively rare H. medicinalis [4, 37, 54, 55, 60]. Kutschera and Elliott  analyzed the behavior of adult H. medicinalis, but could not find differences with respect to its sister taxon H. verbana. Morphological and molecular data demonstrate that commercially available medicinal leeches are generally not H. medicinalis [35, 56, 60], but rather specimens belonging to the Eastern phylogroup H. verbana [61, 62], which is predominantly bred in leech farms and used as a modern ‘medicinal’ stock.
Turkey is rich in wetlands and known to support at least two species of medicinal leech, H. medicinalis and H. verbana. Prior to ~2000, it was believed that medicinal leeches from Turkey’s wetlands were only H. medicinalis [21, 31]. Molecular characterization of Turkish leeches was not performed until the turn of the century, however, and leeches from the Kızılırmak and Yesilirmak Deltas on the Black Sea coast, comprising the majority of leech specimens destined for export, have proven to be to H. verbana [4, 51, 55].
Mapped localities of all Hirudo species show extensive, belt-shaped ranges extending from east to west. To establish the distribution of Hirudo species in Turkey, one of the major exporters of medicinal leeches worldwide, we sampled broadly in three representative localities within the western, eastern and southeastern regions of Turkey. Our data identifies a new species for the genus, H. sulukii n. sp., that forms a basal evolutionary branch among European medicinal leeches and sheds light on the evolutionary history of the genus.
Specimen collection and maintenance
Leech specimens collected throughout Turkey (Kara Lake, Beyaz Cesme Marsh, Uluabat Lake, Segirkan wetland, Balik Lake, Sülüklü Lake) were transported to Fırat University, Fisheries Faculty (Elazig, Turkey) and maintained in separate 600 L fiberglass tanks based on collection location. Tank bottoms were elevated with peat soil ~10 cm on one side to create a terrestrial to aquatic continuum. Leeches were fed one adult frog (e.g., Pelophylax ridibunda) blood meal per month (others have utilized mammalian blood), and typically survived 2+ years in the laboratory. Specimens were fixed in 70 % ethanol for molecular analysis and some were fixed with 10 % formaldehyde in PBS for dissection. External traits of live specimens were observed by stereomicroscopy. Preserved specimens were dissected dorsally and ventrally, with representative sketches of internal morphology derived directly from the type specimen.
Tissue samples from live specimens were obtained by placing the leech in a 10 % ethanol sedating solution until it was unresponsive to touch. Approximately half of the caudal sucker was removed with a scalpel, and tissue cuttings were immediately processed using the E.Z.N.A.™ Tissue DNA kit (Omega Bio-Tek) following the manufacturer’s instructions. Whenever possible, tissue from postmortem specimens was taken from the caudal sucker to avoid contamination from gut contents.
DNA sequence amplification of target genes
Primers used for PCR amplification and DNA sequencing
5'- CGGTAATTCCAGCTCCAATAG -3'
Apakupakul et al. (1999) 
Apakupakul et al. (1999) 
Palumbi, 1996 
Simon et al. 
Folmer et al. 
Folmer et al. 
DNA sequencing and editing
Purified PCR products were shipped to GeneWiz, Inc. (South Plainfield, NJ) for Sanger DNA sequencing using an ABI 3730xl DNA analyzer. Each PCR product was sequenced in both directions using amplification primers, and sequence chromatograms were viewed and manually adjusted in ChromasPro (Technelysium, Queensland, Australia) or BioEdit . Sequence alignments were made with MUSCLE  or CLUSTAL W [29, 38]. Accession numbers for all CO1, 12S and 18S sequences are listed in Suppl. Data (Table 1).
Maximum-likelihood (ML) analyses were performed for all DNA comparisons, using the pipeline sequence MEGA 7  to align corresponding sequences from multiple individuals or homologous DNA across species, Gblocks  for alignment curation, PhyML  for tree building and TreeDyn  for tree drawing, as configured in the Phylogeny.fr platform . The aLRT statistical test (approximation of the standard Likelihood Ratio Test; ) embedded in PhyML determined branch support values. Default settings were used for all parameters.
Bayesian Inference (BI) analysis was performed on the combined data set (morphological parameters, 18S, 12S, COI in Nexus format) in MrBayes v. 3.2.1x64 [48, 49]. Data were partitioned for 18S and 12S, and by codon position for COI. ModelTest  via FindModel was used to determine the optimal model of evolution for each gene under the Akaike Information Criterion (AIC; ). The general time reversible (GTR) model with a gamma distributed rate parameter was used for COI, 12S and 18S. Two analyses were run simultaneously with all parameter sets unlinked by partition for two million generations each, sampling every 100 generations, with a burn-in achieved by <50,000 generations. Setting the burn-in to 500,000 generations left a total of 7413 trees sampled for assessment of posterior probabilities. Gaps were treated as missing data, and default settings were used for all other parameters.
Collection field sites in Turkey and specimen descriptions. Depositions in the Academy of Natural Sciences, Philadelphia, PA (ANSP) and Cukurova University Parasitology Museum, Adana, Turkey (CUPM)
Hirudo sulukii n. sp.
ANSP G1 19489
ANSP G1 19488
Collection field sites in Turkey and specimen descriptions. Depositions in the Academy of Natural Sciences, Philadelphia, PA (ANSP) and Cukurova University Parasitology Museum, Adana, Turkey (CUPM)
Beyaz Cesme Marsh
HV1, HV2, HV3, HV4, HV5, HV6, HV7
Specimens were scored for morphological characters according to Borda and Siddall , Utevsky and Trontelj , Klemm , Sawyer , Nesemann and Neubert , Saglam  and Govedich et al. , Elliott and Dobson  (Additional file 1). By these criteria, 10 leeches were identified as H. verbana, while six specimens did not match characters described for any known Hirudo species. Specifically, external pigmentation was unique, along with internal distinctions of the epididymis and vagina (see below).
Hirudo sulukii n. sp
Despite similarities between Hirudo sulukii n. sp. and other Hirudo species, the former can be distinguished from its closest relatives using internal and external features. Hirudo sulukii n. sp. differs from H. medicinalis and H. orientalis by the form of black spots on the dorsal, paramedian stripes of the body. Hirudo sulukii n. sp. has black, segmentally-arranged united ellipsoid and elongated spots, and dorsal lateral margins of body a pair of zigzagged black dorsolateral longitudinal stripes (Fig. 4a). The ventral coloration pattern of H. sulukii n. sp. has a variable number of irregular spots (Fig. 4b); H. orientalis has black, dorsal rounded or quadrangular spots while H. medicinalis has elongated spots. The marginal spots of H. medicinalis are fused to form distinct black stripes. The ventral of H. medicinalis has an irregular dark mesh-like pattern while that of H. orientalis is more regular, formed by segmentally-arranged pairs of light markings on a predominantly black background. Hirudo verbana has broad, diffuse paramedian stripes orange in color. The ventral pattern of H. verbana is unicolored greenish to yellow, bounded by a pair of black ventrolateral stripes. Hirudo troctina has a pair of zigzag-shaped, black ventrolateral longitudinal stripes . Hechtel and Sawyer  considered external pigmentation to be not only the most useful, but also arguably the best character to distinguish species of Hirudo.
In this study we used the approach of Hechtel and Sawyer  and Utevsky and Trontelj  regarding the size of the epididymis in relation to the ejaculatory duct. The epididymes of Hirudo sulukii n. sp. (Fig. 6) and H. orientalis are medium-sized. In contrast, the epididymes of H. verbana are relatively small, whereas H. troctina and H. medicinalis have massive epididymes . The vagina of Hirudo sulukii n. sp. is relatively long tubular and arc formed (Fig. 6), while in H. orientalis the vagina is tubular and evenly curved. The former two species do not show the central swelling and sharp folding typical for H. verbana. In H. medicinalis, the vagina can have two conditions: straight and tubular, or terminally curved . Hirudo troctina has a bulbous vagina .
Moquin-Tandon  described at least five species of Hirudo including H. verbana and H. medicinalis, but later concluded that they were all varieties of the same leech species. The medicinal leech, H. sulukii n. sp., considered here was determined to be morphologically different than all species described by Moquin-Tandor [40, 41].
Pairwise distance matrix of Hirudo specimens
Maximum Likelihood and Baysian Inference analyses yielded trees with concordant topologies and strong support for H. sulukii as a basal branch of the European medicinal leeches. Relationships between H. medicinalis, H. verbana and H orientalis were less conclusive, consistent with confusion regarding their morphological identification [45, 56]. The relatively small size of H. sulukii, unique dorsal and ventral pigmentation patterns, and internal anatomy (e.g., small and pointed atrium, medium-sized epididymis, relatively long tubular and arc formed vagina) are distinguishing features of this previously undescribed leech. Note that H. sulukii has thus far been collected only from relatively high elevation field sites (i.e., Kara Lake-Adiyaman 1233 m, Sülüklü Lake-Gaziantep 877 m, and Segirkan wetland- Batman 525 m), and its small size in comparison with other Hirudo species may reflect an adaptation to this environment (e.g., reduced foraging season/food supply), as suggested for other annelid species (e.g., ).
Previously, only two medicinal leeches were thought to occur in Turkey, H. verbana and H. medicinalis, while a total of five are currently described throughout Eurasia. The range of H. verbana occurs to the south of H. medicinalis in an almost parapatric fashion with little overlap [5, 32, 42, 43, 51]. The former is subdivided into an Eastern (southern Ukraine, North Caucasus, Turkey and Uzbekistan) and Western phylogroup (Balkans and Italy) that do not overlap, suggesting distinct postglacial colonization from separate refugia [61, 64]. Easternmost records are from Samarqand Province in Uzbekistan [61, 64, 65], resulting in an east-to-west extent of ~4600 km. Leeches supplied by commercial facilities belong to the Eastern phylogroup, originating mostly from Turkey and the Krasnodar Territory in Russia, two leading areas of leech export.
Hirudo medicinalis is distributed from Britain and southern Norway to the southern Urals and probably as far as the Altai Mountains, occupying the deciduous arboreal zone [6, 12, 16, 21, 22, 31, 43, 51, 52, 59, 63, 68]. Hirudo orientalis is associated with mountainous areas in the sub-boreal eremial zone and occurs in Transcaucasian countries, Iran and Central Asia, while H. troctina has been found in northwestern Africa and Spain in the Mediterranean zone . Hirudo verbana and H. medicinalis have recently experienced range expansions while H. orientalis has remained geographically isolated within arid and alpine areas of Central Asia and Transcaucasia .
Species of Hirudo have had broad applications in medicine, ranging from reconstructive surgeries (e.g., facial, finger reattachment, ear flap) to anticoagulants/analgesics secreted from salivary glands [2, 24]. Thus the discovery of a new Hirudo species, particularly a basal member of this phylogroup, has considerable value in the context of medical potential. Specifically, natural variants of known bioactive factors (e.g., hirudin, antistasin, etc.) are logical candidates to explore for their potentially enhanced or novel pharmaceutical properties. The current study has prompted a more systematic survey of Hirudo throughout Turkey and surrounding regions with the collective aims of refining the evolutionary history of the genus, facilitating conservation efforts, and identifying species that may expand the repertoire of medicinal applications for this important Hirudinid genus.
By phylogenetic and morphological criteria, specimens collected from Kara Lake of Adiyaman, Sülüklü Lake of Gaziantep and Segirkan wetland of Batman in Turkey comprise a new species, Hirudo sulukii. Geographic isolation by the Taurus Mountain chain has likely contained H. sulukii within the regional sampling area. By ML and BI analyses, H. sulukii n. sp. forms a basal evolutionary branch of Eurasian medicinal leeches, preceded by a deeper ancestral split with the Asian medicinal leech. H. nipponia. Phylogeographic interpretations of the genus identify a European Hirudo “explosion” during the upper Miocene followed by geological events (e.g., Zanclean flood, mountain building) that likely contributed to range restrictions and regional speciation of extant members of the European clade.
We thank Mark Siddall for helpful comments.
Supported by The Scientific and Technological Research Council of Turkey (TUBITAK) to NS, and Busch Biomedical and GAIA grants to DHS.
Availability of data and material
Holotype and paratype Hirudo sulukii specimens deposited in the Academy of Natural Sciences, Philadelphia, PA, USA (catalogue ANSP G1 19488, 19489). Additional paratypes deposited in Cukurova University Parasitology Museum, Adana, Turkey (catalogue CUPM-HIR/2016-1). All DNA sequences deposited into GenBank (Additional file 1: Table S1).
NS collected leech specimens, conducted experimental analyses including specimen dissections, and contributed to writing the manuscript; RS conducted DNA analyses and contributed to writing; SAL assisted with DNA analyses, phylogenies and writing; DHS oversaw experimental analyses and writing of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Ethical approval is not required at Rutgers University for research conducted on invertebrates such as medicinal leeches used in our study.
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