Parasitology Research 98, 61-66.

Parasitol Res (2005) 98: 61–66
DOI 10.1007/s00436-005-0017-7
ORIGINA L PA PER
Serge Y. Utevsky . Peter Trontelj
A new species of the medicinal leech (Oligochaeta, Hirudinida,
Hirudo) from Transcaucasia and an identification key
for the genus Hirudo
Received: 24 May 2005 / Accepted: 8 September 2005 / Published online: 28 October 2005
# Springer-Verlag 2005
Abstract A recent molecular phylogenetic study has suggested that the genus Hirudo contains a neglected species
previously known as the orientalis coloration type of the
medicinal leech Hirudo medicinalis. In this paper, the new
species is formally described as Hirudo orientalis sp. n. It
can most readily be identified by the grass green coloration
of the dorsum, segmentally arranged pairs of black quadrangular or rounded dots on its paramarginal dorsal stripes
and similarly arranged, but less regular light-colored markings on the predominantly black venter. It has mediumsized epididymes and an evenly coiled vagina. H. orientalis
is known from Transcaucasia, Iran, and Uzbekistan. It is
widely used in medicine as the “medicinal leech.” Very
little is known about its exact distribution, specific habitat,
and conservation status. The paper contains an identification key to all species of the genus Hirudo.
Introduction
Throughout nearly 2,000 years of documented history, the
medicinal leech (Hirudo medicinalis Linnaeus, 1758) has
received considerable attention in various fields of human
activity. Its ectoparasitic, hematophagous feeding habit has
been used and abused for medical purposes for centuries. In
biological classification, it constitutes the taxonomic basis
of the class Hirudinea. In modern times, it has been widely
applied in neurophysiological, behavioral, and develop-
S. Y. Utevsky (*)
Department of Zoology and Animal Ecology,
V. N. Karazin Kharkiv National University,
Kharkiv 61077, Ukraine
e-mail: [email protected]
Tel.: +38-507-619578
Fax: +38-572-587373
P. Trontelj
Biotechnical Faculty,
Department of Biology,
University of Ljubljana,
P.O. Box 2995, SI-1001 Ljubljana, Slovenia
mental genetic studies. Its salivary glands’ secretion has
been the subject of biochemical and pharmaceutical research. In brief, the medicinal leech may be regarded as one
of the best-studied animals in respect of its morphology,
physiology, and behavior (Sawyer 1986), and it has become a standing character in textbooks on zoology and
biology. Leech collecting for medicinal purposes in the
19th century, recent collecting pressure, and a general loss
and pollution of wetland habitats have caused a dramatic
decline of H. medicinalis throughout its geographical range
(Elliott and Tullett 1984; Sawyer 1981). The international
concern for its conservation is reflected by international
conventions and regulations (summarized in Trontelj and
Utevsky 2005).
Despite the high conservation concern, medical attention, and biological interest devoted to the medicinal leech,
there has been confusion regarding the taxonomic status
of different morphological forms. Some recent molecular
systematic research (Trontelj et al. 2004; Trontelj and
Utevsky 2005) corroborated the validity of two neglected
species (Hirudo verbana Carena, 1820, and Hirudo troctina
Johnson, 1816) and suggested that a variety from Transcaucasia and Iran, which has never been recognized as a
species, does deserve specific status. Stschegolew and
Fedorova (1955) treated the latter as a variety of the form
serpentina (=H. medicinalis). Kobakhidze (1946) was the
first who noted that there were two coloration varieties of
the medicinal leech in Georgia. Among practitioners, it was
known as the Persian or Georgian leech (also mentioned by
Lukin 1976). Shevkunova and Kristman (1962) first applied the trinomial Hirudo medicinalis orientalis for this
supposed variety. However, article 15.2. of the International Code of Zoological Nomenclature (International
Commission on Zoological Nomenclature 1999) specifically treats names of varieties or forms published after 1960
as infra-subspecific, and as such as not regulated by the
Code. Thus, the name ‘orientalis’ for the new species has
yet to be made formally available by a proper species
description. The aim of the present paper is to describe the
new species Hirudo orientalis sp. n., corresponding to the
variety H. medicinalis orientalis sensu Shevkunova and
62
Kristman (1962), in a comparative manner based on morphological and recently published molecular data.
Materials and methods
The specimens described herein were obtained from a
pharmacy in Kharkiv, Ukraine, in February 2004. They
were certified for medical use, and all of them originate
from Azerbaijan. Leeches were fixed in 96% ethanol or
10% formalin after relaxation in weak ethanol. Preserved
specimens were pinned under tap water and dissected along
the dorsal midline to reveal diagnostic features of the male
and female reproductive systems. The anterior sucker and
adjacent trachelosome were cut along the ventral midline to
examine the jaws. Only mature specimens were dissected.
A narrow fascia was cut along the ventral midline to reveal
ganglia that indicated central rings of segments. The drawings were made freehand with the aid of an ocular micrometer. The specialized terminology used in this article
follows that of Sawyer (1986) and Hechtel and Sawyer
(2002). The description is based on 28 specimens.
For comparative purposes, we examined specimens of
H. medicinalis and H. verbana collected in ponds near
Geniyivka and Komsomolsk, Kharkiv Region, Ukraine, in
2000 and 2004. The examined specimens of H. orientalis
sp. n. have been deposited in the National Museum of
Natural Sciences (NMNS), Kyiv, Ukraine.
DNA sequences of the mitochondrial genes for 12S
rRNA (12S rDNA) and the cytochrome oxidase subunit
one gene as well as nuclear ribosomal internal transcribed
spacer two (ITS2) were used to determine species-specific,
autapomorphic substitutions and genetic distances between
the most closely related species. The sequences are available from a foregoing study (Trontelj and Utevsky 2005)
under the following GenBank accession numbers: AY763
170 (ITS2), AY763163 and AY768704 (12S rDNA),
AY763154 [cytochrome oxidase subunit one (COI)] for
H. orientalis; AY763166 (ITS2), AY763156–AY763159
(12S rDNA), AY763148 and AY 763149 (COI) for H.
medicinalis; AY763167 (ITS2), AY763160 (12S rDNA),
AY763150 and AY763151 (COI) for H. verbana.
Etymology Orientalis means ‘oriental,’ ‘eastern,’ and describes the range of the species relative to the range of the
type species H. medicinalis. Although with respect to the
Far Eastern Hirudo nipponia, this name is not entirely
consistent, we choose to keep it because it has been in
informal use among local practitioners for decades (Figs. 1–2).
The remaining material is in the collection of the first
author.
Diagnosis
Large leeches, length up to 108 mm including suckers;
pigmentation of dorsum grass green with two pairs of
orange longitudinal stripes; lateral margins with a longitudinal yellow stripe; one annulus of each segment with
quadrangular or rounded dark spots on paramarginal dorsal
longitudinal stripes; venter black, with metameric pairs of
light-colored markings; jaws monostichodont, papillated;
no pharyngeal ridges terminating between jaws; epididymes medium-sized; vagina with small caecum; no vaginal
duct; vagina tubular, evenly coiled.
Description
External characters
The following are the external characters: length up to
108.0 mm, maximum body width 10.0, width of anterior
sucker 4.0, width of posterior sucker 5.5 mm; gonopores,
separated by five annuli; annulation, complete somite fiveannulate; body surface, covered with numerous papillae;
Systematic account
H. orientalis sp. n.
Type locality The specimens were obtained from aquaculture; it is known that they originate from the Agdam
District, central Azerbaijan.
Holotype One adult individual was preserved in formalin.
The NMNS catalog number is AN-41.
Paratypes Three adult individuals were preserved in
ethanol. The NMNS catalog numbers are AN-41/1, AN41/2, and AN-42/3.
Fig. 1 External characters of H. orientalis sp. n. a Dorsal view of
the entire specimen. b Midbody segment, dorsal view. c Midbody
segment, lateral view. d Midbody segment, ventral view. e Anterior
sucker, ventral view. f Clitellum, ventral view. fg Female gonopore,
p penis. a–d, f Holotype. e Paratype
63
tissue bound to the cephalic face of vagina. Entire female
reproductive system located between ganglia XII and XIII.
Molecular characters H. orientalis is characterized by 30
autapomorphic nucleotide substitutions in two mitochondrial and one nuclear DNA sequence (Table 1). Measured
by these sequences, its genetic distance (uncorrected percentage of nucleotide divergence) to both of its closest
relatives is about 5% (Table 2).
Differential remarks
Fig. 2 Internal characters of H. orientalis sp. n. a Dissected anterior
sucker, ventral view, paratype. b Dorsal view of reproductive system
and right lateral view of vagina, holotype. a Atrium, co common
oviduct, e epididymis, eb ejaculatory bulb, g ganglion in segment
XI, j jaw, o ovisac, ps penis sheath, s sulcus, t testisac, v vagina, vc
vaginal caecum, vd vas deferens
eyes, five pairs; sulcus, present as a narrow, distinct groove
running from the crypt of the median dorsal jaw to the
dorsal rim of the anterior sucker; pigmentation, background of dorsal surface grass green, two orange paramedian stripes thin and fragmented, two orange paramarginal
stripes broad and encompassing black segmentally arranged quadrangular or rounded spots, lateral margins of
body with yellow stripes encompassing black, segmentally
arranged rounded spots; the black spots are located at the
level of the ganglion on the central annulus (a2) of each
segment; venter predominantly black, with metameric pairs
of light greenish markings.
Internal characters
Jaws Trignathous, monostichodont, papillated.
Male reproductive system Atrium: large, bulbous with a
glandular cover, located at ganglion in segment XI; penis
sheath, a long broad duct bent anteriorly, not reaching
ganglion in segment XII. Epididymes: medium-sized, discoid, tightly packed masses of ducting standing upright on
either side of the atrium and located between ganglia in
segments XI and XII. Ejaculatory bulbs: fusiform, well
developed, and not larger than epididymes the dorsocefalic faces of which they circle. Vasa deferentia: thin
ducts running from epididymes posteriorly. Testisacs: ovoid,
approximately 1.5 times the size of ovisacs, located
posterior to ganglion in segment XIII.
Despite the fact that H. orientalis is very similar to its
closest relatives H. medicinalis (Fig. 3), H. verbana
(Fig. 4), and H. troctina in external and internal characters,
it can easily be distinguished from the allied forms using
some manifest features. The pigmentation is the most
helpful character in identifying these leeches. H. orientalis
differs from H. verbana by having thin, deep orange
colored paramedian stripes, whereas H. verbana has broad,
diffuse paramedian stripes which are pale orange in color.
The venter of H. verbana is unicolored greenish to yellow,
bounded by a pair of black ventrolateral stripes. H.
orientalis and H. medicinalis can be distinguished by the
form of black spots on the dorsum and on the margins of
the body. In H. orientalis, the black dorsal spots are rounded
or quadrangular, whereas in H. medicinalis, they are
elongated. The marginal spots of H. medicinalis are coalesced to form distinct black stripes. The dark ventral
pigmentation forms an irregular mesh-like pattern in H.
medicinalis, whereas the ventral coloration pattern of H.
orientalis is more regular, formed by segmentally arranged
pairs of light markings on a predominantly black background. H. orientalis and H. troctina appear very similar in
their dorsal pigmentation, but can readily be distinguished
by their ventral coloration patterns. The most distinctive
feature of H. troctina is a pair of zigzag-shaped, black
ventrolateral longitudinal stripes. It should be noted that
Hechtel and Sawyer (2002) considered external pigmentation to be not only the most useful, but also arguably the
best character to distinguish species within this genus.
Further details on the coloration of different Hirudo species
can be found in their paper, as well as in Moquin-Tandon’s
(1846) and Nesemann and Neubert’s (1999).
There are some differences in the characters of the
reproductive system. We use the approach proposed by
Table 1 Number of autapomorphic (species-specific) nucleotide
positions in three closely related Hirudo species for three different
genes and the combined dataset
Species
Female reproductive system Vagina: an upright, long, evenly
curved tube entering directly into ventral body wall posterior to ganglion in XII. Common oviduct enters the
vagina subterminally at a small vaginal caecum. Ovisacs:
globular, small. Common oviduct: a thin duct forming
several loops and covered with a thick layer of glandular
H. orientalis
H. medicinalis
H. verbana
12S rDNA
10
5
9
COI
ITS2
Combined
17
12
16
3
0
3
30
17
28
All three sequences are approximately 550–650 base pairs long
ITS2 internal transcribed spacer two
64
Table 2 Uncorrected genetic distances and standard errors (in
percent) between pairs of three closely related Hirudo species for
three different genes and the combined dataset
Pairwise comparison 12S rDNA COI
H. orientalis–H.
medicinalis
H. orientalis–H.
verbana
H. medicinalis–H.
verbana
ITS2
Combined
5.4±0.9
7.8±1.1
0.8±0.4
5.0±0.6
5.6±1.0
8.8±1.1
1.4±0.5
5.7±0.7
4.7±0.9
8.6±1.0
1.0±0.4
5.1±0.6
Hechtel and Sawyer (2002), who estimated the size of
the epididymis in relation to the ejaculatory duct. In H.
orientalis, the epididymes are medium-sized. Conversely,
the epididymes of H. verbana are relatively small, whereas H. troctina (see Hechtel and Sawyer 2002) and H.
medicinalis have massive epididymes. The vagina of H.
orientalis is tubular and evenly curved. It does not show
the central swelling and sharp folding typical for H.
verbana (Fig. 4). In H. medicinalis, the vagina can have
two conditions: straight and tubular, or terminally curved
(Fig. 3). H. troctina has a bulbous vagina (see Hechtel and
Sawyer 2002). Internal characters should be used as
Fig. 4 H. verbana Carena, 1820. a Midbody segment, dorsal view.
b Midbody segment, lateral view. c Midbody segment, ventral view.
d Reproductive system, dorsal view. a Atrium, co common oviduct,
e epididymis, eb ejaculatory bulb, g ganglion in segment XII, o
ovisac, ps penis sheath, v vagina
diagnostic features only in fixed specimens, and if the
natural pigmentation is not preserved.
To summarize the differences between H. orientalis and
its congeners, the following identification key is proposed.
It should be noted that the Far Eastern species H. nipponia
was not subject to the same systematic scrutiny as the other
Hirudo spp., and that its inclusion in the key is mainly
based on characters mentioned by Whitman (1886).
1. Dorsum with five longitudinal yellow stripes
H. nipponia Whitman (1886)
Dorsum brown olive; venter yellow olive; vagina a
straight tube.
- Dorsum with two or four orange or reddish stripes
or rows of spots
2. Dorsum with two broad, diffuse longitudinal orange
stripes; venter unicolored greenish to yellow with a
pair of black marginal stripes
H. verbana Carena, 1820
Dorsum with reticulate pattern; vagina, a sharply
folded, elongated tube; epididymis small, not much
larger than ejaculatory bulb.
- Dorsum with narrow longitudinal orange or reddish stripes; venter with dark pattern
Fig. 3 H. medicinalis Linnaeus, 1758. a Midbody segment, dorsal
view. b Midbody segment, lateral view. c Midbody segment, ventral
view. d Clitellum, ventral view. c Reproductive system, dorsal view.
f Straight vagina of another specimen. a Atrium, co common
oviduct, e epididymis, eb ejaculatory bulb, fg female gonopore, g
ganglion in segment XI, mg male gonopore, o ovisac, ps penis
sheath, v vagina, vc vaginal caecum
3. Venter black, with metameric pairs of light-colored
markings
H. orientalis sp. n.
Dorsum grass green with quadrangular metameric
or rounded black dots; vagina an evenly curved
tube; epididymis medium-sized, somewhat larger
than ejaculatory bulb.
65
- Venter with irregularly arranged and sized black
markings
4. Dorsum with quadrangular metameric black dots;
ventral marginal stripes zigzag-shaped
H. troctina Johnson, 1816
Dorsum green; vagina bulbous, upright and not
folded; epididymis massive in relation to ejaculatory bulb.
- Dorsum with elongated metameric black spots;
ventral marginal stripes straight if present
H. medicinalis Linnaeus, 1758
Dorsum olive green; vagina terminally curved or
straight tube; epididymis massive in relation to
ejaculatory bulb.
Discussion
Organisms like the fruit fly and the house mouse that are
used as models in various fields of science or are commercially exploited and bred are usually taxonomically
very clearly defined. In this respect, the medicinal leech
appears to be an exception. That a leech from a pharmacy
turned out to belong to a new species shows us how
taxonomy (or so-called α-taxonomy) is still neglected.
The problem which previous workers have faced since
Linnaean times is the apparent lack of characters that can
be used to delimit species. Some leech taxonomists have
treated, often implicitly, coloration as a misleading character. According to this point of view, all different coloration types represent just variations of one and same
species H. medicinalis (e.g., Lukin 1976). Recent molecular systematic investigations (Trontelj et al. 2004; Trontelj
and Utevsky 2005) have corroborated the coloration pattern as the crucial clue to distinguish between good biological species of the genus Hirudo: H. medicinalis, H.
troctina, H. verbana, and H. orientalis sp. n. Knowing this,
the identification of medicinal leech species is relatively
straightforward. It was always possible to unequivocally
call the species among hundreds of specimens from our
collections, although the external coloration varies considerably within a species.
The molecular data presented by Trontelj and Utevsky
(2005) offer a relatively secure basis for the distinction of
four Western Palearctic medicinal leech species, because
genetic variation within species lags for over an order of
magnitude behind genetic divergence between even the
most closely related species. Nevertheless, some relationships between species are less clearly supported, and so is
the sister relationship between H. orientalis and H. medicinalis (Fig. 5). For comparative purposes, these two
species might be tentatively considered as being the closest
relatives, but the alternative hypotheses of either of them
standing sister to H. verbana should not be ruled out.
Fig. 5 Phylogenetic relationships of medicinal leech species
inferred from combined nuclear and mitochondrial DNA sequences
using parsimony. Numbers on branches are support values obtained
by various methods: parsimony bootstrap proportions, maximum
likelihood bootstrap proportions, and Bayesian posterior probabilities, respectively. Note the relatively low support for the sistergroup hypothesis between H. orientalis and H. medicinalis. From
Trontelj and Utevsky (2005), strongly simplified
It would be interesting to know more about the ecology
and distribution of all four Western Palaearctic medicinal
leech species. In particular, since the medicinal leech sensu
lato is considered endangered in many countries and
treated as such by international legislation (IUCN, CITES,
Berne Convention, EU Habitat Directive). Further, health
regulations of some countries specifically approve H. medicinalis for various forms of leech therapy. In practice,
however, other species are used at least as often.
Whereas at present it is not possible to say anything
about specific ecological requirements of different Hirudo
species, their distribution can be inferred indirectly by what
has been said about the distribution of coloration types or
forms. According to the literature, H. orientalis was found
in Georgia, Armenia, Azerbaijan, Iran (Lukin 1976;
Stschegolew and Fedorova 1955; Utevsky et al. 1998),
and Uzbekistan (Abdullaev, personal communication).
H. medicinalis occurs in western and central Europe
(Nesemann and Neubert 1999), Ukraine (Lukin 1976),
and Lithuania (Zapkuvenè 1972). H. verbana is known
from the eastern Mediterranean region and the Balkans
(Nesemann and Neubert 1999), as well as Moldova,
Ukraine, the Krasnodar Territory (Russia), and Armenia
(Lukin 1976; Utevsky et al. 1998). H. troctina is known
to be from Morocco, Tunisia, and Algeria (Hechtel and
Sawyer 2002). It is unknown to what degree the current
distribution has been influenced by range losses and by
introduction of animals to previously uninhabited areas.
Acknowledgements Our thanks go to Vladimir Samoylov
(Kharkiv, Ukraine) for furnishing some of specimens and to
Abdumalik Abdullaev (Samarkand, Uzbekistan) for the information
on the distribution of medicinal leeches in Uzbekistan.
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