Erpobdellid leeches (Annelida, Clitellata, Hirudinida ...

Zootaxa 3681 (4): 440–454 www.mapress.com / zootaxa / Copyright © 2013 Magnolia Press

ISSN 1175-5326 (print edition)

Article

ZOOTAXA ISSN 1175-5334 (online edition)

http://dx.doi.org/10.11646/zootaxa.3681.4.7 http://zoobank.org/urn:lsid:zoobank.org:pub:682CDDA0-FD5E-4F81-B1C5-44C68849FB74

Erpobdellid leeches (Annelida, Clitellata, Hirudinida) from Tunisia: New records with the description of a new Trocheta species RAJA BEN AHMED1,5, ALEKSANDER BIELECKI2, JOANNA M. CICHOCKA2, SAÏDA TEKAYA1, MAŁGORZATA GORZEL3& ABDUL HALIM HARRATH1, 4 1

Université de Tunis El-Manar, Faculté des Sciences de Tunis, UR11ES12 Biologie de la Reproduction et du Développement animal, 2092 Tunis, Tunisia E-mail: [email protected]; [email protected] 2 Department of Zoology, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 5, 10-719 Olsztyn, Poland. E-mail: [email protected]; [email protected] 3 Department of Physiotherapy, University of Vincent Pol, Choiny 2, 20-816 Lublin, Poland 4 College of Science, King Saud University, Saudi Arabia. E-mail: [email protected] 5 Corresponding author. E-mail: [email protected]

Abstract Up to now in Tunisia, freshwater Hirudinida are represented by two mainly haematophagous families: Hirudinidae and Glossiphonidae, and a predatory one: the family Erpobdellidae. The present study provides new information on the diversity and taxonomy of erpobdellid leeches. Identification was based, in addition to morphological data, on the length of sperm ducts and the lengths of ovisacs in relation to the neurosomite (ns) and on the shape and size of the male atrium. Five taxons are found. Two subspecies are reported for the first time in the country: Dina punctata punctata Johansson, 1927 and Dina punctata maroccana Nesemann and Neubert, 1994. Tunisian populations of two species, Erpobdella testacea (Savigny, 1820) and Trocheta africana Nesemann and Neubert, 1994, are described, with records of new localities. The new Trocheta tunisiana n. sp. is discovered and described in detail. Trocheta species live in springs in elevated areas while Erpobdella seem to prefer low altitude reservoirs. A comprehensive comparison of the three genera is presented. The disparity between the actual systematics and phylogeny is discussed. This study gives also a detailed distribution of the five species in the north of Tunisia with notes on ecological preference of the genus Dina. Finally a key for the determination of freshwater erpobdellid species from Tunisia is proposed. Key words: Hirudinida, Erpobdellidae, new records, new species, taxonomy, Tunisia

Introduction Erpobdellid leeches are predators of aquatic invertebrates having abandoned the blood feeding habits of their ancestors (Siddall and Burreson 1998). Among this group, some species have long been investigated as a model organism for both ecological studies of invertebrate species interactions (Seaby et al. 1995; Zerbst-Boroffka 1999) and as indicator species for freshwater toxicology (Wicklum and Davies 1996; McNicol et al. 1997; Zaranko et al. 1997). Species of the Erpobdellidae family are most common in North America and Europe, whereas salifid leeches (e.g. species of Salifa and Barbronia) are more common in Africa and Asia (Soos 1969; El Shimey 1996). The hirudinean fauna, particularly the Erpobdellidae family of the west Mediterranean area and especially of the Maghreb, is still relatively unknown. In fact, little has been published on the erpobdelliforms of this area. Blanchard (1908) published notes on Dina quadristriata from Tunisia, which have been assigned later to Trocheta africana (Ben Ahmed and Tekaya 2009). Nesemann and Neubert (1994) described two new African taxa, Trocheta africana from Tunisia and Dina punctata maroccana from Morocco. Recently, Ben Ahmed et al. (2008) recorded Erpobdella testacea for the first time in Tunisia and thus in Africa. The present study reports our extensive surveys of erpobdellid leeches from localities in northern Tunisia, resulting in the description of one new species, new records for Dina punctata punctata Johansson, 1927 and Dina punctata maroccana Nesemann and Neubert, 1994, and description of Tunisian populations of Erpobdella testacea (Savigny, 1820) and Trocheta africana Nesemann and Neubert, 1994.

440 Accepted by Boris Sket: 2 May 2013; published: 24 Jun. 2013

Material and methods Specimens were collected from many localities in the northwest of Tunisia (Fig 1). The geographical coordinates of the collecting sites were recorded with a GPS. A map showing the distributions of erpobdellid leeches in Tunisia using an ArcView 3.2® (ESRI) and Adobe® Illustrator® CS5 (Adobe Systems Inc.) software was given (Fig 2). Light-sensitive free-living species were found attached to the underside of a variety of objects such as rocks and any submerged vegetation in the water courses. They were gently picked by hand or with forceps and kept in jars containing the same water from reservoirs where they were collected. In the laboratory, leeches were photographed using Nikon “Coolpix” digital camera, and a preliminary identification was made, based on external features. To study the external and the internal features, worms were first relaxed in weak ethanol (10%), then, when they stopped moving, leeches were transferred to 70% ethanol for being dissected. Specimens preserved in 70% ethanol were observed using an OLYMPUS SZ-ST stereomicroscope equipped with an OLYMPUS DP 10 camera. Measurements of the most characteristic structures of the reproductive system (ovisacs and vas deferens) in relation to midbody neurosomites (ns) of the studied species were given (Table 1). The description of the atrium was given based on five dissected specimens from each species. For the new species, ten specimens were dissected. Museum Depository: The material examined was deposited in the Muséum National d’Histoire Naturelle (MNHN) in Paris, France and in the author’s collection at the Faculty of Science of Tunis.

Results FAMILY ERPOBDELLIDAE BLANCHARD, 1894 Genus: Erpobdella De Blainville, 1818 Erpobdella testacea (Savigny, 1820) Material examined. 325 specimens collected in: Elkhirba dam, Bizerte (37°16'354” N, 10°09'552” E), Port Prince dam, Cap Bon, Korbus (36°51'162’’ N, 10°39' 404’’ E), Lebna dam (36°44'326'' N, 10°55'255'' E) and Marsh in Sejnen, Bizerte (37°03’ N, 09°13’ E). Description of examined specimens. The length of narcotized and preserved specimens can reach 40 mm; the width at mid-body region is up to 3 mm. The anterior sucker is longitudinally elliptical and the posterior sucker is circular. The dorsal surface of the living specimens is reddish brown in colour with minuscule darker specks situated dorsally (Fig. 3a). The ventral surface is brighter than the dorsal (Fig. 3b). The mid-body segment consists of five equally long annuli. Pharyngeal stylets are absent. The head has four pairs of eyes. In the majority of examined specimens the gonopores are separated by 4 annuli with the occasional occurrence of variation in their position. In fact it may reach in some specimens 4.75 annuli. The male reproductive system consists of numerous and globular testes that form voluminous and grape-like testisacs present on each side of the nerve cord and reach close to the posterior sucker. The vasa deferentia are about 7 ns long and they run from XII to XIX ganglia. They are located on the latero-ventral sides and connected to the atrium with the two ejaculatory ducts. The latter forms a loop, which is located in front of ganglia XI. The wide male atrium is 0.5 neurosomite (ns) in length and is located almost half way between the ganglia XI and XII. The atrium is small and the cornua are greatly expanded forming obtuse angle between them. They bend towards each other rather than to the ventral side. The bursa is relatively big (Fig. 4a, b). The female reproductive system consists of two symmetric, U-shaped ovisacs situated on either side of the nerve cord. The ovisacs begin two-third of the way between the ganglia XII and XIII and end half way between the ganglia XV and XVI. They are connected to the pocket by oviducts. Ecology and distribution. The occurrence of E. testacea in Tunisia is restricted to lowland stagnant waters (50–65 m ASL). To date this species had been recorded from three reservoirs in the north of the country and from a small marsh. In the Lebna dam E. testacea is present in a large number all year round. Erpobdella testacea is a Palearctic species. In the Mediterranean region it is present in northern Italy and Greece (Nesemann 1997) and northern Tunisia (Ben Ahmed et al. 2008). It was recorded in Croatia, Montenegro, Bosnia and Herzegovina (Sket 1968). ERPOBDELLID LEECHES FROM TUNISIA

Zootaxa 3681 (4) © 2013 Magnolia Press ·

441

FIGURE 1. (a–f): Some sites of leech collection in Tunisia: a: Lebna Dam; b: Aïn Essaniya, Siliana; c: Aïn Soltane, Jendouba; d: Aïn Nfaja; e: National Parc ElFaïja; f: Aïn Sidi Saleh, Bizerte; g: T. africana collected from a leaf of Quercus siber; h: Dina punctata maroccanna swallowing a worm (Lumbricus. sp).

442 · Zootaxa 3681 (4) © 2013 Magnolia Press

BEN AHMED ET AL.

FIGURE 2. Distribution of erpobdellid leech species (Annelida, Clitellata, Hirudinida) in the North of Tunisia.

Remarks. While our specimens show a variation in the distance between gonopores which may reach 4.3–4.7, Agapow and Bielecki (1992) found a constant distance of 4 annuli between gonopores in the specimens studied in Poland. In contrast, Manoleli (1972) mentioned that in the majority of specimens examined in Romania, gonopores are separated by 3.5 annuli.

Genus: Dina Blanchard, 1892 Dina punctata punctata Johansson, 1927 Material examined. 137 specimens collected in: Aïn Amdoun, Béja (36°46'216“ N, 09°56’821” E), Aïn Touta (36°88’403“ N, 09°53’513” E), Aïn Sidi Rhouma (36°17’575” N, 08°87’473” E), Aïn Oued Ramil, Kef (36°11’539” N, 08°63’863” E), Aïn Soltane, Siliana (35°48’424” N, 09°21’588” E), Aïn Tfifila (35°51’215” N, 09°43’385” E), Aïn Essaniya, Siliana (35° 46’141”N, 09°26’450” E), Aïn Zakkar, Siliana (36°01’298” N, 09°32’181” E), Aïn Serkha, Fernana (36°62’593” N, 08°71’408” E), Aïn Zoulèl, Bullarijia (36°56’531” N, 08°78’953” E), Aïn Sidi Saleh, Bizerte (37° 12' 302'' N, 10° 04' 002 ”E). Description of examined specimens. The length of preserved leeches ranges from 38 to 45 mm; the width of the mid-body region is 3–5 mm. Both anterior and posterior suckers are transversally elliptical. The colour of living specimens is brown to green. The dorsal surface is covered by yellow spots that are arranged transversally on annulus a2, with rough surface due to the presence of small papillae (Fig. 3c). On the dorsal side is a pair of faded paramedian stripes. The ventral surface is grayish in colour (Fig. 3d). The somites are divided to four short annuli and an elongated one (Fig. 6b). Head with four pairs of eyes. The gonopores are separated by two annuli, with prominent male gonopore. ERPOBDELLID LEECHES FROM TUNISIA


443

The male reproductive system consists of numerous and globular testes that form voluminous, grape-like structures of testisacs situated latero-ventrally. The sperm duct has a length of 7 ns, reaching the XX ganglion. The ejaculatory duct forms a preatrial loop. The relatively narrow atrium is 0.7 ns in length. Cornua are slightly expanded, bending to the ventral side. They are sharply coiled to connect with the ejaculatory ducts. The bursa is relatively big and enlarged (Fig. 4.d, e, f). The ovisacs are paired convoluted organs lying mid-ventrally; they start at ¼ part of the distance between the ganglia XII and XIII, and ends at the ganglia XVI. Ecology and distribution. Dina punctata punctata is the most commonly observed leech occurring in almost the entire examined territory (north of Tunisia). It has only been recorded in small brooks and stagnant, often temporal water bodies connected to springs in mid to high elevations (265–1010 m). They are semi-aquatic annelids. As they are absent in the dry season, we suppose that these speimens bury deep into the soil during this period. They appear again in the winter to reproduce.

Dina punctata maroccana Nesemann and Neubert, 1994 Material examined. 23 specimens collected in: Aïn Nfaja 24 km before Séjnéne, Mateur (37°02’291” N, 09°26’219” E). Description of examined specimens. Dina punctata maroccana is very similar to D. punctata punctata. It can be clearly distinguished from the nominal subspecies found in Europe by the shape of its body which is exceptionally dorso-ventrally flattened, and by the presence of sharp lateral fringes (keels) at the post-clitellar region. The length of preserved specimens ranges from 40–60 mm, and the width of the mid-body region is around 5 mm. The anterior sucker is longitudinally elliptical and the posterior sucker is transversally elliptical. The dorsum of living specimens is green to black in colour and is covered with prominent yellow spots (Fig. 3e) and by small papillae and thus appears rough. The specimens from Tunisia have more than 8 spots in a2. The ventral surface of the body is grayish (Fig. 3f). Each somite is divided on five annuli, the first four are short and the fifth is elongated (Fig. 6b). The eyes are arranged in four pairs. The gonopores are separated by two annuli. The male reproductive system consists of numerous globular testes that form voluminous grape-like testisacs that almost reach the end of the leech body. The sperm ducts form loops that reach half distance between the ganglia XI and XII ganglion. The sperm duct has a length of 13.5 ns and thus it reaches almost XXVI ganglion. The relatively wide atrium is located at the ganglia XII and is almost 0.5 ns in length. The cornua are greatly expanded, sharply coiled and bending to the ventral side. The bursa is stout and wide (Fig. 4g, h, i). The female reproductive system is composed of two elongated convoluted ovisacs forming a "V". They have a length of 3.5 ns. The ovisacs start at the ¼ of the distance between the ganglia XII and XIII ganglion, and end at ¾ of the distance between the ganglia XV and XVI. Ecology and distribution. To date D. punctata maroccana had been recorded from only one site: a brook (230 ASL) connected to a water spring, where it is abundant. The species is a semi-aquatic annelid, having similar behavior as D. punctata punctata. The leeches were also collected from moist soil, destroying the earthworm, Lumbricus sp. (Fig. 1h). Dina punctata maroccana has been previously observed in Morocco and never before found in Tunisia (Nesemann and Neubert 1994). The current distribution of this subspecies has likely been under-estimated due to its a semi-aquatic lifestyle and insufficient surveillance of its preferred habitats. Additional records can be expected, especially in the northern part of Tunisia. Thus, we recommend a certain degree of protection for this leech until further studies can establish its true status in the country.

Genus: Trocheta Dutrochet, 1817 Trocheta africana Nesemann and Neubert, 1994 Material examined. 97 specimens collected in: National Parc El Faïja (36°30’172” N, 08°19’081” E), Aïn Soltane, Jendouba (36°31’261” N, 08°20’151” E), river elFeïja (36°48’631” N, 08°30’771” E).


BEN AHMED ET AL.

FIGURE 3. Photos of live erpobdellid specimens.; a–b: E. testacea; c–d: D. punctata punctata; e–f: D. punctata maroccana; g–h: T. africana; D.F—dorsal view (this aspect looks brighter than the ventral one because of the uneven illumination conditions); V.F—ventral view; Scale bar: 0.6 cm.

ERPOBDELLID LEECHES FROM TUNISIA


445

FIGURE 4. Ventral, dorsal and lateral views of male atria of mature specimens: a–c: E. testacea; d–f: D. punctata punctata; g– i: D. punctata maroccana; j–l: T. africana; m–o: T. tunisiana n. sp.


BEN AHMED ET AL.

Description of examined specimens. The length of preserved specimens reaches up to 70 mm and the width is around 6 mm. Both anterior and posterior suckers are transversally elliptical. The caudal sucker of preserved specimens is narrower than the maximum body width. Lateral keels are present. The colour of living specimens is black. Four dark narrow longitudinal stripes are visible in the smooth dorsal surface (Fig. 3g). The area between the inner paramedian stripes is less pigmented than the dorsal surface. The ventral surface is pale grey (Fig. 3h). Annulation: somites with eight annuli, the first two of which are short, followed by three longer ones and another three short annuli. Head with four pairs of eyes. The gonopores are separated by two annuli. The male pore is situated in XII b2/a2. Whereas the female one is in furrow XII b5/c11. The male reproductive system consists of numerous globular testes that form voluminous, grape-like testisacs that reach the end of the leech body. The sperm duct has 9.75 ns in length, beginning at XII and ends close to XXII ganglion. The atrium, located at the ganglion XII, is 0.5 ns in length. The bursa is relatively wide and well rounded. The cornua are thick and curved towards the middle (Fig. 4j, k, l). The female reproductive system consists of a pair of well-developed and convoluted ovisacs showing anteriorly a 1.75 ns long ‘lateral’ loop and then lying medio-ventrally. The ovisacs are 3.33 ns in length, running from a ¼ part of the way between the ganglia XII and XIII until just before the ganglia XVI. From the ovisacs appear two oviducts that run ventrally. Ecology and distribution. Presently, Trocheta africana is the only species of the genus Trocheta that has been reported from North Africa. This species prefers stagnant water or slow running rivers or brooks. It occurs mainly in the higher regions of northern Tunisia (805–868 ASL). The specimens were found attached to the underside of rocks and stones and the dead leaves of Quercus suber (Fig. 1g). It has been discovered in Tunisia (in a stream near Hammam Bourguiba, a mountain stream between Thibar and Téboursouk, Béja) and in Algeria. Remarks. In the original description of T. africana made by Nesemann and Neubert (1994) the examined specimens are described as small leeches. In fact, the length of preserved specimens ranged from 15 to 28 mm. In this study, the length of examined specimens ranged from 50 to 70 mm. Notwithstanding, there is no detailed description of the ovisacs condition we believe that the specimens studied by Nesemann and Neubert (1994) were juveniles, because they were collected at the end of February. The specimens examined in the present study were collected in December and they were mature, because we could observe oocytes inside the ovisacs.

Trocheta tunisiana n. sp. Holotype north-west Tunisia, spring Zaga in Béja (36°58’965” N, 9°5’693” E), December 2008, body length 50 mm, maximal width 5.2 mm, deposited in Muséum National d’Histoire Naturelle, Paris (catalogue number W245), preserved in ethanol 70%. Paratypes the same locality, data, and depository as holotype, two mature specimens, body length 48 mm, maximal width 5 mm, preserved in ethanol 70%. Other material examined Collected during the period extending from December 2007 to December 2009. Spring Oued El Madin (36°90’107” N, 9°16’501” E); stream Oued el Melih Ouechtéta (36°97’467” N, 09°01’214” E); spring Aïn Sobh Jendouba (36°57’173” N, 08°54’565” E); spring Touiaytia, Aïn Draham (36°74’456” N, 08°58’686” E). Etymology. The specific epithet is derived from the name of the country from which the species was described. Description of examined specimens. The largest preserved specimen measured 50 mm in length and 5.2 mm in width. The front of the body is broad and rounded compared to T. africana; both anterior and posterior suckers are longitudinally elliptical and the posterior sucker is as wide as the widest part of the body. The head has four pairs of eyes. In the post-clitellar region, lateral keels are less prominent than in T. africana. The colour of living specimens is light brown. Dorsally, there are four longitudinal stripes (Fig. 5a, b).The ventral surface is always somewhat lighter than the dorsum. Annulation is typical for the Trocheta species. The complete somite is divided into eight annuli, the first two of which are short, followed by three longer ones and another three short annuli (Fig. 5c). The gonopores are separated by two annuli, the prominent male pore is situated in XII b2/a2, whereas the inconspicuous female one is in furrow XII b5/c11. The pharynx lacks jaws whereas pseudognaths are present (Fig 6 b).The digestive system is constructed typically for erpobdellid leeches. The testes are globular, numerous and form grape-like structures of testisacs. They begin at 1/3 of the distance between XVIII and XIX ganglion and



447

reach close to the posterior sucker. The vas deferens ends at 1/3 of the distance between the ganglia XVIII and XIX. The sperm ducts form a loop (Fig. 6a). The atrium is located at the ganglia XII. The length of the male atrium is approximately 1/3 ns. It has relatively narrow cornua and a broad insertion in the atrial body, which is slender and narrow (significantly longer than wide) and slightly expanded. It is a little oblique in relation to the bursa and curves to the ventral side. The bursa is not big (Fig. 4m, n, o). The ovisacs are paired, elongated convoluted structures, showing anteriorly a 1.5 ns large ‘lateral’ loop and then lying mid-ventrally. They have 4 ns in length and they are arranged as loops, first they go posteriorly up to XVI ganglion and then turn back anteriorly up to the XII ganglion (Fig. 6a). Within the ovisacs of mature specimens, there are no visible oocytes observed which is probably due to the fact that the specimens were after reproduction. The paper by Sket (1968 fig. 47–48) appeared to be very helpful in our interpretation of the reproductive system of T. tunisiana n. sp.

FIGURE 5. a: Dorsal view of T. tunisiana n. sp.; b: dorsal view of both T. tunisiana n. sp. (double arrow) and T. africana (arrow); c: annulation pattern in T. tunisiana n. sp. Scale bar : in a : scale bar = 6mm ; in b : scale bar = 8mm ; in c : scale bar = 10mm.


BEN AHMED ET AL.

FIGURE 6. a: Reproductive system of T. tunisiana n. sp.: atrium (at), ovisacs (o), preatrial loops (pr.l), sperm ducts (s), testisacs (t); b: anterior sucker of T. africana in the left (after Nesemann and Neubert, 1994) and T. tunisiana n. sp. in the right; arrows point to the pseudognathes; c: Schematic annulations type and gonopores of both Tunisian species of Trocheta.

Remarks. The new taxon was assigned to the genus Trocheta based on the number and the subdivision of annuli per somite. In fact, our specimens possess a mid-body somite consisted of eight unequal annuli. Moreover, the ovisacs are long and may take 4ns, showing a large lateral loop in anterior part of ovisac course.



449


BEN AHMED ET AL.

Trocheta tunisiana n. sp. can easily be distinguished from T. africana by its colour and size. The latter species is a relatively large leech that is mostly black in colour whereas T. tunisiana n. sp. is a medium-sized leech that is reddish brown in colour. Anatomically, the atrium in T. tunisiana n. sp. is smaller in size and different in shape than that of T. africana. The relative lengths of the ovisacs and sperm ducts are also different (Table 1). Moreover, the anterior sucker is more elongated in the new species than in T. africana (Fig 6b). The collection of specimens of T. africana and T. tunisiana n. sp. from the same region of Tunisia (northwest; Fig. 2) and during the same period (December) revealed that, although the specimens of both species were sexually mature, they were in different phases of the life cycle (respectively, before and after breeding). Trocheta tunisiana n. sp. differs from T. subviridis Dutrochet 1817, the type species of the genus, and from T. falkneri (Nesemann and Neubert, 1996) by the distance between its gonopores (six to nine annuli in T. subviridis and four to five in T. falkneri). It also differs from T. cylindrica Örley, 1886 (syn. T. bykowskii Gedroyć, 1913, see Košel 2004) by the varied colour of the body (grey and flesh coloured with lighter or darker brownish shade). Moreover, the integument of T. cylindrica is very transparent, making the internal structure well visible. Trocheta haskonis Grosser, 2000 is a giant leech compared with the T. tunisiana n. sp. having a body length up to 220 mm. In T. pseudodina Nesemann, 1990 the surface of the body is covered by papillae; similar to some species of the genus Dina in body coloration, there are also dark spots on dorsal side, which are arranged in transverse lines. On the basis of this comparison we conclude that T. tunisiana n. sp. represents a new species. Ecology and distribution. Trocheta tunisiana n. sp. has been collected in smaller brooks and springs occurring in the mid and higher elevations (usually over 350 ASL). It is distributed in the northwest of Tunisia; we suggest that it should, probably, occur also in Algeria.

Taxonomy The taxonomy of genera and species within erpobdellid leeches has been for a long time the subject of prolonged dispute or debate. Since the establishment of the genus Dina by Blanchard (1892) the generic status of erpobdellids and, in particular, the separation of Erpobdella and Dina is surrounded by considerable confusion. Recently, molecular study conducted in Erpobdellidae leeches demonstrated that morphological taxonomic characters are insufficient to define genus and higher taxa within the family (Siddall 2002). Two taxonomic characters chiefly characterize the traditional generic subdivision in erpobdellids: the number or subdivision of annuli per somite (Sawyer 1986; Neubert and Nesemann 1995; Nesemann and Neubert 1996) and the form of the male atrium (Nesemann 1997). Dina, Trocheta and Erpobdella are considered to be the most species-rich genera of the family Erpobdellidae. They all possess a pre-atrial loop of the ejaculatory duct and are thus distinguished on the basis of their annulate pattern. In fact, the formula of annulation in Erpobdella leeches is b1+b2+a2+b5+b6. All annuli are undivided and are of equal length. However, some annuli in Dina and Trocheta are distinctly longer and subdivided in the course of maturation. The formula of annulation in Dina is as: b1+b2+a2+b5+c11+c12, and in Trocheta is as: c1+c2+b2+a2+b5 +c11+d23+d24 (Trontelj and Sket 2000).The last two genera are often referred to the Trochetinae subfamily. A molecular phylogenetic analysis has revealed a sister relationship between Trocheta and Erpobdella rather than between Trocheta and Dina. Furthermore, the pattern of annulation has proved to be inappropriate for the identification of Dina and Trocheta (Trontelj and Sket 2000). A similar observation was made by Bielecki (1997) in the family Piscicolidae and has been mentioned by Williams and Burreson (2006). There are two possible ways to rectify the taxonomic error: the first is to follow Siddall’s (2002) opinion that the genera Dina, Mooreobdella, Nephelopsis and Trocheta should be abolished and synonymized with the genus Erpobdella. Only a thorough revision with specimens from type localities, sequenced at two or more independent loci, and studied anatomically could provide the grounds to do so. This leaves us with the second – possibly provisional – solution: that in the absence of a better solution the traditional taxonomy should be retained, at least provisionally. It should be kept in mind however that the genera Dina and Trocheta might be in fact non-monophyletic and any conclusions based on this premise have to be reconsidered. The description of species of the genus Trocheta using only anatomic and morphologic characters is difficult due to their simplified anatomy. It is important to perform a study that combines morphology, and molecular approach. Morphological (including morphometry) data shows that there are no anatomic characters (i.e. their presence or absence) that can help in species identification. Species are differentiated by groups of quantitative



451

characters (for example number of eyes, number of annuli between gonopores, subdivision of annuli per somite, colour etc.). The anatomical data collected, showed that the most satisfactory characters that could be used to differentiate between species are the shape and the size of the male atrium. Salifid leeches (e.g. species of Salifa and Barbronia) are common in Africa and Asia (Soos 1969; El Shimey 1996). In fact, they were recorded in Egypt with the identification of a new species of Barbronia and two species of salifid leeches, known also in eastern and southern Africa (El Shimey 1987). These two genera have not been reported from Tunisia yet, and we do not think that they have been overlooked in our actual collection.

Distribution The river of Medjerda, the largest river in Tunisia by volume, serves as an ‘avenue’ for leech distribution. Our study focused on the various kinds of water bodies in the North of Tunisia that provide optimal conditions for most species of leeches that occur in the country. Tunisian erpobdellid leeches can be classified into a group of species with stable and strong populations that were recorded from many sites throughout the studied area (D. punctata punctata). Another group including species with weak populations that are only recently recorded in Tunisia, within an increasing number of localities. For these species, there are either no historical records or they had been reported only once in the country prior to the present study (i.e. E. testacea, D. punctata maroccana, T. tunisiana and T. africana). Dina punctata punctata is reported in this paper to be newly found in Tunisia and North Africa. Moore (1939) recorded D. lineata in Morocco but we suggest that he referred to the D. punctata punctata or to the subspecies D. punctata maroccana because we think that the D. lineata doesn’t exist in the occidental part of the Mediterranean. In our opinion the biology and ecology of erpobdellid leeches is very interesting. The leeches are distributed in varied environments. This is especially noticeable within the species of the genus Dina that have settled habitats with extreme conditions of temperature, moisture, lighting, oxygenation, pH levels, salinity, and so on. Recently, it has been reported a new species of leeches with an unusual body form, Croatobranchus mestrovi Kerovec, Kučinić and Jalžić, 1999. Based on anatomical and molecular data, this species has been found closely related to species of the genus Dina (Sket et al. 2001). Related to this species is D. absoloni Johansson, 1913, a troglobiotic leech that is found in caves (Sawyer 1986). Dina lineata (O. F. Müller, 1774) prefers water reservoirs, often drying, similar to D. punctata punctata and D. punctata maroccana. Dina stschegolewi (Lukin and Epshtein, 1960) favors estuaries (Bielecki 1978, 1995, ). Dina apathyi Gedroyc, 1916 occurs in waters with low pH, which is a characteristic of low lying moors (Lukin 1976). Another species resembling Dina is the leech Archaeobdella esmonti Grimm, 1876, which is problematic from a taxonomic point of view because many authors believe it to be a Dina species (Lukin 1976). It lives in specific habitat, mud in brackish waters, and its caudal sucker reduced into a plaque-like structure (Lukin 1976).

A key for freshwater Erpobdellids species of Tunisia We propose the following key for identification of Tunisian Erpobdellidae 1.

2. 3. -

Leeches with two to eight eyes placed dorsally on the head; when there are eight, these are placed in two longitudinal rows. Mouth small and jaws absent. Total number of annuli less than 80 annuli or over 200 . . . . . . . . . . . . . Order: Rhynchobdellida Body dorsoventrally flattened, triannulate segment; the only leeches to carry their youngs . . . . . . . . ..Family: Glossiphonidae Leeches with eight, ten, or more eyes, some of which may be placed laterally on the head ; if there are eight eyes, these are placed in two transverse rows and proboscis absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Order: Arhynchobdellida...2 Pharynx with pseudognaths, testes arranged in numerous grape-like testisacs . . . . . . . . . . . . . . . . . . Family: Erpobdellidae ...3 Pharynx short, mouth armed with toothed jaws; eyes five pairs forming lateral crescents; testes in large sacs arranged segmentally in pairs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Family: Hirudinidae Smaller to medium-sized leeches with cylindrical or slightly flattened body, five annuli per somite, all of them with the same length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus: Erpobdella Dorsal surface reddish brown; gonopores separated by four to five annuli. . . . . . . . . . . . . . . . . . . . . . . . . . Erpobdella testacea Medium-sized to large leeches; animal with lateral keels; annuli b6 always elongated; five to eight annuli per somite . . . . . 4


BEN AHMED ET AL.

4. 5. 6. -

Annulus b6 homonomously subdivided in two annuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus: Dina... 5 Annuli b1 and b6 heteronomously subdivided; five short and three longer annuli per somite. . . . . . . . . . . Genus: Trocheta... 6 Ground colour is brown to green, yellowish spots are arranged transversally on annuli a2; lateral keels present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dina punctata punctata Dark blackish ground colour, more than 8 prominent yellowish spots on each annulus, body form dorsally flattened and ventrally rounded, sharp lateral keels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dina punctata maroccana Large leeches, ground colour of the body black, four dark longitudinal stripes dorsally, lateral keels prominent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Trocheta africana Medium-sized leeches, ground colour of the body light brown, four dark longitudinal stripes dorsally, lateral keels not prominent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trocheta tunisiana n. sp.

Acknowledgments The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project NoRGP-VPP-164. We also thank “Unité de Recherche Biologie de la Reproduction et du Développement” and the DGRSRT Tunisia for financial support. We would like to thank Professor Fathia Zghal for her precious advice.

References Agapow, L. & Bielecki, A. (1992) A systematic study on the complex species Erpobdella testacea (Savigny, 1820) (Hirudinea, Erpobdellidae). Genus, 3 (4), 185–199. Ben Ahmed, R., Tekaya, S. & Harrath, H. (2008) Etude préliminaire des Hirudinées en Tunisie: Description et systématique (Clitellata, Hirudiniea). Bulletin de la société zoologique de France, 133 (1–3), 81–91. Ben Ahmed, R. & Tekaya, S. (2009) Etat des connaissances sur la Biogéographie des Hirudinées du pourtour de la Méditérranée. Bulletin de la société zoologique de France, 134 (1–2), 99–124. Bielecki, A. (1978) Herpobdella (Dina) stschegolewi Lukin et Epstein, 1960 – nowy gatunek pijawki dla fauny Polski. Przeglad Zoologiczny, 22 (2), 140–143. Bielecki, A. (1995) The effect of salinity on leech (Hirudinea) communities of the lake Jamno. Przeglad Zoologiczny, 29, 23– 35. Bielecki, A. (1997) Fish leeches (Hirudinea: Piscicoidae: Piscicolinae) of Poland in relation to the Palearctic piscicolines. Genus (2), 223–375. Blanchard, R. (1892) Sur la présence de la Trocheta subviridis en Ligurie et description de cette Hirudinée. Atti della Società Linguistica di Scienze naturali e geografiche, 3 , 1–31. Blanchard, R. (1908) Hirudinées. In: Kerville, H.G. de. (Ed.), Voyage Zoologique en Khroumirie (Tunisie), B. Baillère et fils, Paris, pp. 307−310 El Shimey, N.A. (1996) Revision of the genus Barbronia Johansson,1918 (Hirudiniea: Erpobdelliformes: Barbronidae) in Egypt. Zoology in the Middle East, 12, 99–104. http://dx.doi.org/10.1080/09397140.1996.10637695 ELShimey, N.A. (1987) Ecological and biological studies on freshwater Hirudinea. Ph.D. Thesis, Assiut University, Assiut, Egypt (unpublished thesis), 224 pp. Košel, V. (2004) Taxonomic position of two species of Trocheta (Hirudinea) described from Central Europe. Biologia, 59, Suppl. 15, 25–27. Lukin, E.J. (1976) Pijavki presnych i solenovatych vodojemov. Fauna SSSR, Pijavki Izdat. Nauka, Leningrad, 484 pp. Manoleli, D. (1972) Contributions concernant la systématique et la distribution du genre Erpobdella de Blainville 1818 (Hirudinoidea, Erpobdellidae) en Roumanie. Tarvaux du Museum d’Histoire Naturelle, 17, 1–7. McNicol, D.K., Mallory, M.L., Mierle, G., Scheuhammer, A.M. & Wong, A.H.K. (1997) Leeches as indicators of dietary mercury exposure in non–piscivorous waterfowl in Central Ontario, Canada. Environmental Pollution, 95, 177–181. http://dx.doi.org/10.1016/S0269-7491(96)00125-X Moore, J.P. (1939) Leeches (Hirudinea) from the Atlas Mountains of Morocco. Annals and Magazine of Natural History, 3, 80– 87. Nesemann, H. & Neubert, E. (1994) A new species of Trocheta (Hirudinea, Erpobdellidae) from France (Pyrenees, Nivelle River basin). Lauterbornia, 26, 27–30. Nesemann, H. & Neubert, E. (1996) New data to the leeches of the subfamily Trochetinae (Hirudinea, Erpobdellidae), Miscellanea Zoologica Hungarica, 9, 19–28. Nesemann, H. (1997) Egel und Krebsegel (Clitellata: Hirudinea, Branchiobdellida) Ôsterreichs. Gedruckt mit Unterstützung des Bundesministeriums für Umwelt, Jugend und Familie, Wien, 107 pp. Neubert, E. & Nesemann, H. (1995) A new species of Batracobdella from Turkey. Zoology in the Middle East, 11, 109–111.



453

Sawyer, R.T. (1986) Leech Biology and Behaviour .Vol. 3. Clarendon Press, Oxford, 1065 pp. Seaby, R.M.H., Martin, A.J. & Young, J.O. (1995) The reaction time of Leech and triclad species to crushed prey and the significance of this for their coexistence in British lakes. Freshwater Biology, 34, 21–28. http://dx.doi.org/10.1111/j.1365-2427.1995.tb00419.x Siddall, M.E. & Burreson, E.M. (1998) Phylogeny of Leeches (Hirudinea) based on mitochondrial cytochrome c oxidase subunit I. Molecular Phylogenetics and Evolution, 9, 156–162. http://dx.doi.org/10.1006/mpev.1997.0455 Siddall, M.E. (2002) Phylogeny of the leech family Erpobdellidae (Hirudinida: Oligochaeta). Invertebrate Systematics, 16, 1– 6. Sket, B. (1968) Zur Kenntnis der Egel-Fauna (Hirudinea) Jugoslawiens. Academia Scientiarum et Artium Slovenica CL. IV Pars Historiconaturalis, Diss. 9 (4), 127–178. Sket, B., Dovč, P., Jalžić, B., Kerovec, M., Kučinić, M. & Trontelj, P. (2001) A cave leech (Hirudinea, Erpobdellidae) from Croatia with unique morphological features. Zoologica Scripta, 30, 223–229. http://dx.doi.org/10.1046/j.1463-6409.2001.00065.x SoỏS, A. (1969) identification key to the leech (Hirudinoidea) genera of the world with a catalogue of the species.III. Family: Erpobdelidae. Acta Zoologica Academiae Scientiarum Hungaricae, 12(3–4), 371–407. Trontelj, P. & Sket, B. (2000) Molecular re-assessment of some phylogenetic, taxonomic and biogeographic relationships between the leech genera Dina and Trocheta (Hirudinea: Erpobdellidae) inferred from restriction-site data of the 18S ribosomal gene and ITS2 region. Journal of Zoological Systematics and Evolutionary Research, 37, 85–93. Wicklum, D. & Davies, R.W. (1996) The effects of chronic cadmium stress on energy acquisition and allocation in a freshwater benthic invertebrate predator. Aquatic Toxicology (Amsterdam) 35, 237–252. http://dx.doi.org/10.1016/0166-445X(96)00795-3 Williams, J.I. & Burreson, E.M. (2006) Phylogeny of the fish leeches (Oligochaeta, Hirudinida, Piscicolidae) based on nuclear and mitochondrial genes and morphology. Zoologica Scripta, 35 (6), 627–639. http://dx.doi.org/10.1111/j.1463-6409.2006.00246.x Zaranko, D.T., Griffiths, R.W. & Kaushik, N.K. (1997) Biomagnification of polychlorinated biphenyls through a riverine food web. Environmental Toxicology and chemistry, 16, 1463–1471. http://dx.doi.org/10.1002/etc.5620160720 Zerbst-Boroffka, I. (1999) Osmotic adaptation of the endemic fauna to the ancient freshwater Lake Baikal. Naturwissenschaften, 86, 330–333. http://dx.doi.org/10.1007/s001140050627


BEN AHMED ET AL.