Checklist and Distribution of Marine and freshwater leeches (Annelida, Clitellata, Hirudinea) in Tunisia with i dentification key

In this study 13 leech species from Tunisia are list d. They belong to 2 orders, 2 suborders, 4 families and 11 genera. The paper includes also data about hosts and habitat s, dis ribution in the world and in Tunisia. Faunis tic informations on leeches were found in literature and in the results of recent surveys conducted by the authors in the North East and the South of the country. The objectives of this study were to summarize historical and recent taxonomic d ata, and to propose an identification key for species signalize d. This checklist is to be completed, taking into a ccount the hydrobiological network of the country especially t he North West region, which may reveal more species in the future.


Introduction
Available information on the distribution, taxonomy, and ecology of Tunisian leeches has been scattered throughout various historical (Blanchard 1891, 1908, Megnin 1891, Seurat 1922) as well as recent papers (Ben Ahmed et al. 2008a, 2008b, 2008c, Ben Ahmed & Tekaya 2009, Ben Ahmed et al., 2013, Nesemann & Neubert 1994).None of these papers have focused on the country's fauna as a whole.Furthermore, until 2006, only 5 leech species have been reported for Tunisia.Also, the taxonomy of Mediterranean marine leeches is poorly known, and information on Tunisian fauna is limited to a single species recently recorded by Ben Ahmed et al. (submitted paper).The principal limitation on availability of material is because marine leeches are difficult to obtain with standard sampling equipment.
This paper lists 12 freshwater and one marine leech species and the list is expected to get much longer in the future.

Material and Methods
The information and data summarised herein have been obtained from previously published papers and from extensive material collected by the authors during the period 2006-2012.Freshwater leeches were collected from rivers, drainage basin, springs and streams (Fig. 1).Leeches were found attached to the underside of a variety of objects, such as rocks and submerged vegetation.The material examined was deposited in the first author's collection at the Faculty of Science of Tunis.A Tunisian marine leech, attached to the fish Symphodus tinca (Linnaeus, 1758), was collected from the lagoon of Bizerte.
In the laboratory, the living leeches were first photographed using a Nikon Coolpix digital camera, then a preliminary identification was made based on external features as that several specimens could be identified using

Research Article
external morphology only.Other leeches requiring the internal structure study, were narcotised, dissected, preserved in 70% ethanol and then identified using an Olympus SZ-ST stereomicroscope.

Systematic account
In total, 13 species of Hirudinea representing four families (Table I) are now known to occur in Tunisia based upon records in the literature and/or specimens collected during our seven-year study (2006)(2007)(2008)(2009)(2010)(2011)(2012).Detailed accounts on these leeches are presented below.Habitat: Found under stones, it seems to prefer stagnant to slow flowing water bodies often of artificial origin such as drainage basin.The presence of aquatic molluscs appears to be important.
In Tunisia (Fig. 5B), this species was reported for the first time by Ben Ahmed et al. (2008a) in Lebna drainage basin (36.44326 N, 10. 55 255 E).In the present study, we add its presence in Port Prince drainage basin (36.51162 N, 10. 39404 E).It seems that this species has a limited distribution and up till now only known from two localities in Nabeul governorate in the North East of the country.

Diagnosis:
The body is dorsoventrally flattened.The dorsal surface is covered by papillae with three distinct longitudinal rows of larger papillae.The body colour is brown.The head bears two separate eyes.The caudal sucker is large.The genital pores are separated by two annuli and seven pairs of crop caeca are present.

Distribution:
This species was described from Algeria.Viguier (1879) attributed this leech to the genus Batracobdella.Seurat (1922) recorded it under the name Helobdella algira from Algeria and Tunisia but without citing localities.It has been collected from the skin of several Amphibian Anura species (Discoglossus pictus, Pelopphylax ridibundus) and Urodeles (Hydromantes genei).Soỏs (1969) reported the following distribution: North Africa, Portugal, Spain, Balearic Islands, Corsica and the Crimean Peninsula.Minelli (1979) mentioned the same distribution, but added its presence in Sardinia.Nesemann & Neubert (1999) mentioned that this rare species is only known from North West Africa and from the western Mediterranean.They also noted that there are records from Morocco, Algeria, Portugal, Spain and Corsica.B. algira is widespread on the Iberian Peninsula, but not common.

Diagnosis:
The body is flattened and lanceolate.The general colouration is gray.The head bears one pair of eyes.A brown plaque (scute) is present on the dorsal side in the neck region.The gonopores are separated by a single annulus.

Habitat:
The species was collected under stones, especially in stagnant and slowly running waters.Associated fauna: chironomids and Gammarus.
Distribution: Helobdella stagnalis is a cosmopolitan species.It is common in Europe and fairly frequent in Asia and in North and South America.It is poorly represented in India and in Africa.It was reported from Morocco (Moore 1939) and recently from Egypt by Gouda & El-Shiemy (2004).Additional records from South Africa have been reported by Oosthuizen & Siddall (2003).
In Tunisia (Fig. 5A), this species was collected under emergent stones by Ben

Genus Placobdella (Blanchard, 1893)
Placobdella costata (F.Müller, 1846) (Fig. 2D) Diagnosis: P. costata is a flattened species.The colour of living specimens is green to brown.Seven longitudinal rows of numerous papillae are present on the dorsal face.The mouth pore is situated on the anterior rim of the cranial sucker.The latter bears two unseparated eyes.The crop has seven pairs of crop caeca and the genital pores are separated by two annuli.
Habitat: Found in small groups under rocks and under various objects such as plastic bottles.It colonizes several habitats (drainage basins, oueds and springs ...).An ectoparasitic association with the turtle Emys orbicularis was noted (Ben Ahmed & Tekaya 2009).
Distribution: P. costata was described from the Crimean Peninsula, under the name Clepsine costata.Rousseau (1912) recorded it from Southern Europe (France, Italy and Crimean Peninsula).Soỏs (1969) recorded that this species is widely distributed in Europe (except North and Northwest Europe) and North Africa.Minelli (1979) found it in Italy (Rome, Sassari and Padova).In the Middle East, this species has been recorded from Lake Hula in Israel (Bromley 1989).Nesemann & Neubert (1999) have characterized P. costata as a Mediterranean species extending eastwards to the Ukraine, south-eastwards to Greece, Turkey and the Levant, and to the southern Arabian Peninsula.In West and Central Europe, this species was recorded from southern England, the Netherlands, Poland and Germany (Nesemann & Neubert 1999).Recently, Sağlam (2001) collected P. costata in discharge channels at Elazig in Turkey, which correspond to the first record for the country.More recently, Vamberger & Trontelj (2007) recorded it for the first time in Slovenia.
In Tunisia (Fig. 5C), this species was collected by Henri Gadeau de Kerville in Jendouba governorate: the valleys of el Omor (Tabarka) and El Lebga (Aïn Drahim), being identified by Blanchard (1908) as Placobdella catenigera.Ben Ahmed et al. (2008a) have recorded it in Nabeul governorate (Lebna drainage basin).In this paper, we add its presence in a new locality of this later governorate: Port Prince drainage basin (36.51162N, 10.39404E); in a marsh in Jendouba governorate (36.30N, 8.47E) and in a source in El Kef governorate (36.1056N, 8.4253E).

Diagnosis:
The body is dorsoventrally flattened and nearly translucent.The colour of living specimens is green to grey.The dorsal body surface shows four rows of longitudinal papillae.The head bears four pairs of eyes and the genital pores are separated by four annuli.The species is known as bloodsucker of birds.
Habitat: Found under stones, it seems to prefer stagnant or slow flowing water bodies most often of artificial origin such as drainage basins.The presence of aquatic birds appears to be important.
Distribution: This species was described as Hirudo tessulata from Lake Frederiksdahl, Denmark.Soós (1969) and Minelli (1979) reported it from the Palaearctic region and South America.In the Middle East, T. tessulatum was reported in Palestine and Lebanon by Blanchard (1893) and Moore (1944).Bromley (1994), in his investigation in the Middle East, did not find this leech and suggested that it may have disappeared in this region.Recently, Jueg (2008a) has collected it in Central Asia (Kyrgyzstan) and Spain.The species was already reported by Johansson (1927) in Spain.
In Tunisia (Fig. 5D), this species was recorded in the North East of the country in the governorate of Nabeul in two stations: Lebna drainage basin (36.44326N, 010.5525,5E) by Ben Ahmed et al. (2008a) and in Port Prince drainage basin (36.51162N, 10.39404E) (this study).

Diagnosis:
The maximum length is 3 cm.The body has obvious paired pulsatile vesicles.The colour of living specimens is yellow-orange.The oral sucker has one pair of eyes.The posterior sucker is very small and is without ocelli.The male and female gonopores are separated by one annulus.This parasitic species was found in the gill cavity of teleosts.
Habitat: This leech lives exclusively in the gill chambers of a variety of teleost fish in warm temperate and tropical seas worldwide.The mature leeches leave the fish to lay eggs, after which they die (Sawyer 1986;Williams et al. 1994).
Distribution: Trachellobdella lubrica has been reported on marine fishes from the Mediterranean, west coast of Africa, Caribbean, Gulf of Mexico, South Carolina and near the Hawaiian Islands (Sawyer 1986).
In Tunisia (Fig. 5F), the species was collected from the fish Symphodus tinca (Linnaeus 1758) in the lagoon of Bizerte.

Diagnosis:
The colour of living animals is grass green.The dorsal surface shows six longitudinal bands of orange and black spots.The venter is greenish to yellow with large black spots.Laterally, there are two marginal longitudinal zigzag lines.This species has three muscular jaws.The anterior sucker has a parabolic arc of 10 eyespots and the genital gonopores are separated by five annuli.
Habitat: Found under stones and other submerged objects.It seems to prefer drainage basins and marshes.It was not found in springs.
Distribution: Seurat (1922) recorded it from Algeria (in Mitidja) and reported its presence in Spain, Portugal and Italy (Sardinia).

Diagnosis:
The dorsal side shows two different color patterns: the typical pattern is green to brown with four interrupted black lines, while some specimens have an additional median orange band.Marginally, there are two orange stripes.The ventral side of the body is dark brown.The anterior sucker has five pairs of parabolic eyes, the posterior sucker is very large and the genital pores are separated by five annuli Habitat: Eurytopic species, common in the springs, oueds and drainage basins.
Distribution: L. nilotica (Syn.Bdella nilotica Savigny, 1822) was the first species discovered by Savigny, who accompanied Napoleon's expedition to Egypt.This species was attributed by Moquin-Tandon (1846) to the genus Limnatis because the name Bdella was preoccupied by a genus of Arachnida.Blanchard (1908) and Seurat (1922) mentioned the following distribution for L. nilotica: Egypt, Turkestan, Middle East, southern Italy (Sicily) and Spain (Balearic and Canary islands).Nesemann & Neubert (1999) mentioned it as a circum-Mediterranean species, occurring in the Ukraine and the Crimean Peninsula, in the Middle East, the Arabian Peninsula and eastern Africa.

Diagnosis:
The dorsal surface of the living specimens is reddish brown in colour with minuscule darker specks.The ventral surface is brighter than the dorsal.The mid-body segment consists of five equally long annuli.The head has four pairs of eyes.Gonopores are separated by 4 annuli.

Habitat:
The occurrence of E. testacea in Tunisia is restricted to lowland stagnant waters (50-65 m asl.).

Diagnosis:
The colour of living specimens varies from reddish brown to greenish.The dorsum has a rough surface due to the presence of numerous small papillae and has yellow spots arranged transversally on annulus a2.The somites are divided into four short and one long annuli.The head has four pairs of eyes arranged semi-circular along the anterior edge of the head.The gonopores are separated by 1.5 to 2 annuli.Habitat: It was found in small ditches and in temporary streams.
Distribution: Dina lineata was described from Spain and two varieties notata and punctata was distinguished.Therefore it is believed that the geographical distribution of D. punctata requires confirmation.Prior to Johansson (1927) the variety punctata was not known and most recent authors did not discriminate both varieties.Also, the taxonomy of the genus Dina in the western Mediterranean requires revision.Indeed, Minelli (1979) reported that D. lineata recorded from Italy is probably D. punctata.Also Jueg (2008), during his investigations in the Iberian Peninsula, found that D. punctata was very common in this area and D. lineata is totally absent.The latter suggested that D. lineata reported for the Iberian Peninsula by García-Más & Jiménez (1984) and García-Más et al. (1998) is Dina punctata.Recently, Nesemann & Neubert (1994) described a new subspecies from Morocco (D. punctata maroccana) and they suggest that some of the specimens described as Dina lineata by Moore (1939) from Morocco can be considered to be conspecific to their new subspecies.We think that in the eastern Mediterranean, D. punctata maroccana may be confused with D. punctata.Until further evidence, the taxonomic status of Dina lineata and the geographic distribution of Dina punctata remain uncertain.
In Tunisia (Fig. 6A), it was found in almost all governorates (Ben Ahmed et al. 2013)

Diagnosis:
The colour of living specimens varies from greenish brown to blackish.The dorsum is covered with prominent yellow spots and by small papillae and, thus, appears rough.Each somite is divided into five annuli; the first four are short and the fifth is long.The body is dorso-ventrally flattened.The eyes are arranged in four pairs, as in the nominate subspecies.The gonopores are separated by two annuli.
Habitat: This leech was found in a stream.It is semi-aquatic, with behaviour similar to that of D. punctata punctata.The associated fauna includes planarians (Dugesia sicula (Lepori, 1948)), gastropods and isopods.

Diagnosis:
The colour of living specimens is black.Four dark narrow longitudinal stripes are visible in the smooth dorsal surface.The area between the inner paramedian stripes is less pigmented than the rest.Lateral keels are present.The ventral surface is pale grey.Annulation: somites with eight annuli, the first two of which are short, followed by three longer ones and another three short annuli.Head is with four pairs of eyes.The gonopores are separated by two annuli.
Habitat: This species prefers stagnant water or slow running rivers or brooks.It occurs mainly in the higher regions of northern Tunisia (805-868 m asl.).The specimens were found attached to the underside of rocks and stones and underneath the dead leaves of Quercus suber.

Diagnosis:
The colour of living specimens is light brown.Dorsally, there are four longitudinal stripes.The ventral surface is always somewhat lighter than the dorsum.The head has four pairs of eyes.In the postclitellar region, lateral keels are present.Annulation is typical for 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.The gonopores are separated by two annuli.
Habitat: Trocheta tunisiana has been collected in smaller brooks and springs occurring in the mid and higher elevations (usually over 350 m asl.

Discussion and Concluding remarks
The check-list of leeches (Hirudinida) of Tunisia discussed in this paper includes thirteen species, reflecting historical and recent records published in the literature and specimens from collections by the authors during this seven-year study (2006 through 2012).The following seven of them were collected by the present authors: Alboglossiphonia hyalina, Helobdella stagnalis, Theromyzon tessulatum, Erpobdella testacea, Dina punctata punctata, Dina punctata maroccanna and Trocheta tunisiana.The others (Batracobdella algira, Placobdella costata, Hirudo troctina, Limnatis nilotica and Trocheta africana had been recorded before from Tunisia.Further surveys extended to new governorates are recommended since we believe that many more new species await discovery.For example Glossiphonia complanata (Linnaeus, 1768), cited for the Maghreb by Mann (1978) without indication of locality is a potential species.The 13 species recorded in the present study show Holarctic, Palaearctic and also endemic Maghrebian distribution.Dina punctata, which is considered to be the most common species in Tunisia, is completely absent in the eastern Mediterranean (Ben Ahmed and Tekaya 2009) and thus it shows a western Mediterranean or Alboran distribution.Similarly, Batracobdella algira and Limnatis nilotica are common in Tunisia occurring in a wide range of water bodies, such as drainage basin, springs and marshes.On the other hand, the actual distribution of these two species shows that they are present mostly in the circum-Mediterranean regions.The genus Hirudo Linnaeus, 1758 is composed of five species: the European medicinal leech (m.l.) H. medicinalis, the Mediterranean m.l.H. verbana, the Caucasian m.l.H. orientalis, the African m.l.H. troctina, and the East Asian m.l.H. nipponia.Hirudo troctina is also a common species in Tunisia and it is considered as an interesting biogeographic case worth mentioning.In fact, it is the sole species of the genus in Africa and thus it represents an endemic Maghrebian distribution.Also Trocheta africana is the only species of the genus in Africa and seems to be restricted to the western part of Tunisia.This species shows a Tuniso-algerian distribution as it was recorded to date only in these two countries.Perhaps this is also true for Trocheta tunisiana.T. tessulatum is very rare and only known from two drainage basin s in the eastern part of Tunisia.This species has a Holarctic distribution.Its introduction via birds from Europe to North Africa is quite possible.Alboglossiphonia hyalina and Erpobdella testacea have a wide distribution in Europe and might represent an example of Palaearctic distribution.Both species, as well as the cosmopolitan Helobdella stagnalis, are rare in Tunisia and only known from a few localities.

A key for identification of Tunisian leeches species
We propose the following key for identification of Tunisian Leeches.
The body is flattened and uniform yellowish coloured.The rounded head has three pairs of eyes.The dorsal surface is more or less rough by small regularly arranged papillae.The genital pores are joined and open in a common pore.Ectoparasite of freshwater Gastropods, sometimes penetrating into the mantle cavity.

Figure 5 .
Figure 5.The distribution of selected leech species (families Glossiphoniidae, Piscicolidae and Hirudinidae) in Tunisia based upon literature and specimens collected during this study.The symbol indicates the governorate in which the species is found, and not the exact location.

Figure 6 .
Figure 6.The distribution of selected leech species (Family Erpobdellidae) in Tunisia based upon literature and specimens collected during this study.The symbol indicates the governorate in which the species is found, and not the exact location.