The oligotrophic karstic Lake Ohrid is located in the western part of the Balkan Peninsula on the Macedonian-Albanian frontier. With a limnological age of 2-5 million years it is considered to be one of the oldest lakes in Europe (Albrecht and Wilke 2008). The lake is characterized by a high degree of biodiversity and endemicity in several groups of organisms (Stanković 1960, Albrecht and Wilke 2008). With more than 210 known endemic species it is probably the most biodiverse lake in the world, at least when one takes surface area into account (Albrecht and Wilke 2008). Due to its peculiarities, Lake Ohrid is considered to be a key site for biodiversity and speciation research (Albrecht and Wilke 2008).

The first studies on the triclad fauna of the Ohrid area date back to the 1920’s with the first description of several new species of Phagocata Leidy, 1847 and Dendrocoelum Örsted, 1844 (cf. Stanković and Komárek 1927). Further important researches carried out during the 20th century, mainly by Stanković (1938, 1960, 1969) and Kenk (1978), contributed to a better knowledge of this very interesting planarian fauna. In his valuable monograph, Stanković (1960) in particular pointed out the extraordinary biogeographical situation of the endemic triclads in the Ohrid region.

In this paper we report on a new species of freshwater planarian of the genus Dugesia, forming the first fully documented species description for this genus in the Ohrid area. Our morphological species delimitation was supported by complementary molecular, karyological, and cytogenetic data available from the literature. Therefore, our species delineation is based on a truly integrative approach. Further, we provide a short account on the biogeographical patterns in freshwater planarians and their degree of endemicity in the Ohrid region.

Planarians were collected in 1995 from the southern section of Lake Ohrid basin, near the town of Çërravë, along the Pogradec-Korçë road, ca. 10 km south-east of Pogradec, at an altitude of ca. 800 m asl (Fig. 1). The animals were found under pebbles and among vegetation in a rivulet, flowing along a steep meadow, joining a tributary stream of the lake. All individuals (n = 20) were asexual at collection. The collected specimens were transferred to the laboratory, reared in glass bowls under semi-dark conditions at 18 +/- 2 °C and fed with fresh beef liver.

After having been kept in the laboratory for about one year, during which the strain notably increased in numbers due to asexual reproduction by fission, approximately 30% of the specimens shifted from the fissiparous reproductive mode towards a tendency to sexualize, i.e. to develop reproductive organs. These sexualized animals displayed the characteristic features of ex-fissiparous individuals: large body size, deve-lopment of the copulatory apparatus, hyperplasic ovaries.

For morphological study sexualized specimens were fixed for 24 hours in Bouin’s fluid, dehydrated in a graded ethanol series, cleared in toluene, and embedded in synthetic paraffin. Serial sections were made at intervals of 5–7 μm and were stained with Harris’ haematoxylin-eosin, Mallory’s trichrome, or Pasini’s reagent.

The material is deposited in the Naturalis Biodiversity Center, Leiden, The Netherlands (collection code: ZMA), and in the Giacinta A. Stocchino collection (CGAS), University of Sassari.

Dugesia superioris differs from its congeners in particular in (a) the dorsal course of the ejaculatory duct, with its sub-terminal opening, (b) the asymmetrical openings of the oviducts into the bursal canal, and (c) the openings of vasa deferentia at about halfway along the seminal vesicle.

For the genus Dugesia a dorsal course of the ejaculatory duct was reported for the first time by Stocchino et al. (2005) for the endemic Sardinian species Dugesia hepta Pala, Casu & Vacca, 1981. However, in this species the opening of the duct is located laterally on the right side, near the tip of the penis papilla. Moreover, this species is characteri-zed by a ventro-lateral penial fold, which is absent in the new species. Dugesia superioris therefore represents the second species of the genus showing a dorsal course of the ejaculatory duct. Further, another important difference between Dugesia hepta and Dugesia superioris is the haploid chromosome number, which counts n = 7 in the former (Pala et al. 1981) and n = 8 in the latter (Deri et al. 1999, see below).

A subterminal opening of the ejaculatory duct, as found in Dugesia superioris, occurs in no less than 26 species of Dugesia: Dugesia bakurianica Porfirjeva, 1958, Dugesia biblica Benazzi & Banchetti, 1972, Dugesia leporii Pala et al., 2000, and Dugesia sicula Lepori, 1948, from the Western Palaearctic; Dugesia aethiopica Stocchino et al., 2002, Dugesia arabica Harrath & Sluys, 2013, Dugesia astrocheta Marcus, 1953, Dugesia lanzai Banchetti & Del Papa, 1971, Dugesia lamottei De Beauchamp, 1952, Dugesia neumanni (Neppi, 1904) and Dugesia myopa De Vries, 1988b from the Afrotropical Region; the other 15 species are distributed in the Oriental Region, Eastern Palaearctic and Australasian Region, viz. Dugesia andamanensis (Kaburaki, 1925), Dugesia austroasiatica Kawakatsu, 1985, Dugesia batuensis Ball, 1970, Dugesia bengalensis Kawakatsu, 1983, Dugesia burmanensis (Kaburaki, 1918), Dugesia deharvengi Kawakatsu & Mitchell, 1989, Dugesia indica Kawakatsu, 1969, Dugesia indonesiana Kawakatsu, 1973, Dugesia japonica Ichikawa & Kawakatsu, 1964, Dugesia leclerci Kawakatsu & Mitchell, 1995, Dugesia lindbergi De Beauchamp, 1959, Dugesia nannophallus Ball, 1970, Dugesia novaguineana Kawakatsu, 1976, Dugesia tamilensis Kawakatsu, 1980, and Dugesia uenorum Kawakatsu & Mitchell, 1995. However, in all of these species the ejaculatory duct is ventrally displaced, except for Dugesia bakurianica in which the ejaculatory duct is central. Therefore, a dorsal course of the ejaculatory duct and a subterminal opening of the duct represents a new diagnostic combination in the genus Dugesia.

The Pogradec population had already been subjected to karyological, cytogenetic, and phylogeographic studies before anything was known about the anatomy of the specimens (see above). All of these analyses pointed to a situation that this Dugesia population differs considerably from congeneric populations. Therefore, it was unsurprising that the anatomy of the Pogradec animals suggested also that they represent a new species. As a result of the cumulation of the evidences from these independent datasets, the present delineation of the new species is based on a truly integrative approach to taxonomy.

Studies on the phylogeny of Dugesia (Sluys et al. 1998 and references therein) considered the asymmetrical penial papilla to constitute an important taxonomic feature. However, this asymmetry related to the apomorphic presence of a ventral ejaculatory duct. Our present study shows that in future analyses this asymmetry needs to be specified by adding a third character state to character (1) (Sluys et al. 1998, p. 277 and Table II), i.e. ejaculatory duct located dorsally.

An asexual population of Dugesia sp. was collected in 2006 by R. Manconi from Voskopojë, an Albanian locality situated south-west of Lake Ohrid. Unfortunately, we have been unable to ascertain the taxonomic status of this population due to the lack of sexual specimens (Stocchino and Manconi, pers. obs.). However, according to the phylogeographic analysis of Lázaro et al. (2009) this population is molecularly identical to the Pogradec population and therefore should be assigned also to Dugesia superioris. It is noteworthy that the Voskopojë locality is outside of the Ohrid basin and therefore signals a wider distribution of Dugesia superioris.

That Kenk (1978) identified his Dugesia material from Ohrid (NMNH 55294) as Dugesia gonocephala is hardly surprising in view of the fact that at that time many European populations were assigned to Dugesia gonocephalasensu lato. The precise anatomy of Dugesia gonocephalasensu stricto was only resolved by De Vries and Ball (1980) and De Vries (1984a, 1986). A comparison with Kenk’s specimen quickly learns that this animal does not conform to Dugesia gonocephala because it does not exhibit the muscular ridges, the elongated penis papilla, or the two penial folds (cf. De Vries and Ball 1980, De Vries 1984a). In the presence of a small dorsal penial fold and a central ejaculatory duct the animal resembles Dugesia benazzii Lepori, 1951, Dugesia elegans De Vries, 1984, Dugesia taurocaucasica (Livanov, 1951) and Dugesia effusa, the latter recently described from the Greek island Chios (Sluys et al. in prep.). Dugesia benazzii from Corsica and Sardinia is characterized by a pointed diaphragm and a penial fold, the position of which is variable but which is usually located dorsally; the size of the penial fold is also variable (Lepori 1951, De Vries 1984b). In Dugesia benazzii ectal reinforcement is restricted to the region of the oviducal openings, the latter being symmetrically arranged. In contrast, in the NMNH 55294 specimen the oviducts open asymmetricaly into the bursal canal, while the ectal reinforcement extends further on the bursal canal.

Dugesia elegans from Rhodes differs from NMNH 55294 in the presence of a much larger seminal vesicle, a stubbier diaphragm, and the situation that its bursal canal epithelium is infranucleated (De Vries 1984a).

The penial fold of Dugesia taurocaucasica is considerably larger than the one in NMNH 55294, while the fold is also traversed by the abundant secretion of cyanophilic glands, which discharge through the lining epithelium of the penial fold. Furthermore, in Dugesia taurocaucasica the ectal reinforcement layer on the bursal canal extends for a considerable distance towards the copulatory bursa (Porfirjeva and Dyganova 1987).

The species Dugesia effusa differs from NMNH 55294 in the presence of a short, valve-like diaphragm, a large intrabulbar seminal vesicle, a highly glandular penis papilla, and symmetrical oviducal openings into the bursal canal.

Therefore, the Dugesia specimen NMNH 55294 may well represent a new species. However, on the basis of only the presently available material we refrain from describing it as new. Furthermore, the asymmetrical openings of the vasa deferentia into the seminal vesicle of this animal represents a highly unusual condition for a species of Dugesia and needs to be checked on additional material.

Present data support Stanković’ (1960) suggestion that two faunistic complexes may be distinguished in the Lake Ohrid region, viz. (1) lacustrine endemic forms and (2) inhabitants of other waters outside of the lake with a wider distributional range. In the Ohrid region it is evident that there is a very low degree of exchange between the lacustrine endemic fauna and the non-endemic fauna. Further, the distribution of triclads in the Lake Ohrid area supports the situation for the lake’s fauna in general, namely that endemism occurs at different spatial scales, ranging from species endemic to some parts of the lake to species endemic to the whole Ohrid basin (Albrecht and Wilke 2008).