Integrative taxonomy of a new species of planarian from the Lake Ohrid basin, including an analysis of biogeographical patterns in freshwater triclads from the Ohrid region (Platyhelminthes, Tricladida, Dugesiidae) Author Stocchino, Giacinta Angela Author Sluys, Ronald Author Deri, Paolo Author Manconi, Renata text ZooKeys 2013 313 25 43 http://dx.doi.org/10.3897/zookeys.313.5363 journal article http://dx.doi.org/10.3897/zookeys.313.5363 1313-2970-313-25 Dugesia superioris sp. n. Figs 1-4; Table 1 Material examined. Holotype: ZMA V.Pl. 7153.1, Cerrave , Pogradec District ( 40°50'56"N , 20°42'60"E ), Lake Ohrid basin, Albania, August 1995, coll. P. Deri and N. Mazniku, one set of sagittal sections on 50 slides (stained in Harris' haematoxylin-eosin). Paratypes: CGAS Pla 6. 1, ibid., sagittal sections on 43 slides (stained in Harris' haematoxylin-eosin); CGAS Pla 6. 2, ibid., sagittal sections on 12 slides (stained in Mallory's trichrome); CGAS Pla 6. 3 ibid., transverse sections on 135 slides (stained in Pasini's reagent); ZMA V.Pl. 7153.2, ibid., transverse sections on 131 slides (stained in Harris' haematoxylin-eosin); CGAS Pla 6. 4, ibid., transverse sections on 60 slides ( stained in Harris' haematoxylin-eosin); ZMA V.Pl. 7153.3, ibid., horizontal sections on 21 slides (stained in Harris' haematoxylin-eosin). Diagnosis. Dugesia superioris is characterized by the presence of the following features: dorsal course of the ejaculatory duct; subterminal opening of the ejaculatory duct; asymmetrical openings of the oviducts into the bursal canal; openings of vasa deferentia at halfway along the seminal vesicle; plump penis papilla; small diaphragm; triploid chromosome complement of 24 + 1B-chromosomes. Description. Body size of living fissiparous specimens ranged from 7-10 mm in length and 1.5-2 mm in width (Fig. 2). Sexualized specimens were about 13-16 mm in length and about 3 mm in width. Two eyes are present in the middle of the head, and unpigmented auricular grooves are marginally placed just posteriorly to the eyes. The colour is uniformly brown dorsally, and pale ventrally. Inner and outer pharyngeal musculature is bilayered, i.e. without an extra, third, outer longitudinal muscle layer. The ovaries are hyperplasic, with several scattered masses at a short distance behind the brain, filling up the entire dorso-ventral space. A degenerative condition is clearly evident in the ovaries, in that maturation of the oocytes is regular up to the beginning of the diplotene stage, whereas diplotenic oocytes show progressive cytoplasm vacuolation, followed by collapse of the entire cell content and by cell necrosis. Figure 2. Dugesia superioris . Habitus of a living fissiparous specimen. The anterior portion of the infranucleated oviducts is expanded to form a seminal receptacle that arises in the middle of the ovarian masses at a poorly defined position, dependent upon the hyperplasic condition of the ovaries. The oviducts run ventrally in a caudal direction up to the vaginal area and open asymmetrically into the distal section of the bursal canal. The right oviduct opens dorsally to the left one. The latter opens very close to the point where the canal communicates with the common atrium (Fig. 3). The very abundant shell glands open at the level of the left oviducal opening. Figure 3. Dugesia superioris . Holotype ZMA V.Pl. 7153.1, sagittal reconstruction of the copulatory apparatus (anterior to the left). The testes are situateddorsally and extend from just anterior to the ovaries to the posterior end of the body. The testes generally are under-developed in that the majority of germ cells are represented only by spermatogonia (ca. 90%). In only some specimens, and then in only a few follicles, mature sperms are present. However, in all cases anomalies were observed, such as irregularly shaped spermatids and spermatozoa. Vitellaria are located between the testes and the intestinal branches. The large sac-shaped copulatory bursa is lined by a columnar, glandular epithelium bearing basal nuclei and it is surrounded by a thin layer of muscles. From the mid-posterior wall of the bursa the bursal canal runs in a caudal direction, to the left of the copulatory apparatus. Posteriorly to the gonopore the bursal canal recurves antero-ventrally and, subsequently, opens into the posterior section of the atrium. The bursal canal is lined by a pleated epithelium with cylindrical, infranucleated, and ciliated cells and is surrounded by a thin, subepithelial layer of longitudinal muscles, followed by a thicker layer of circular muscle. Ectal reinforcement is absent (Figs 3, 4C). At its distal section, near the atrium, the bursal canal shows several deep folds. Figure 4. Dugesia superioris . Photomicrographs of the copulatory apparatus. A Holotype ZMA V.Pl. 7153.1, sagittal section showing the penis bulb and the penis papilla with the ejaculatory duct B Paratype CGAS Pla 6. 3, transverse section of the penis papilla and the ejaculatory duct surrounded by numerous glands C Paratype CGAS Pla 6. 3, transverse section of the bursal canal. The moderately developed penis bulb, rich in glands, consists of intermingled longitudinal and circular muscle fibres. It houses an elongated seminal vesicle, which extends through the entire length of the penis bulb. The anterior half of the seminal vesicle is tubular in shape, while its distal, posterior section is considerably expanded. The vasa deferentia penetrate the antero-lateral wall of the penis bulb and open separately and symmetrically into the seminal vesicle at a position about halfway along the vesicle. No spermiducal vesicles were observed in any of the specimens examined. The seminal vesicle, lined with a flat epithelium and surrounded in its distal, posterior section by layers of circular muscle fibres, opens into the ejaculatory duct via a small diaphragm. The latter, located at the base of the penis papilla, receives the openings of very abundant bulb glands. The blunt penis papilla is lined with an infranucleated epithelium that is underlain with a thin subepithelial layer of circular muscles fibres, followed by a layer of longitudinal muscle fibres. The ejaculatory duct follows a dorsally displaced course through the penis papilla and has a sub-terminal opening. The spacious lumen of the ejaculatory duct is lined by a cuboidal, infranucleated epithelium that is surrounded by a layer of longitudinal musclesand receives the abundant secretion of penis papillaglands; in the majority of examined specimens the ejaculatory duct contained an empty spermatophore (Fig. 4A, B). Both the bulb glands and the penis papilla glands secrete globules that stain purple in Pasini's reagent (Fig. 4B). The acentral, dorsally displaced ejaculatory duct makes the penis papilla asymmetrical, with the ventral part being thicker than the dorsal one (Figs 3, 4A, B). The genital atrium is lined by an infranucleated epithelium that is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres. The common atrium communicates with a gonoduct that is lined by a columnar epithelium, which receives the openings of very abundant cement glands; the gonoduct communicates with the ventral gonopore (Fig. 3). Etymology. The specific epithet is derived from the Latin superius, located at a higher position, and alludes to the dorsally displaced course of the ejaculatory duct in the penis papilla. Geographical distribution. Known from the type locality and, most likely, also from a second Albanian locality, viz. Voskopoje (see below). Table 1. Checklist of Tricladida from the Lake Ohrid hydrographic basin.<br/>

Taxa	Lacustrine habitat	Adjacent waters of lake Ohrid	Endemic species	References
Dugesiidae Ball, 1974
Dugesia Girard, 1850
Dugesia superioris	_
Dugesia	_	Kenk 1978
Dugesia gonocephala	_	_	Stankovic 1960
Schmidtea Ball, 1974
Schmidtea lugubris	_	_	Stankovic 1960 Kenk 1978
Dendrocoelidae Hallez, 1892
Dendrocoelum Oersted , 1844
Dendrocoelum adenodactylosum	_	Stankovic and Komarek 1927 Kenk 1978
Dendrocoelum albidum	_	+	Kenk 1978
Dendrocoelum cruciferum	_	+	Stankovic 1969 Kenk 1978
Dendrocoelum decoratum	_	+	Kenk 1978
Dendrocoelum dorsivittatum	_	+	Kenk 1978
Dendrocoelum jablanicense	_	+	Stankovic and Komarek 1927 Stankovic 1960 Kenk 1978
Dendrocoelum komareki	_	+	Stankovic 1969 Kenk 1978
Dendrocoelum lacteum	_	Stankovic and Komarek 1927 Arndt 1938 Stankovic 1960 Kenk 1978
Dendrocoelum lacustre	_	+	Stankovic 1938 Stankovic 1969 Kenk 1978
Dendrocoelum lychnidicum	_	+	Stankovic 1969 Kenk 1978
Dendrocoelum maculatum	+	Stankovic 1960 Kenk 1978
Dendrocoelum magnum	_	+	Stankovic 1969 Kenk 1978
Dendrocoelum minimum	_	+	Kenk 1978
Dendrocoelum ochridense	_	+	Stankovic 1960 Kenk 1978
Dendrocoelum sanctinaumi	+	Stankovic 1960 Kenk 1978
Dendrocoelum sinisai	_	+	Kenk 1978
Dendrocoelum translucidum	_	+	Kenk 1978
Planariidae Stimpson, 1857
Crenobia Kenk, 1930
Crenobia alpina montenegrina	_	Stankovic and Komarek 1927 Stankovic 1960 Kenk 1978
Phagocata Leidy, 1847
Phagocata maculata	+	Stankovic 1938 Stankovic 1960 Kenk 1978
Phagocata ochridana	+	Stankovic and Komarek 1927 Stankovic 1960 Kenk 1978
Phagocata stankovici	+	Reisinger 1960 Kenk 1978
Phagocata undulata	+	Stankovic 1960 Kenk 1978
Planaria Mueller , 1776
Planaria torva	_	_	Stankovic 1960 Kenk 1978
Polycelis Ehrenberg, 1831
Polycelis tenuis	_	Stankovic 1960 Kenk 1978