Dendrocoelum obstinatum Stocchino & Sluys, 2017

Stocchino, Giacinta Angela, Sluys, Ronald, Kawakatsu, Mahasaru, Sarbu, Serban Mircea & Manconi, Renata, 2017, A new species of freshwater flatworm (Platyhelminthes, Tricladida, Dendrocoelidae) inhabiting a chemoautotrophic groundwater ecosystem in Romania, European Journal of Taxonomy 342, pp. 1-21 : 3-13

Dendrocoelum obstinatum Stocchino & Sluys , sp. nov.

urn:lsid:zoobank.org:act:28FF68D5-C4C2-42F0-8DD3-223D2E32CCA0

Figs 1–5 , Tables 1–2

Diagnosis

Dendrocoelum obstinatum Stocchino & Sluys , sp. nov. is characterized by: a male atrium extending ventrally to form a cervix-like structure projecting into the common atrium; a proximal tract of the bursal canal with an almost non-existent lumen; testes extending to the far posterior end of the body; a Balkan type of adenodactyl, with a length approximately equal to that of the penis; the penis being in a dorsal position and the adenodactyl located ventrally; the proximal half of the bursal canal being surrounded by a subepithelial layer of longitudinal muscle, while its distal half is surrounded by a subepithelial layer of circular muscles, followed by a layer of longitudinal fibres.

Etymology

The specific epithet is derived from the Latin adjective ‘obstinatus’, firm, stubborn, obstinate, and alludes to the fact that the species lives in subterranean waters without hydrogen sulphide but is also able to live in and withstand sulfidic groundwaters.

Material examined

Holotype

ROMANIA: Movile Cave , 43°49′36″ N, 28°33′43″ E, southeastern Dobrogea plateau, 5 Apr. 2011, coll. S.M. Sarbu, sagittal sections (sag. sect.), 4 slides ( ZMA V.Pl. 7264.1 ).

Paratypes

ROMANIA: same data as for holotype, sag. sect., 4 slides ( ZMA V.Pl. 7264.2); same data as for holotype, sag. sect., 4 slides ( ZMA V.Pl. 7264.3); same data as for holotype, 1 entire immature specimen preserved in ethanol ( ZMA V.Pl. 7264).

Other material

ROMANIA: Movile Cave, 43°49′36″ N, 28°33′43″ E, southeastern Dobrogea plateau, 11 Apr. 2011, coll. S. M. Sarbu, sag. sect., 6 slides ( ZMA V.Pl. 7265.1); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 7265.2); same data as previous, 5 entire immature specimens preserved in ethanol ( ZMA V.Pl. 7265). – Movile Cave, 43°49′36″ N, 28°33′43″ E, southeastern Dobrogea plateau, 15 Sep. 1992, coll. S. M. Sarbu, sag. sect., 3 slides ( ZMA V.Pl. 1750.1); same data as previous, sag. sect., 2 slides, immature specimen ( ZMA V.Pl. 1751.1). – Movile Cave, 43°49′36.28″ N, 28°33′43.67″ E, southeastern Dobrogea plateau, 19 Dec. 1992, coll. S. M. Sarbu, sag. sect., 2 slides, immature specimen ( ZMA V.Pl. 1752.1). – Neţoi street #1 well, 43°49′11.27″ N, 28°34′12.79″ E, Mangalia, southeastern Dobrogea plateau, 28 Aug. 1992, coll. S. M. Sarbu, sag. sect., 4 slides ( ZMA V.Pl. 1746.1); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1746.2); same data as previous, sag. sect., 2 slides ( ZMA V.Pl. 1746.3); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1746.4); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1746.5). – Neţoi street #1 well, 43°49′11.27″ N, 28°34′12.79″ E, Mangalia, 21 Jun. 1993, coll. S.M. Sarbu, sag. sect., 5 slides ( ZMA V.Pl. 1753.1); same data as previous, sag. sect., 5 slides ( ZMA V.Pl. 1754.1); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1754.2); same data as previous, sag. sect., 1 slide ( ZMA V.Pl. 1754.3). – Neţoi street #1 well, 43°49′11.27″ N, 28°34′12.79″ E, Mangalia, southeastern Dobrogea plateau, 5 Apr. 2001, coll. S.M. Sarbu, sag. sect., 3 slides ( ZMA V.Pl. 7266.1); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 7266.2); same data as previous, sag. sect., 4 slides ( ZMA V. Pl. 7266.3); same data as previous, one entire immature specimen preserved in ethanol ( ZMA V.Pl. 7266); same data as previous, sag. sect., 5 slides ( CGAS Pla 11. 1); same data as previous, sag. sect., 5 slides ( CGAS Pla 11. 2); same data as previous, sag. sect., 4 slides ( CGAS Pla 11. 3); same data as previous, 6 entire immature specimens preserved in ethanol ( CGAS Pla 11). – Horia, Cloşca & Crişan street # 13 well, 43°49′18.65″ N, 28°33′23.05″ E, Mangalia, southeastern Dobrogea plateau, 2 Jun. 2013, coll. S.M. Sarbu, sag. sect., 13 slides ( CGAS Pla 12. 1); same data as previous, sag. sect., 14 slides ( CGAS Pla 12. 2), same data as previous, 11 entire immature specimens preserved in ethanol ( CGAS Pla 12). – Aleea Cetăţii street # 1well, 43°48′52.95″ N, 28°35′03.37″ E, Mangalia, southeastern Dobrogea plateau, 28 Aug. 1992, coll. S.M. Sarbu, sag. sect., 10 slides ( ZMA V.Pl. 1747.1); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1747.2); same data as previous, sag. sect., 8 slides ( ZMA V.Pl. 1747.3); same data as previous, sag. sect., 7 slides ( ZMA V.Pl. 1747.4); same data as previous, sag. sect., 11 slides ( ZMA V.Pl. 1747.5); same data as previous, horizontal sections (hor. sect.), 4 slides ( ZMA V.Pl. 1747.6). – Aleea Cetăţii street # 1 well, 43°48′52.95″ N, 28°35′03.37″ E, Mangalia, southeastern Dobrogea plateau, 2 Jun. 2013, coll. S.M. Sarbu, sag. sect., 8 slides ( CGAS Pla 13. 1); same data as previous, sag. sect., 6 slides ( CGAS Pla 13. 2); same data as previous, sag. sect., 7 slides ( CGAS Pla 13. 3); same data as previous, sag. sect., 8 slides ( CGAS Pla 13. 4); same data as previous, hor. sect., 6 slides ( CGAS Pla 13. 5); same data as previous, sag. sect., 6 slides ( CGAS Pla 13. 6); same data as previous, 21 entire immature specimens preserved in ethanol ( CGAS Pla 13). – General Dragalina street # 10 well, 43°49′12.66″ N, 28°34′07.68″ E, Mangalia, southeastern Dobrogea plateau, 28 Aug. 1992, coll. S.M. Sarbu, sag. sect., 5 slides ( ZMA V.Pl. 1748.1); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1748.2); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1748.3); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1748.4); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1748.5); same data as previous, sag. sect., 2 slides ( ZMA V.Pl. 1748.6); same data as previous, sag. sect., 5 slides ( ZMA V.Pl. 1748.7); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1748.8); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1748.9); same data as previous, hor. sect., 4 slides ( ZMA V.Pl. 1748.10); same data as previous, transverse sections (transv. sect.), 7 slides ( ZMA V.Pl. 1748.11). – 2 (Doi) Mai well, 43°47′19.77″ N, 28°34′34.82″ E, southeastern Dobrogea plateau, 19 Nov. 1993, coll. S.M. Sarbu and D. Dancau, sag. sect., 22 slides ( ZMA V.Pl. 1755.1); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1755.2); same data as previous, sag. sect., 5 slides ZMA V.Pl. 1755.3. – 2 (Doi) Mai well, 43°47′19.77″ N, 28°34′34.82″ E, southeastern Dobrogea plateau, Romania, 11 Apr. 2011, coll. S.M. Sarbu, sag. sect., 4 slides ( ZMA V.Pl. 7267.1); same data as previous, sag. sect., 5 slides ( ZMA V.Pl. 7267.2); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 7267.3); same data as previous, sag. sect., 5 slides ( CGAS Pla 14. 1); same data as previous, sag. sect., 6 slides ( CGAS Pla 14. 2); same data as previous, sag. sect., 3 slides ( CGAS Pla 14. 3); same data as previous, sag. sect., 3 slides ( CGAS Pla 14. 4); same data as previous, sag. sect., 6 slides ( CGAS Pla 14. 5); same data as previous, sag. sect., 3 slides ( CGAS Pla 14. 6); same data as previous, hor. sect., 2 slides ( CGAS Pla 14. 7); same data as previous, 3 entire immature specimenspreservedinethanol(CGASPla14).–Limanuwell, 43°48′00.22″N, 28°31′34.82″E,southeastern Dobrogea plateau, 28 Aug. 1992, coll. S.M. Sarbu, sag. sect., 5 slides ( ZMA V.Pl. 1749.1); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1749.2); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1749.3); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1749.4); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1749.5); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1749.6); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1749.7); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 1749.8); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1749.9); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1749.10); same data as previous, hor. sect., 4 slides ( ZMA V.Pl. 1749.11); same data as previous, transv. sect., 7 slides ( ZMA V.Pl. 1749.12). – Limanu well, 43°48′00.22″ N, 28°31′34.82″ E, southeastern Dobrogea plateau, 3 Apr. 2011, coll. S.M. Sarbu, sag. sect., 3 slides ( ZMA V.Pl. 7268.1); same data as previous, sag. sect., 3 slides ( ZMA V.Pl. 7268.2); same data as previous, 3 entire immature specimens preserved in ethanol ( ZMA V.Pl. 7268); same data as previous, sag. sect., 3 slides ( CGAS Pla 15. 1); same data as previous, sag. sect., 2 slides ( CGAS Pla 15. 2); same data as previous, sag. sect., 2 slides ( CGAS Pla 15. 3); same data as previous, transv. sect., 6 slides ( CGAS Pla 15. 4); same data as previous, transv. sect., 3 slides ( CGAS Pla 15. 5); same data as previous, 12 entire immature specimens preserved in ethanol ( CGAS Pla 15). – Limanu well, 43°48′00.22″ N, 28°31′34.82″ E, southeastern Dobrogea plateau, 11 Apr. 2011, coll. S.M. Sarbu, sag. sect., 2 slides ( ZMA V.Pl. 7269.1); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 7269.2); same data as previous, one entire immature specimen preserved in ethanol ( ZMA V.Pl. 7269); same data as previous, hor. sect., 2 slides ( CGAS Pla 16. 1); same data as previous, hor. sect., 2 slides ( CGAS Pla 16. 2); same data as previous, 7 entire immature specimens preserved in ethanol ( CGAS Pla 16). – Albeşti well, 43°48′45.63″ N, 28°25′37.05″ E, southeastern Dobrogea plateau, 6 Apr. 2011, coll. S.M. Sarbu, sag. sect., 7 slides ( ZMA V.Pl. 7270.1); same data as previous, sag. sect., 8 slides ( ZMA V.Pl. 7270.2); same data as previous, sag. sect., 8 slides ( ZMA V.Pl. 7270.3); same data as previous, 20 entire immature specimens preserved in ethanol ( ZMA V.Pl. 7270). – Vama Veche well, 43°45′07.10″ N, 28°34′20.48″ E, southeastern Dobrogea plateau, 17 May 1993, coll. S.M. Sarbu and D. Dancau, sag. sect., 8 slides ( ZMA V.Pl. 1756.1); same data as previous, sag. sect., 8 slides ( ZMA V.Pl. 1756.2); same data as previous, sag. sect., 7 slides ( ZMA V.Pl. 1756.3); same data as previous, sag. sect., 5 slides ( ZMA V.Pl. 1756.4); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1756.5); same data as previous, sag. sect., 7 slides ( ZMA V.Pl. 1756.6); same data as previous, sag. sect., 5 slides ( ZMA V.Pl. 1756.7); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1756.8); same data as previous, sag. sect., 4 slides ( ZMA V.Pl. 1756.9); same data as previous, sag. sect., 6 slides ( ZMA V.Pl. 1756.10); same data as previous, sag. sect., 7 slides ( ZMA V.Pl. 1756.11); same data as previous, hor. sect., 4 slides ( ZMA V.Pl. 1756.12); same data as previous, transv. sect., 8 slides ( ZMA V.Pl. 1756.13).

Description

Live animals unpigmented, typically whitish, and lacking eyes. Preserved mature specimens measured 3–8 mm in length, and 1–2 mm in width. The anterior end is truncated, with the middle part of the frontal margin convex, and is provided with a pair of rounded lateral lobes ( Fig. 2 ).

The subterminal anterior adhesive organ is moderately developed and consists of a shallow cup made up of infranucleated epithelial cells, which are pierced by numerous openings of erythrophil glands. The musculature associated with this organ consists of a more strongly developed section of the usual ventral longitudinal body musculature ( Fig. 4B ).

In specimens from Movile Cave (holotype ZMA V.Pl. 7264.1, ZMA V.Pl. 7264.2, ZMA V.Pl. 7264.3, ZMA V.Pl. 7265.1, ZMA V.Pl. 7265.2 and ZMA V.Pl. 1751.1), and in all specimens from Neţoi well, the main gut branches, their diverticula, as well as pharyngeal pouch house numerous specimens of a gregarine protozoan probably belonging to the genus Monocystella Valkanov, 1934 (I. Desportes, Muséum national d’Histoire naturelle, Paris, pers. comm.) ( Fig. 5A ).

The pharynx is located in the posterior half of the body and measures about 1/6 th of the body length ( Fig. 2 ). Its internal muscle zone consists of a very thick layer of intermingled circular and longitudinal fibres. A subepithelial layer of longitudinal muscles, followed by a layer of circular fibres forms the thin outer zone of muscles.

The ventral ovaries are located at ¼ th of the distance between the brain and the root of the pharynx, and at 1/10 th of the distance between the brain and the posterior end of the body. The oviducts originate from the postero-lateral part of the ovaries and are provided with a very slight expansion at their anterior end, thus forming the tuba. The oviducts run posteriorly, recurve posterior to the gonopore and, subsequently, fuse to form a common oviduct. The common oviduct runs anteriorly to the right side of the bursal canal to open into the distal, ventro-posterior part of the male atrium, representing the cervix-like protrusion of the latter (see below). The common oviduct receives numerous openings of erythrophil shell glands along ¾ of its entire length ( Figs 3C , 4A, C ).

Well developed, rounded resorptive vesicles are present at least along the oviducts of ZMA V.Pl. 7265.1 from Movile Cave, ZMA V.Pl. 1749.8 from the Limanu well, ZMA V.Pl. 1756.4, ZMA V.Pl. 1756.7 from the Vama Veche well and ZMA V.Pl. 1747.1 from the Aleea Cetăţii well. Each vesicle communicates with the oviduct through a short, narrow ductule. Sperm is present, both in the oviducal lumen and in the short interconnecting ductules.

The well developed testes are numerous and basically dorsal in position. In ZMA V.Pl.7270.1, ZMA V.Pl. 7270.3 (Albeşti well), CGAS Pla 12. 1 (Horia Cloşca and Crişan well), and ZMA V.Pl. 1756.9 (Vama Veche well) testes are present also in ventral position, while some follicles are situated in the middle of the body. The testes extend from a short distance behind the ovaries to far into the posterior end of the body.

The sperm ducts form well-developed spermiducal vesicles, packed with sperm, between the mouth and the anterior level of the penis bulb ( Fig. 3B ). Thereafter the ducts curve to the dorsal side, in some cases (ZMA V.Pl. 1749.8, ZMA V.Pl. 1749.9, ZMA V.Pl. 1749.10 and ZMA V.Pl. 1749.12 from the Limanu well, as well as ZMA V.Pl. 1755.1 from 2 (Doi) Mai well) reaching the dorsal part of the body, and, subsequently, separately penetrate the penis bulb from the antero-lateral sides to open symmetrically and closely together into the anterior section of the lumen of the penis papilla ( Fig. 3B ). In specimens ZMA V.Pl. 7268.2, and ZMA V.Pl. 1749.7 from Limanu well the right vas deferens enters the penis bulb ventrally to the left one.

The copulatory apparatus occupies the anterior half of the postpharyngeal region. The copulatory bursa, situated just behind the pharynx, is sac-shaped and occupies the entire dorso-ventral space of the body. The bursa is lined with a cuboidal to columnar glandular epithelium and is surrounded by a layer of longitudinal muscles ( Figs 3 , 4A ). The bursa contains a mass of sperm that is not enveloped by a spermatophore. This mass is not homogeneous, but within it scattered rod-like structures are recognizable. These structures have a sclerotic-like appearance, are of various lengths and more intensely stained as compared to the rest of the mass of sperm ( Fig. 5C ). Besides the holotype, such rod-like structures are present in ZMA V.Pl. 7265.1 and ZMA V.Pl. 7265.2 from Movile Cave and also in the copulatory bursa of CGAS Pla 13. 1 from the Aleea Cetăţii well and in ZMA V.Pl. 7270.1 from the Albeşti well. In ZMA V.Pl. 7269.1 from the Limanu well the sperm mass inside the copulatory bursa is characterized by circular structures with a multilayered concentric organization ( Fig. 5D ).

The bursal canal runs posteriorly to the left of the penis. In almost all specimens examined the lumen of first tract of the canal is so much reduced that it can be considered to be almost non-existent, after which it gradually widens slightly, the canal subsequently turning ventrally, becoming more wide and opening into the common atrium ( Fig. 3A ). The wall of the bursal canal consists of a nucleated epithelium, which gradually changes from being cuboidal in the proximal tract to become cylindrical in the more distal section of the canal. The epithelial cells are provided with very long cilia that fill the bursal canal lumen from its proximal tract to ca ¾ of its length; in the distal tract of the canal, however, the cilia are much more sparse. The proximal half of the canal is surrounded by a subepithelial layer of longitudinal muscle, while its distal half is surrounded by a subepithelial layer of circular muscles, followed by a layer of longitudinal fibres ( Fig. 3A ).

The penis is located dorsally to the adenodactyl ( Figs 3B , 4A ), while the bursal canal is situated to the left of the midline of the body. The penis is approximately of the same length as the adenodactyl. In some specimens, such as in ZMA V.Pl. 7270.3 from the Albeşti well, the penis papilla is longer than the adenodactyl.

The muscular penis bulb is rather small; the papilla is about three times as large as the penis bulb. The penis papilla is covered with a thin epithelium at its apical part that becomes thicker at the basal part of the papilla. This epithelium is underlain by a thick layer of circular muscle, which is absent at the very tip of the penis papilla. The penis papilla is slightly asymmetrical, with the ventral part being somewhat larger than the dorsal one because of the dorsally displaced position of the lumen of the penis papilla ( Figs 3B , 4A ).

In the majority of the specimens examined the penis papilla is conical, however, its shape is variable and thus it may be more or less elongated or shortened, depending on the state of contraction. This implies also a certain variability in the length, width, and position of the papilla lumen, in that in some animals

it is displaced towards the center of the penis papilla, in contrast to the more dorsal position in most of the specimens examined.

The penis is rich in glands, which in the holotype are located mainly in the penis bulb and in the ventral part of the penis papilla. Some extrabulbar glands are also present. All of these glands open into the penis lumen, which is full of secretion, as is the male atrium. In all specimens examined it was impossible to detect traces of spermatophores, neither in the penial lumen, nor the male atrium, nor inside the copulatory bursa ( Figs 3 , 4A ).

The adenodactyl is very large and consists of a free papilla and a well-developed bulbar part. In the holotype and in many other specimens examined the apical part of the adenodactyl is thrust out of the body ( Figs 3B , 4A ), a condition very likely due to preservation artefacts. The bulb consists of intermingled rows of longitudinal and circular muscle, bounded by a thin layer of longitudinal fibres. The very small lumen of the adenodactyl is lined by a layer of ciliated cells and it is surrounded by a well developed zone of mesenchymatic tissue. Through this section of the mesenchyme runs a thick layer of fine circular muscle fibres. Ectally and internally to this zone of circular muscles runs a thin layer of longitudinal muscle fibres ( Figs 3B , 4A ). In the specimens stained in Mallory-Heidenhain, this layer of fine circular fibres is pale blue, while the longitudinal fibres stain red and the intermingled muscles stain bright blue. Many erythrophilic glands open into the adenodactyl lumen.

In ZMA V.Pl. 7265.2 from Movile Cave a nematode specimen infected the bulb of the adenodactyl and stained red with Mallory-Heidenhain ( Fig. 5B ).

The male atrium is lined by a columnar, nucleated epithelium and is surrounded by a subepithelial layer of circular muscles, followed by a layer of longitudinal fibers. The male atrium communicates with the common atrium through a cervix-like protrusion, which extends ventrally to some extent, with the result that the lumen of the common atrium is virtually non-existent. In this cervix-like protrusion the male atrium receives the opening of the common oviduct, which is lined by a nucleated epithelium ( Figs 3C , 4A, C ).

Geographical distribution

Exclusively known from the Movile Cave, wells in the town of Mangalia, and wells in the villages of Limanu, 2 (Doi) Mai, Albeşti and Vama Veche in Romania.

Habitat

Movile Cave

This is a land-locked cave and represents the first chemoautotrophically based groundwater ecosystem ever described. This cave system is developed in oolitic and fossil-rich limestone of Sarmatian age (ca 12.5 Ma). It consists of a network of upper dry cave passages, ca 200 m long, and a lower level cave ca 40 m long, partially flooded by thermomineral waters rich in hydrogen sulphide, forming a lake and some air pockets. Geophysical investigations, water chemistry, and stable isotope data indicate that this maze of fissures is part of an extensive sulfidic groundwater aquifer that spreads out over an area of ca 70 km 2 (cf. Sarbu 2000 and references therein). The thermal sulfidic groundwaters at Mangalia ascend along natural geological faults from a confined aquifer at a depth of 200–400 m (this pressurized water with a temperature of 25°C contains H 2 S, CH 4, NH 4 + and lacks O 2) under the Sarmatian limestones and flood the superficial carbonate bedrock up to the groundwater level. The submerged portion of the cave contains microbial mats composed of chemoautotrophic sulfide-oxidizing bacteria as well as fungi, which float on the water surface and grow on the limestone walls. The subterranean ecosystem is based entirely on food produced in situ by these chemoautotrophic organisms (cf. Sarbu 2000 and references therein).

Flatworm specimens were collected along the shore of the lake where water temperature is constantly 21°C all year round, with absolutely no fluctuations; pH value is 7.29 ( Table 1 ). The worms were found gliding on sediment in very shallow water and never went any deeper than one centimetre, due to the fact that oxygen is present only at the surface and in the first one millimetre of water, while all of the deep water is completely anoxic ( Riess et al. 1999). Planarians were seen moving upside-down, thus having access to oxygenated water. Only a few flatworm specimens were found per collection.

Planarians were found associated with a diverse invertebrate fauna, consisting of Nematoda (5 species), Rotatoria (2 species), Hirudinea (1 species), Aphanoneura (2 species), Gastropoda (1 species), Ostracoda (1 species), Copepoda (3 species), Amphipoda (3 species), Isopoda (1 species), Heteroptera (1 species) (cf. Sarbu 2000; Brad et al. 2015). Dendrocoelum obstinatum Stocchino & Sluys , sp. nov. is one of the three top predators in the aquatic community of the sulfidic groundwaters at Mangalia, together with the leech Haemopis caeca Manoleli, Klemm & Sarbu, 1998 and the heteropteran Nepa anophtalma Decu, Gruia, Keffer & Sarbu, 1994 (cf. Sarbu & Popa 1992).

Sulfidic wells

Planarians were collected from four old, hand-dug wells in the town of Mangalia. These wells are 0.8 to 2.5 km far from the cave ( Table 1 ). Depth of the wells varies from 14 to 19 m. Water temperature range is 18–19°C. The temperature of the wells depends on the distance to the place where the fault system brings up the warm water (see above) to the surface and how long it takes the waters to flow through the surface limestones. The pH value is around 7.0 with non-significant variations. Both in the cave and the sulfidic wells, pH values near neutrality depend on the great buffering capacity of the carbonate bedrock. Although H 2 S oxidizes and forms sulphuric acid, this is immediately neutralized by the bicarbonate in the water ( Sarbu 2000).

Planarian specimens were found only at the water surface. In the past all of these wells were used for drinking water. Planarians were not found in many other wells in the town that were also sampled. In the General Dragalina, Horia, Cloşca & Crişan and Aleea Cetăţii wells planarians were found associated with Niphargus dancaui Brad, Fiser, Flot & Sarbu, 2015 ( Brad et al. 2015).

Non-sulfidic wells

Planarians were collected from four old, hand-dug wells in the villages of Limanu, 2 (Doi) Mai, Albeşti and Vama Veche, localized at a distance ca 3.5 to ca 11 km from Movile Cave. Specimens were seen moving on the limestone walls and descending to a depth of more than 1 m. Water temperature of the non-sulfidic wells is ca 13°C, which is the mean annual temperature of southern Dobrogea ( Table 1 ).

Figures

Fig. 1. Geographic distribution of freshwater planarians of the genus Dendrocoelum recorded from Romania. ▲: unspecified locality of D. lacteum Müller, 1774. Rectangular inset (bottom) corresponds with enlarged area, showing the collection sites of D. obstinatum Stocchino & Sluys, sp. nov. indicated by blue stars (see also Table 1).

Fig. 2. Dendrocoelum obstinatum Stocchino & Sluys, sp. nov. Sketch of the ventral view of a preserved (Bouin’s fluid) specimen from Movile Cave.

Fig. 3. Dendrocoelum obstinatum Stocchino & Sluys, sp. nov., holotype (ZMA V.Pl. 7264.1). A. Sagittal reconstruction of the female copulatory apparatus (anterior to the left). B. Sagittal reconstruction of the male copulatory apparatus (anterior to the left). Only the terminal portions of the spermiducal vesicles are drawn. C. Sagittal reconstruction of the male copulatory apparatus with the cervix-like protrusion (anterior to the left).

Fig. 4. Dendrocoelum obstinatum Stocchino & Sluys, sp. nov., holotype (ZMA V.Pl. 7264.1). A. Microphotograph of the copulatory apparatus. B. Microphotograph of the anterior adhesive organ with the associated ventral muscles. C. Microphotograph of the cervix-like protrusion of the male atrium, with the opening of the common oviduct.

Fig. 5. Dendrocoelum obstinatum Stocchino & Sluys, sp. nov. A. Holotype (ZMA V.Pl. 7264.1), microphotograph of gregarine protozoans in a gut diverticulum. B. Specimen from the Movile Cave, microphotograph of a nematode infecting the bulb of the adenodactyl (ZMA V.Pl. 7265.2). C. Holotype, microphotograph of the mass of sperm inside the copulatory bursa with the rod-like structures. D. Specimen from the Limanu well, microphotograph of the mass of sperm inside the copulatory bursa, with a multilayered concentric organization of the circular structures (ZMA V.Pl. 7269.1).