A NEW SPECIES OF CASPIA CLESSIN ET W . DYBOWSKI , 1887 ( GASTROPODA : TRUNCATELLOIDEA : HYDROBIIDAE ) IN THE DANUBE OF BULGARIA

Species of the genus Caspia Clessin et W. Dybowski, 1887 are only known from brackish waters of the Caspian Sea and the Azov-Black Sea Basins. The first freshwater species of Caspia is described from the Danube. Inclusion of Caspia in the Hydrobiidae is discussed. C. makarovi Anistratenko, 2013 is synonymised with C. knipowitschii Anistratenko, 2013. KeyworDs: Hydrobiidae, Caspia milae Boeters, Glöer et Georgiev n. sp., Danube, Bulgaria


INTRODUCTION
The freshwater mollusc fauna of the Danube, the border river of Bulgaria and Romania, has been well known for a long time.The river and its delta are inhabited by many operculate snails representing at least nine families (Table 1).According to cioBoiu (2010: 3) also spring snails of the family Hydrobiidae can be found there (Table 2).At least for one of them, that is Bythinella austriaca (Frauenfeld, 1857), cioBoiu (2010: 1) defined the habitat as "on rocky banks".
Despite the fact that during the last decades continuous investigations contributed to the knowledge of the Bulgarian and Romanian freshwater mollusc fauna, no previously unrecorded operculates were observed in the Danube and its delta.It is true that already in 1956 Grossu mentioned a species of Caspia Clessin et W. Dybowski, 1887 for the Bulgarian coast; consequently, GoliKov & staroBoGatov (1966: 354) wondered if this species might inhabit the Danube.Later, anistratenKo (2007a: 795, figs 1.1-7) presented maps of the Azov-Black Sea Basin with estuarine areas such as that of the Danube, and added a photograph of Caspia sp., as one of the species known to inhabit at least some of the estuaries of the Basin.Whereas Grossu (1956) listed this species as Caspia gmelinii Clessin et W. Dybowski, 1887 (Figs 1-6) and later (in Grossu 1986) as Caspia g. aluschtensis (Golikov et Starobogatov, 1966), GoliKov & staroBoGatov (1966) named it C. ma karovi (Golikov et Starobogatov, 1966).However, the presence of a representative of Caspia in the Danube was never reported.This is all the more surprising that recently a previoulsy unknown representative of Caspia was found at the Danubian Island Vardim.

CHARACTERISTICS OF THE GENUS CASPIA CLESSIN ET W. DYBOWSKI, 1887
Caspia comprises two subgenera (see below).Its type species was described from the Caspian Sea based on conchological characters only.Overall, the knowledge of morphological, anatomical, biological and ecological characters and behaviour of species of Caspia is scanty.The existing information can be briefly summarised as follows:

MATERIAL AND METHODS
The snails were collected at the Island Vardim (Fig. 7), situated in the Danube about 500 km up of the Black Sea (Fig. 8) with a sieve or by hand, sorted on site from live material and preserved in 75% ethanol.The dissections and measurements of the genital organs and shells were carried out using a stereomicroscope (Zeiss); the photographs were taken with a digital camera system (Leica).The measurements are given as minimum-mean-maximum values.

Remark:
We have not seen shells with a thickened palatal and basal margin of the aperture, that is those with completed growth as distinctly visible as in C. gmelinii (Figs 1 and 3).Soft parts: Ommatophores with a median black stripe, head and mantle except for its white margin with scattered black pigment (Fig. 18).Gill with 12-13 lamellae (n = 1 ♀, 2 ♂♂) (Fig. 17).Intestine in the roof of mantle cavity shaped like a very flat U (Fig. 19).
Anus at a distance of about two times the width of the unpigmented and slightly thickened mantle margin behind its edge, in females only slightly protruding beyond the complex of albumen and capsule glands.
Penis: In contracted specimens the unpigmented short, blunt, broad penis is bent towards the opening of the mantle cavity; in top view its left side shows a semicircular protrusion which continues on the lower side as a lamella; the median penial duct runs to the penis tip and opens at a pointed protrusion (Figs 13,17).4).Furthermore, in C. milae n. sp. the h : d ratio is 1.84 and the height ratio of body whorl to shell is ca.0.70, whereas in C. g. aluschtensis the h : d ratio is more than 2 and the body whorl : shell height ratio is less than 0.67.Habitat and distribution: Right bank of the point of the Vardim Island (Fig. 7), situated on the Danube about 500 km upstream of its estuary to the Black Sea (Fig. 8).The snails were collected on a heterogeneous substratum formed by large stones (boulders), coar-se gravel and fine gravel (10 : 60 : 30%).The water parameters, measured on 28 August 2013, are listed in Table 5.It cannot be excluded that C. milae n. sp.inhabits the Danube also downstream of the type locality, to the Danube Delta, and its limans such as Lacu Razelm, since the drawing published by Grossu (1986: 277, fig. 109 contrary to Grossu 1956: 122, fig. 60) shows a shell without a thickened lip.

DISCUSSION IDENTITY OF CASPIA AND ITS SUBGENERA
Caspia comprises two subgenera, the nominate subgenus and Clathrocaspia Lindholm, 1929(w. DyBowsKi 1887-1888, linDHolM 1929).The type species of the nominate subgenus is C. baerii.According to the original description, its shell is 1.6 mm high, very weakly radially striated and shows a very weak spiral stria below the suture.
The type species of Clathrocaspia is C. pallasi Clessin et W. Dybowski, 1887.Its shell was originally described (w.DyBowsKi 1887-1888) as 1.8-1.9mm high, spirally striated, with about eight striae on the body whorl.In addition to the spiral striation, the original description of C. (Clathrocaspia) gmelinii (w.DyBowsKi 1887-1888) states that its shell, 1.6 mm in height, is very weakly radially striated.The renal oviduct in C. (Clathrocaspia) gmelinii is known to be provided with a proximal receptaculum (RS2) but no bursa or distal receptaculum (RS1) (sitniKova & staroBoGatov 1998, anistratenKo 2013).The reticulate shell sculpture and the female genitalia with  only one receptaculum but no bursa place C. milae n. sp. also in Clathrocaspia.The shells of all three species are provided with a strong columellar callus.Unfortunately, no shell photographs of C. baerii or C. pallasi are available, and none of them has been anatomically examined.More studies on the poorly known genus Caspia with its two subgenera, and especially their type species, are required.Moreover, we failed to obtain at least photographs of the holotypes of Pyrgula (Caspia) gmelinii aluschtensis Golikov et Starobogatov, 1966 and Caspia gmelinii stanisla vi Alexenko et Starobogatov, 1987 which are allegedly deposited at the Institute of Zoology, Russian Akademy of Sciences (RAS) in St. Petersburg.

FAMILY ASSIGNMENT
The renal oviduct of representatives of the Hydrobiidae can be provided with a proximal receptaculum (RS2), a distal receptaculum (RS1) and a bursa below the distal receptaculum.When the proximal receptaculum is present, it is shaped like a more or less elongated sac which is connected with the oviduct by a short receptaculum duct.In females of Caspia milae n. sp.there is no bursa or distal receptaculum (RS1), and only the proximal receptaculum (RS2) is present.It has the form of an elongated smooth sac connected with the oviduct by a very short duct.
CASPIA KNIPOWITSCHII AND C. MAKAROVI anistratenKo (2013: 56) listed several characters to discern between these two species (Table 6) 1) he gave values of protoconch measurements for only two specimens of each species; they may not be representative and useful as reliable diagnostic characters.Furthermore, except for the protoconch maximum diameter, the ranges of measurements in Table 6, including the apical angle, are identical or overlap.Finally, the radulae of the two species have not been compared, whereas the radulae of C. knipowitschii and C. gmelinii are "almost identical" (anistratenKo 2013: 58).The difference in the spiral striation seems to be insignificant, since both species are "similar in … ornamentation patterns".Here, we assume that C. makarovi is a junior synonym of C. knipowitschii, however the status of both species should be studied in detail.1), the renal oviduct is provided with a bursa in addition to a single receptaculum (RS1).The Black Sea's circulation patterns are primarily controlled by the basin topography and fluvial inputs, which result in a strongly stratified vertical structure.The deep waters of the Black Sea do not mix with the upper layers, so that over 90% of the deeper Black Sea volume is anoxic water.The surface water leaves the Black Sea with a salinity of 17 psu.Grossu (1986: 277) mentioned a depth of 20-25 m for C. gmelinii aluschtensis in the Black Sea.However, it is not obvious whether really live animals were found at those depths.The same applies to GoliKov & staroBoGatov's (1966: 354) (GittenBerGer et al. 1998: 82).The Black Sea tides of the semidiurnal type have the maximum amplitude of only 12 cm, but at the coast offshore winds can rise the Black Sea level up to 60-80 cm and winds from the land decrease the sea level by 60 cm.Consequently, the water level in the estuaries of the rivers flowing into the Black Sea may be subject to tides with amplitudes of up to 140 cm (BonDar 2007: 47).
The above considerations do not seem to apply to the habitat of C. milae n. sp. which lives in the Danube about 500 km upstream of the Black Sea coast, far from the estuary.

Fig. 7 .
Fig. 7. Type locality: sampling site of Caspia milae n. sp.(Island Vardim, view towards Danube) separated by a moderately deep suture (Figs 9-10); walls with a lattice-like sculpture formed by spiral ridges crossed by longitudinal lines or striae (Fig.11); the thickness of the spiral ridges on each whorl decreases from the upper to the lower suture, in frontal view forming pronounced steps to very flat waves; about eight spiral ridges on the body whorl above the corner of the aperture (Fig.11); this sculpture weakens towards the aperture and is replaced by growth lines; suture of the last quarter of body whorl ascends slightly, indicating maturity; aperture obliquely ovate with slightly pointed upper corner; peristome sharp; umbilicus completely closed by the lobed columellar border of the aperture; parietal border completely fused with shell wall and connected with a spiral filamentous thickening, which connects the suture inside the whorls; the columellar margin runs nearly straight down before passing into the basal margin.Measurements: heigth 2.15-2.50mm (mean 2.33, SD 0.117, n = 9), diameter 1.20-1.35mm (mean 1.28, SD 0.061, n = 8).Operculum spiral, without any hook, horn-coloured.
Female genitalia: The unpigmented renal oviduct turns from the gonopericardial duct towards the distal wall of the stomach, forming a V-like loop, and from there, forming another V-like loop, runs straight towards the complex of albumen and capsule glands; the second loop carries a large ovate receptaculum (proximal receptaculum = RS2); distal receptaculum (RS1) and bursa are missing.Sex ratio: The sex ratio among the eight dissected animals was 2♂ : 6♀ (BOE 3330).Diagnosis: Reticulate (lattice like) sculpture of the shell and the female genitalia with only one receptaculum but with no bursa place the new species within Clathrocaspia Lindholm, 1929, a subgenus of Caspia, a genus of the Hydrobiidae.Differential diagnosis: 1.Compared to C. knipowitschii (= C. makarovi; see below) the umbilicus is covered by a lobed col-umellar margin and not narrow and slit-like to closed (anistratenKo 2013: 54, 56); furthermore, in C. milae n. sp. the transition from the columellar to the basal margin is less regularly rounded than in C. knipowitschii.In C. kni po wi tschii the protoconch has 1.25-1.30and its diameter is 0.28-0.35mm, whereas in C. milae n. sp. the number of whorls is only 1.00-1.20,however the diameter is 0.38-0.42mm (Table 3).Finally, in the Black Sea region, C. knipowitschii inhabits the deltas of the Dnieper River and the Dniester River.C. milae.sp. was found in a different environment: about 500 km upstream of the Danube estuary.

Figs 15- 21 .
Figs 15-21.Caspia milae n. sp.(paratypes, BOE 3330): 15-16 -female shells (15 -same ♀ as for 18 and 19; 16 -same ♀ as for 21); 17 -male (head with penis and gill exposed through slit mantle); 18-21 -females (18 -body with head covered by mantle and with operculum, first whorls of visceral sac removed, 19 -body and mantle cavity opened to show renal oviduct, complex of albumen and capsule glands and intestine, partially broken away, 19-21 -renal oviduct with receptaculum).Scale bars -1 mm for figs 15-16 and 0.5 mm for figs 17-21.Abbreviations: AN -anus, EY -eye spot, FP -faecal pellet, GI -gill, GP -gonoporus, IN -intestine, MB -mantle border, OP -operculum, PE -penis, RO -renal oviduct, RS -receptaculum, VD -vas deferens : both species "are similar in size, general shape and ornamentation patterns [...].They differ in the dimensions of the protoconch" [i.e.] "the protoconch of C. makarovi is slightly smaller than that of C. kni powitschii" […] "in the proportions and degree of inflation of the whorls.The shell of C. makarovi is more slender and has slightly more inflated whorls than C. knipowitschii: the apical angle in C. makarovi is 40-45° compared with 45-50° in C. knipowitschii."In his table (anistratenKo 2013: 57, table FEMALE GENITALIA As mentioned above, according to sitniKova & staroBoGatov (1998: 1363), as interpreted by anistratenKo (2013: 61), the renal oviduct of Caspia lacks bursa and distal receptaculum (RS1); only the proximal receptaculum (RS2) was found by sitniKova & staroBoGatov (1998) in C. gmelinii and C. gaillardi.This is confirmed by our results.On the other hand, the figures presented by sitniKova & staroBoGatov (1998) are not clear.The receptaculum duct enters the renal oviduct at its distal end (sitniKova & staroBoGatov 1998: 1359, fig.1.12)or it directly enters the complex of albumen and capsule glands together with the renal oviduct(sitniKova & staroBoGatov 1998: 1359, fig.1.13).The receptaculum in their figure is located in the position of the bursa.Besides, according to sitniKova & staroBoGatov (1998) the receptaculum is U-shaped -a shape we did not observe in our specimens.In their opinion(sitniKova & staroBoGatov 1998: 1558)  in females of Turricaspia B. Dybowski etGrochmalicki, 1915, which also inhabit the Danube HABITAT OF CASPIA SPECIES IN THE BLACK SEA BASIN When describing Caspia based on 200 shells of C. baerii, 70 shells of C. gmelinii, 25 shells of C. ulskii and more than 300 shells of C. grimmi presumably from a depth of 0-27 m ["0 bis 15 Faden"] from the Caspian Sea, clessin & w.DyBowsKi (in DyBowsKi 1887-1888: 35, 69) added that they had never seen a live animal.The same applies to the description of C. gaillardi from the Caspian Sea at depths of 0-30 m (taDjalli-Pour 1977).It can be assumed that they dealt with samples of shells brought and deposited by sea currents, probably from estuaries -a view supported by the obervations from the Azov-Black Sea Basin.
report of C. gmelinii in the Caspian Sea at depths of 30-81 m.According to alexenKo & anistratenKo (1998: 60) and also anistratenKo (2013: 52) live C. knipowitschii and C. makarovi could be found in the Dnieper River at depths of 1.5-3 m.It would not be surprising to see that the habitat of Caspia species at least in the Black Sea Basin is indeed restricted to the estuarine areas of large rivers flowing into the Sea.This assumption is in conformity with observations reported by alexenKo & staroBoGatov (1987: 36-37 with tables 1-2) for C. knipowitschii and C. stanisla vi at a depth of 1.1-3.0m in the Dnieper Delta, since their population density decreases towards the river mouth.It is remarkable that of the three species of Caspia from the Azov-Black Sea Basin two are endemic to specific estuaries: C. logvinenkoi to the Don Delta and C. stanislavi to the Dnieper Delta.The conditions of salinity, tides and topographical isolation of the areas inhabited by Caspia resemble those of the habitats of Mercuria in West European estuaries.anistratenKo (2007b: 23, 25) reports a salinity from freshwater to 1 psu for the type locality of C. logvinenkoi, the estuary of the Don River, while in the tidal areas of Dutch estuaries inhabited by Mercuria anatina (Poiret, 1801) the salinity is 0.5-5.4psu

Table 5 .
Water parameters at the type locality

Table 4 .
Shell measurements of different species of Caspia -differentiating characters