Cochlostoma revised: the subgenus Lovcenia Zallot et al. , 2015 (Caenogastropoda, Cochlostomatidae)

. Five species of the subgenus Lovcenia of Cochlostoma (Cochlostomatidae) are recognized, three of which are described as new to science: C. (L.) tropojanum sp. nov., C. (L.) jakschae sp. nov. and C. (L.) lanatum sp. nov. A lectotype is designated for C. (L.) erika (A


Introduction
The most recent comprehensive revision of the genus Cochlostoma Jan, 1830 at the species level dates back to the end of the 19 th century (Wagner 1897).Since then, only restricted taxonomical contributions or revisions concerning species of limited geographical ranges have been published (e.g., Schütt 1977;Bank 1988;Fehér et al. 2001;Fehér 2004).
In a revision of the generic taxonomy of the Cochlostomatidae, Zallot et al. (2015) showed the applicability of genital characters complementary to the traditional conchological data in taxon diagnoses within this family.This approach, combined with the use of molecular phylogenetic methods, has opened new prospects in the taxonomy of Cochlostoma and formed the basis for a revised system at the generic and subgeneric level (Zallot et al. 2015).Using the same methods, the various subgenera of Cochlostoma will be dealt with in a series of articles.Here, a start is made with the subgenus Lovcenia Zallot et al., 2015.

Material and methods
The locality data, summarized in Table 1, are also indicated for each species in the taxonomic section of this paper.The location of the sampling localities is indicated in Fig. 1.Pre-GPS samples are represented as an approximate position.
High resolution images of the shell specimens were made with a Canon EOS 600D camera with an EF-S 60 mm 1:2.8 macro lens and an additional NL-5 close-up lens, mounted on a tripod with a micrometric slide.Using Helicon Focus stacking software, several photographs were combined into a single picture.With the GIMP 2.6.11-GNUsoftware, potentially diagnostic distances and angles were established (see Fig. 2).The distances were measured in pixels and then translated into metric measurements on the basis of the number of pixels per millimetre.This ratio was determined by comparing the height in pixels on the picture and the height in millimetres that was measured directly.
The number of whorls was counted on the photographs in frontal view.The apical whorl, because it is variable in size, was counted in tenths of the height of the whorl before the apical whorl.For convenience, the whorls are numbered from the basal (body whorl) up.Therefore, the 1 st whorl refers to the basal one.

Heights
The tip of the protoconch was aligned with the internal side of the columella visible in the left side of the aperture.The following heights were then measured on this main axis: total height (as above mentioned); height of the 1 st , 2 nd , 3 rd , 4 th whorls; height of the aperture between the highest point of the parietal lobe and the basis; height of the aperture along the main axis; height of the parietal lobe.Zallot et al., 2015, as well as for C. (Turritus) arnautorum (A.J.Wagner, 1906) and C. (T.) mnelense (A.J.Wagner, 1914).Countries: AL = Albania; KS = Kosovo; MAK = Macedonia; MONT = Montenegro.

Widths
On a plane orthogonal to the main axis the following widths were measured: width of the basal whorl; width of the 2 nd whorl; width of the lower and upper suture of the 2 nd whorl; width of the basal protoconch whorl; width of the aperture; maximum width of the lips on the columellar side.Fig. 2. Potentially diagnostic distances and angles. 1 = shell height (H). 2 = height of aperture at the columellar side.3 = height of the first whorl.4 = width of body whorl (Wbw).5 = width of the suture above the body whorl.6 = width of the penultimate whorl.7 = width of the suture above the penultimate whorl.8 = distance between the 5 most central ribs on the 4 th whorl up.9 = diameter of the 4 th whorl up. 10 = aperture width (Wa).11 = width of the lip at the columellar side.12 = aperture height (Ha).13 = rib inclination.14 = suture inclination.15 = aperture inclination.16 = protoconch diameter in upper view (Dp).17 = end of the smooth part of the protoconch.18 = end of the protoconch.
European Journal of Taxonomy 464: 1-25 (2018) 6 The ratio between the width of the 2 nd whorl and the average width of the upper and lower sutures of the 2 nd whorl gives an index of the roundness of the whorl, a feature frequently mentioned in the description of the taxa within Cochlostoma.The ratio between the width of the 1 st and the 4 th whorl is an index of the slenderness of the shell.

Ribs per mm and rib features
The number of ribs per mm was calculated by measuring the distance between the first and the last of the most central 5 ribs of a whorl in frontal view.Because the ribbing is normally different in the apical rather than in the basal whorls, the measurement was repeated on the 1 st (taking the distance on the upper suture of the whorl) and on the 4 th whorl (taking the distance on the lower suture).Because the roundness of the 4 th whorl can distort the distances measured on the photograph, this distance (equal to the chord of the circumference) was translated to the distance along the arc.
The observed rib features (on the ribs of the 4 th whorl) were the following: type of ribbing (regular, irregular, double ribbing); shape of the ribs (sharp, rounded; straight or sinuous); height.

Inclination of the ribs
This was calculated taking the average of the inclination of the 3 most central ribs of the 4 th whorl and the most central rib of the 3 rd whorl with respect to the main axis.The angle between the main axis and the lower suture of the 4 th whorl was also recorded.In this way, the inclination of the ribs can be related either to the main axis or the suture itself.

Inclination of the aperture
In lateral view, the shell was moved into a standard position in order to have the basal point of the aperture aligned with the tip of the protoconch.Then, the angle between the lip of the aperture and the resulting main axis was measured.The inclination of the suture between the 1 st and the 2 nd whorl was also measured, allowing us to relate the inclination of the aperture to it.
To investigate the genital morphology, the lower whorls of the shells of male animals were removed in order to expose the right side of the foot.Shells of females were entirely removed.The body was then fixed in absolute ethanol.Females were positioned in such a way that the ventral side was visible at the level of the bursa copulatrix, between the 2 nd and the 3 rd whorls.In males, the mantle was cut vertically from the front up to the anus and, behind it, the tip of the prostatic gland.The anatomical details of individual specimens were figured with the help of a camera lucida and a stereo microscope.The description of the genitalia (see Fig. 3 for the terminology) begins with the structures that are located in the apical part of the body (i.e., the ovary or the testis).For more details, see Zallot (2002: 95-96) and Zallot et al. (2015).In the 'Material examined' sections, the number of specimens is only provided for type material lots.
When Zallot et al. (2015) presented the first phylogenetic reconstruction for the genus Cochlostoma, only two species of Lovcenia were included, i.e., C. (L.) erika (A.J.Wagner, 1906) and the species described here as C. (L.) lanatum sp.nov.That study was based on only two markers, the nuclear Histone H3 gene (H3) and the mitochondrial 16S rRNA gene (16S).Due to the incomplete overlap of the available samples, the trees based on these two markers were calculated separately.In the present study, more taxa and more populations per taxon are included, and an additional marker, the mitochondrial cytochrome oxidase subunit I (COI) was used.The molecular analysis was performed on the basis of material from 13 different locations.Additionally, we used two populations of two different species belonging to the subgenus Turritus Westerlund, 1883.
The analysed specimens had either been kept in ethanol 70% or were dried animals, i.e., shells containing mummified bodies (Table 1).From specimens in ethanol, DNA was extracted with the DNeasy Blood LGC Genomics (Berlin, Germany).Sequences were assembled and edited using BioEdit ver.7.0.1 (Hall 1999) and deposited in GenBank (Table 1).

Results
We accept five species in Cochlostoma (Lovcenia), three of which are new to science.The molecular phylogenetic reconstruction, based on three markers (H3, 16S, COI), supports the monophyly of the subgenus Lovcenia as well as that of each species of Lovcenia.2).The subgenus is known from a relatively small range that includes W Montenegro, N Albania and W Macedonia.

Diagnosis
Shell slender conical, with more or less regularly spaced riblets, which may vary in prominence.Unlike the species of most subgenera of Cochlostoma, those belonging to Lovcenia can be distinguished conchologically, i.e., by the early ribbing of the protoconch, with fine riblets appearing already after

Description
Shell.Large, with 8½-10 whorls, H/W ratio 2.55.The apical 2.4 whorls form the protoconch, which is smooth on only the initial 0.5 whorl; further on the whorls have fine, rather widely spaced riblets.The protoconch is relatively small, Dp/D4 th 0.55.The shell is more or less light horn-brown, without spots.The teleoconch whorls are sculptured with whitish, rounded but thin ribs, which are uniform in height, spacing and shape; on the body whorl the ribs become more narrowly spaced and weaker while approaching the aperture.The aperture has a well-developed lip, which broadens at the columellar side before curving abruptly back, forming an acute angle and covering the umbilicus.The body whorl widens near the aperture.
Female genitalia.Bursa copulatrix large, with a pedunculus connected proximally; its proximal lobe smaller than the distal one.Short seminal receptacle, without distal oviduct, confined to the ventral side of the body.Visceral oviduct nearly linear while running over the apex of the seminal receptacle.
The junction of the uterine gland and the copulatory duct is far from the connection between the distal oviduct and the pedunculus of the bursa.

Distribution
This species lives on the Njeguši Plateau in Montenegro, where only two locations are known.

Habitat preference
Like the majority of known Cochlostoma, this is an obligate rock-dwelling species.Although it can be found at Popovo Cave together with its congener Cochlostoma (Auritus) auritum (Rossmässler, 1837), this is not syntopy in the strict sense, because there is a difference in their niches.Cochlostoma (A.) auritum lives on the exposed rock surface around the cave entrance, whereas C. (L.) erika is found in the twilight zone inside the cave.Though little is known about its ecological requirements, C. (L.) erika is supposedly bound partly to the epigean environment (i.e., subtroglophile in the sense of Sket 2008).

Remarks
In order to promote nomenclatural stability, a female specimen, which was illustrated in the original description by Wagner (1906: fig. 15a-b), is herein designated as the lectotype.

Diagnosis
The irregular sculpture and its large shell height distinguish C. (L.) dalmatinum from other species of Cochlostoma (Lovcenia).
The protoconch is relatively small, i.e., Dp/D4 th = 0.36-0.46.The shell is more or less dark horn-brown, without spots.The teleoconch whorls are ribbed with whitish ribs that are irregular in height, spacing and shape.Between these main ribs, there are fine riblets, which have the same colour as the shell.There is no difference in rib distances between the upper whorls and the body whorl.The sculpture fades on the last part of the body whorl behind the aperture.The aperture is relatively small (Ha/H = 0.20), with an irregular lip.The lip enlarges on the columellar side before curving abruptly back and covering the umbilicus.The body whorl abruptly enlarges near the aperture, forming a moderately large external lobes.
Female genitalia.Large bursa copulatrix, with a pedunculus connected proximally; its proximal lobe smaller that the distal one.Short seminal receptacle, without distal oviduct and confined to the ventral side of the body.Loops of the visceral oviduct situated over the apex of the seminal receptacle.The junction of the uterine gland and the copulatory duct is situated far from the connection between the distal oviduct and the pedunculus of the bursa.

Distribution
Northwest of the bay of Kotor in Montenegro.Wagner (1897) reported this species from 'Mont Falcone' (= Mt Radoštak), not far from the site where we have recently collected it.As this area is poorly explored and because it is difficult to access, the species may be more widely distributed than believed earlier (see Gittenberger 1976).

Habitat preference
Unlike C. (L.) erika, this species is not bound to cave environments.We have found it on large limestone cliffs, mostly hidden in shady fissures.

Remarks
Due to the lack of material, Zallot et al. (2015) did not assign this species to any of the subgenera of Cochlostoma.The available anatomical and molecular data now unequivocally support its classification in Lovcenia.

Diagnosis
Rather small, with a very slender, spotless and regularly ribbed shell, which has a poorly developed lip, inclined towards the front and broadening at the columellar side, before curving back and covering the umbilicus.In C. (L.) erika and C. (L.) dalmatinum, which are both larger, the columellar lobe differs; the latter species may also be distinguished by its prominent, irregular ribs, which are more uniform in C. (L.) tropojanum sp.nov.Cochlostoma (L.) lanatum sp.nov.differs by its regularly spaced, prominent, sharp ribs on the upper whorls.The geographically separate C. (L.) jakschae sp.nov. is conchologically similar but less slender, with an even narrower lip.It also differs in the female genitalia, i.e., by the less convoluted visceral oviduct.Moreover, C. (L.) jakschae sp.nov.has, relative to its shell size, a smaller protoconch.

Description
Shell.Small, with 8½ whorls, H/W ratio 2.8.The initial 2.4-2.5 whorls form the protoconch, which is smooth for only the initial 0.3-0.5 whorl and finely ribbed with narrowly spaced riblets further on.The spotless shell is light yellowish.The teleoconch whorls are convex (Rr = 1.24) with whitish, rounded, moderately developed ribs.The ribs are regularly spaced, without any variation between the upper whorls and the body whorl.The ribbing is still present on the final part of the body whorl, approaching the aperture.The aperture is relatively small (Ha/H = 0.19-0.20),with a poorly developed lip, inclined towards the front.The lip is broadened at the columellar side before curving back and covering the umbilicus.The body whorl has a small external lobe.
Female genitalia.Bursa copulatrix large, with a pedunculus connected proximally; its proximal lobe clearly smaller than the distal one.Short seminal receptacle, confined to the ventral side of the body and connected with the pedunculus without a distal oviduct.The 3-4 wide and small loops of the visceral oviduct are situated over the apex of the seminal receptacle.The junction of the uterine gland and the copulatory duct is moderately far from the connection between the seminal receptacle and the pedunculus of the bursa.male genitalia.The penis is longer than the body and thin, with a tapering tip.The spermiduct is narrowly twisted.The body spermiduct is straight.There is a sharp and long groove, ending in a wellformed sperm pocket, delimited on the frontal side by an ascending sperm funnel.

Distribution
Mount Shkëlzen in the eastern part of the Prokletije Mts in NE Albania.Known from four nearby locations.

Habitat preference
Like most species of Cochlostoma, these are obligate rock-dwelling snails that can be found on the surface of limestone cliffs or under boulders on rocky alpine grasslands.Zallot, Fehér & Gittenberger sp. nov. urn:lsid:zoobank.org

Description
Shell.Small, with 7½-8½ whorls (H/W ratio 2.42-2.57).The initial 2.4 whorls form the protoconch, which is smooth for the first 0.5-0.7 whorl and then finely costulate with closely spaced riblets.The protoconch is relatively large (Dp/D4 th = 0.73).The shell is more or less dark horn-brown, without spots.The teleoconch whorls are costate, with whitish, slightly irregular in height, rounded ribs.The ribs are quite regularly and narrowly spaced, with 9-10 ribs per mm both on the 4 th whorl and on most of the body whorl.The ribbing is less prominent and denser on the last part of the body whorl approaching the aperture.The peristome is moderately developed; the lip broadens at the columellar side before curving increasingly back and covering the umbilicus.While approaching the aperture, the body whorl enlarges, while forming a moderately developed external lobe.
Female genitalia.Bursa copulatrix very large, with a more or less posterior connection of the pedunculus.The short seminal receptacle is confined to the ventral side of the body.The tortuous loops of the visceral oviduct are situated over the apex of the seminal receptacle.The junction of the uterine gland and the copulatory duct is moderately far from the connection between the distal oviduct and the pedunculus.

Etymology
This species is named after Ms Katharina Jaksch-Mason (NHMW) who found the first individual of this species at Bugarska Peak.

Distribution
This species is known from two mountain ranges that are nearly 200 km apart, i.e., the Galičica Mts in southwestern Macedonia, Ohrid District, and the Prokletije (= Bjeshkët e Nemuna) Mts in northern Albania.It occurs at altitudes above 1700 m.The former area is the southernmost occurrence known for the subgenus Lovcenia.

Habitat preference
Like most species of Cochlostoma, this is an obligate rock-dwelling snail that can be found on limestone cliffs or under boulders on rocky alpine grasslands.

Remarks
The molecular analyses confirm that the morphologically similar but geographically distant populations of the Bugarska Peak and the Pejë Pass are indeed conspecific.Zallot, Fehér & Gittenberger sp. nov. urn:lsid:zoobank.org

Description
Shell.Rather large and slender, with 8½-9¼ whorls (H/W ratio 2.84).The apical 2.2 whorls form the protoconch, which is smooth for only the initial 0.2 whorl and finely ribbed with close riblets further on.The shell is more or less light horn-brown, without spots.The teleoconch is sculptured with rather prominent, sharp ribs, which are widely spaced on the upper whorls and increasingly more narrowly spaced and less sharp towards the aperture.The aperture has a moderately developed lip, enlarged at the columellar lobe, which is abruptly bent inwards to cover the umbilicus.While approaching the aperture, the body whorl abruptly enlarges, forming a rather prominent external lobe.
Female genitalia.Bursa copulatrix large, with a pedunculus connected proximally; its proximal lobe smaller that the distal one.The short seminal receptacle is confined to the ventral side of the body, under (ventrally) the bursa copulatrix.The loops of the visceral oviduct are situated over the apex of the seminal receptacle.The junction of the uterine gland and the copulatory duct is far away from the connection between the pedunculus of the bursa and the distal oviduct.

Distribution
This species is only known from its type locality, the Deleve Cave on the southwestern slope of Mount Veleçik in northern Albania.

Habitat preference
Like the majority of known species of Cochlostoma, this is an obligate rock-dwelling snail.It was found in the twilight zone inside the Deleve Cave.Though little is known about its ecological requirements, it may be restricted to the epigean environment, like C. (L.) erika (i.e., subtroglophile in the sense of Sket 2008).At the Deleve Cave, its niche partitioning with Cochlostoma (Auritus) roseoli (A.J.Wagner, 1901) is similar to that of C. (L.) erika and C. (A.) auritum at the Popovo Cave in Montenegro.Zallot et al. (2015) assigned this species to Cochlostoma (Lovcenia), but with only one male specimen available, its systematic status remained difficult to judge.The shell morphology, in particular the protoconch sculpture, showed affinity to that of C. erika, whereas the molecular data suggested a status as a separate species.Since then, we have been able to collect and dissect female specimens.The structure of the female genitalia supports regarding it as a species on its own.

Discussion
As far as is known, the range of the subgenus Lovcenia extends from the Orjen Mts and the Njeguš Plateau (Montenegro) in the northwest to the Galičica Mts (Macedonia) in the south, with the central area of distribution in the Prokletije Mts.
Cochlostoma (Lovcenia) erika and C. (L.) lanatum sp.nov.are the only subtroglophile species known in the Cochlostomatidae.According to the trees based on concatenated COI, 16S and H3 sequences, these two taxa are not sister species, but relatively low supports of some branches indicate that relationships among the taxa of Lovcenia are not clarified beyond any doubt.Therefore, it seems feasible that the subtroglophile lifestyle evolved only once in this subgenus and was lost secondarily, but it is difficult to say whether the ancestral form of Lovcenia was already a subtroglophile species or not.Cochlostoma (L.) erika and C. (L.) lanatum sp.nov.occur syntopically with other large cochlostomatid species, namely C. (A.) auritus and C. (A.) roseoli.Snails of small species of Cochlostoma (e.g., of the subgenera Wagneriola Zallot et al., 2015or Turritus Westerlund, 1883) and snails of large congeneric species often co-occur, but only very rarely are two large-sized species found syntopically.

Fig. 1 .
Fig. 1.Distribution of the species of Cochlostoma (Lovcenia) Zallot et al., 2015 in the western part of the Balkan Peninsula.
At the same time, two different tree reconstruction methods resulted in partly incongruent topologies concerning relationships within the Lovcenia clade.Cochlostoma (L.) jakschae sp.nov.and C. (L.) tropojanum sp.nov.are sister taxa with high branch support in both of the trees.However, the relationships among the other three species are unclear: in the Bayesian tree C. (L.) dalmatinum (L.Pfeiffer, 1863) is in a basal position, in the ML tree C. (L.) erika is in that position (Fig. 4).For the most widely used marker (COI), the uncorrected p-distances among conspecific populations vary from 0.015 (C.(L.) tropojanum sp.nov.) to 0.021 (C.(L.) jakschae sp.nov.), whereas it varies between species from 0.034 (C.(L.) jakschae sp.nov.and C. (L.) lanatum sp.nov.) to 0.087 for C. (L.) erika and C. (L.) dalmatinum) (Table

Table 1
(continued on next page).List of sampling localities for species of Cochlostoma(Lovcenia)

Table 2 .
Zallot et al., 2015ion divergence between populations and species in Cochlostoma (Lovcenia)Zallot et al., 2015.Mean uncorrected COI p-distances are shown between species (in the lower half of the matrix) and between conspecific population (in the matrix axis).
Table 1 for localities and Table 3 for measurements.
Pfeiffer 1863: 136)was not available for study, but specimens from the type locality were compared (see below) as well as a photograph of a syntype (SMF 160850, ex coll.Parreyss) published byZilch (1958: fig.12).