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

Five species of the subgenus Clessiniella of Cochlostoma (Cochlostomatidae) are recognized, viz. Cochlostoma (Clessiniella) villae (Strobel, 1851), Cochlostoma (Clessiniella) tergestinum (Westerlund, 1878), Cochlostoma (Clessiniella) waldemari (A.J. Wagner, 1897), Cochlostoma (Clessiniella) anomphale Boeckel, 1939 and Cochlostoma (Clessiniella) stelucarum sp. nov. The shells and the genitalia are described and the distributional data are summarized for all the species.


Introduction
The genus Cochlostoma Jan, 1830 includes predominantly rock-dwelling species distributed along the mountain ranges surrounding the Mediterranean Sea. Until recently, the taxonomy of the genus was based only on shell morphology. The lack of clear species-specific shell characters, however, makes it extremely difficult in many cases to assign a population to a certain species. In our previous papers (Zallot et al. 2015(Zallot et al. , 2018 we proved that the structure of the genitalia, together with the analyses of some DNA markers, may facilitate identifications while enabling a sound phylogenetic classification. The results of this approach imply a revision of the shell-based taxonomy, which is still used in the most recent European checklist (Welter-Schultes 2012). Whilst revising each of the subgenera of Cochlostoma, we herewith analyze the subgenus Clessiniella Zallot, Groenenberg, De Mattia, Fehér & Gittenberger, 2015, which includes a group of phylogenetically closely related species inhabiting the eastern Alps and are characterized by a diagnostic structure of the female genitalia.

Material and methods
The sampling localities, mapped in Fig. 1, are also indicated for each species in the taxonomic section of this paper. An approximate position is used for the pre-GPS samples. The availability of female and male specimens from the different sampling localities was extremely various (dry or alcohol preserved, number of male and female specimens) and therefore the level of analysis of each sample was consequently different, as reported in Table 1. The methods for depicting the shells, obtaining measurements and dissecting genitalia have been discussed previously in Zallot (2002: 94-97) and Zallot et al. (2015: 64-67;2018: 2-8). Here, we provide a description of the characters of the shell and of the genitalia that are used for classification.

Shell characters
Ap. incl.
= inclination of the apertural edge in lateral view in respect to the main axis (no. 13 in Fig. 2) H 1 st /4 th = ratio between the height of the 1 st and that of the 4 th whorl. It is an index of the slenderness of the shell Ha/Ht = ratio between the height of the aperture measured along the main axis and the total height Ht = total height: measured along the columella (no. 1 in Fig. 2) Ht/W = ratio between the total height and the width of the basal whorl

LipW
= the apertural lip, on the columellar side, enlarges and flattens (no. 11 in Fig. 2). LipW is the ratio between the projection in frontal view of the maximum width of the lip on the columellar side and the maximum width of the aperture LobeH = projection in frontal view of the height of the external lobe (no. 5 in Fig. 2) Prt = number of whorls of the protoconch. The embryonic shell is recognizable by a change of ribbing and often of the color (Fig. 3) Prt/4 th = ratio between the width of the basal whorl of the protoconch and the width of the shell measured along the 4 th suture. This ratio summarizes the relative size of the protoconch in respect to the whole shell Ribs/mm 1 st = number of ribs per mm on the 1 st whorl. The number of ribs per mm is calculated by measuring the distance between the first and the last of the most central five ribs close to the upper suture of the whorl (no. 15 in Fig. 2) Ribs/mm 4 th = number of ribs per mm on the 4 th whorl. The number of ribs per mm is calculated by measuring the distance between the first and the last of the most central five ribs of a whorl, in frontal view, close to the basal suture of the whorl (no. 16 in Fig. 2) Zallot et al. 2015. 1 = height (H); 2 = height of the 1 st whorl; 3 = height of the 4 th whorl; 4 = height of the aperture (Ha); 5 = height of the external lobe; 6 = width of the 1 st whorl (W); 7 = width of the 1 st suture; 8 = width of the 2 nd whorl; 9 = width of the 4 th whorl; 10 = width of the aperture (Wa); 11 = maximum width of the lip on the columellar side; 12 = width of the basal whorl of the protoconch; 13 = inclination of the aperture; 14 = inclination of the ribs; 15 = distance between the 5 central ribs of the body whorl; 16 = distance between the 5 central ribs of the 4 th whorl. The figure in the lower right corner shows how the whorls (green) and sutures (red) are counted.

Fig. 2. Measurements taken on the shell of Clessiniella
Ribs incl. = inclination of the ribs in respect to the plane orthogonal to the columellar axis (no. 14 in Fig. 2) Smooth Prt = number of protoconch whorls, starting from the tip point that are smooth at 40× magnification (no. 1 in Fig. 3) W = width: measured at right angles to the columella (no. 6 in Fig. 2) W 1 st /4 th = ratio between the width of the 1 st and that of the 4 th whorl. It is an index of the slenderness of the shell W round = ratio between the maximum width of the 2 nd whorl and the width of the shell measured at the 1 st suture. This measure indicates the convexity of the whorl(s), a feature frequently mentioned in the description of the taxa within Cochlostoma Whorls = number of whorls: counted with the shell in front view. The whorls and the sutures are counted bottom-up, so that the basal whorl is the 1 st one and the suture above is the 1 st suture as shown in Fig. 2.  less strong than on the front side of the body whorl; S = as strong as in the front side ( Fig. 4D) Rib prominence = from 0 = ribless shell to 3 = really strong/prominent ribs (Fig. 4C) Spots noticeability = from 0 = spotless shell to 2 = really noticeable spots (Figs 4B,12). The shell spots are areas of the shell that are darker than the background. They are usually reddish/brown.

Nominal or ordinal data
Female genitalia (Figs 5-6) Club-shaped seminal receptacle: seminal receptacle with narrow basis, gradually enlarging towards the apex where it reaches the maximum width: SS = short, club-shaped seminal receptacle; SM = clubshaped seminal receptacle of average length; SL = long, club-shaped seminal receptacle.
Oval-shaped seminal receptacle: seminal receptacle with recognizable basis and apex, with maximum width close to the midline; BS = short, oval-shaped seminal receptacle; BM = oval-shaped seminal receptacle of average length; BL = long, oval-shaped seminal receptacle.
Loops set up: the visceral oviduct, between the final loop and the seminal receptacle, is more or less tortuous and the resulting loops are more or less clustered together ( JUG position: the junction of the uterus gland with the copulatory channel can be more or less far from the connection between the pedunculus of the bursa and the distal oviduct: from 0 = really close to 3 = really far (Fig. 6H).
Loops n.: the visceral oviduct can have a different number of loops. A rough count of them gives a measure of the complexity of the loops path (and of the length of the visceral oviduct in between the final loop and the apex of the seminal receptacle).

Morphological characters
Five species of Cochlostoma, one of which is new to science and here described, show the female genital characters of the subgenus Clessiniella as described by Zallot et al. (2015: 82). In Table 2 we present the measurements of the shell characters of the types and the average, minimum, maximum and standard  deviation values of the characters at the subgenus and species level. In Table 3 we report of each species the features as explained in Fig. 3. A discriminant analysis based on the shell features is presented in Fig. 8. In Figs 9-11 the relations between some of the most relevant shell characters are graphically illustrated.
Female genitalia. The data regarding the female genital features are presented in Table 4.  Table 5. GenBank voucher (H3).

DNA analysis
Female specimens of each species were used for DNA extraction. A Histone fragment (H3) was the only DNA marker we could successfully amplify in the 5 species. New H3 sequences were deposited in GenBank (KX120829-31 and KX120837, see Table 5). They were aligned with the sequences of Clessiniella published by Zallot et al. (2015) (Fig. 3). As Zallot et al. (2015) have found that Cochlostoma s. str. is the sister clade of Clessiniella in the H3 tree, a sequence of Co. (Co.) septemspirale (Razoumowsky, 1789) was used as outgroup. Because of the low resolution, the phylogeny does not show a tree-like structure. Therefore, we illustrated in Fig. 7

Diagnosis of the subgenus
Shell. Ribbed teleoconch with straight ribs, variable in strength and shape. The columellar lobe is curved backwards, covering the umbilicus. The teleoconch spots (if present) form 2 rows on the whorls (often, a 3 rd row of spots is visible on the body whorl): the upper one close to the suture with rectangular spots abapically elongated; the lower one roughly ⅓ up on the whorls (Fig. 12). In some populations the 2 rows of spots merge and there is a continuous vertical darker band on the whorls. The protoconch is dull and robust with fine riblets starting after 1.2 whorls or more from the apex; it is transparent and fragile or the ribbing starts close to the apex in other subgenera.
Female genitalia. There is an apical connection of the pedunculus to the bursa copulatrix, a long seminal receptacle (normally club-shaped), which runs parallel to the pedunculus. The junction of the uterus gland is situated close to the connection between the pedunculus and the distal oviduct. This combination of characters in the female genitalia is most diagnostic to distinguish Clessiniella from the other subgenera.
male genitalia. Apart from its apical part, the penial spermiduct is tortuous. The body spermiduct is protruding (often almost detached from the body) more than in the other subgenera and either straight or more or less tortuous. The posterior side of the penis is wrinkled.
Identification key for the species 1. Body whorl inflated shortly before the lip (Fig. 4A: OB
Other material (fresh specimens from the type locality have also been analyzed)
lip. Moderately developed, and more or less flattened by the internal callus of the aperture. It gently curves backwards at the columellar side, starting rather far from the upper part of the body whorl. On lateral view, the body whorl, approaching the aperture, in most of the specimens barely enlarging (Fig. 4A: FF, Table 3).
Female genitalia. There is a long and relatively thin (as compared to the other species of Clessiniella; see Table 5) seminal receptacle with a curved apex. male genitalia. The penis is long and inflated. The penial spermiduct is slightly twisted and occupies only the central part of the penis. There is a clearly protruding but straight body spermiduct.

Distribution
The species inhabits a relatively small area at the foothills of the eastern Alps. It can also be found on the hills stretching down the Venetian flatland of the "pianura Veneta" (Colli Berici and Colli Euganei). It has been found sympatric with Cochlostoma (Cochlostoma) septemspirale and Cochlostoma (Eupomatias) philippianum in some localities of the province of Verona (Fernando Scarlassara, personal communication).

Habitat preference
The snails are found on rocky cliffs as well as on smaller boulders and dry stone walls in woody places.

Remarks
The range of this species is far from the almost continuous area in the east where the other Clessiniella species are known from. The species has been reported (and still is in the checklist of the Italian fauna -www.faunaitalia.it) as Cochlostoma villae (De Betta & Martinati, 1855) but Bank (1988) found that Strobel (1851) (1851) is poor: "Varieta' generalmente maggiore, coi giri della spira piu' gonfi e colle coste trasversali piu' regolari, e saglienti" = "variety generally bigger, with more rounded whorls and more regular and strong ribs" and follows "P. Villae Spinelli in  It could be, as was common at the time, that the specimens used to describe the species by both Strobel (1851) and De Betta & Martinati (1855) have had a tortuous path, after being collected by Villa in 1849 (as it appears from Strobel's label), given to Spinelli later on, who gave the collector's name to the species (as far as is known, however, Giovanni Battista Spinelli never published the specific epithet villae, probably using the name only in private correspondence). Spinelli eventually gave part of the material to Strobel and part to De Betta (the De Betta material is currently kept at the Museo di Storia Naturale di Verona, see Fig. 21). Boeckel, 1939 Figs 1, 3A, 3A1, 14A, 15A, 16A, 22-24

Diagnosis
Shell quite densely ribbed, spots on the teleoconch generally clearly visible, body whorl inflated shortly before the lip. Visceral oviduct simplified with 1-2 superficial loops at most; seminal receptacle always club-shaped.

Remarks
The holotype and 23 paratypes should be in the Boeckel collection at the Naturkundemuseum Erfurt (Hartmann et al. 2018: 26). However, the shells could not be traced when this article was prepared (C. Unger in mail October 14, 2020).Type locality -Austria, Trögern, way to Vellach.   Boeckel, 1939. Paratypes at the Senckenberg Museum Frankfurt (SMF42967/2).

Description
Shell. The outside of the body whorl is inflated shortly before the apertural lip ( Fig. 4A: OB). The lip is poorly developed; it is almost vertical at the palatal side, and curved backwards and covering the umbilicus at the columellar side. The shells are more densely sculptured and the ribs are weaker than in the other species of Clessiniella. Most of the shells have spots.
Female genitalia. There is a spindle-like seminal receptacle. The visceral oviduct has only 1-2, superficial, loops. male genitalia. The swollen penis is longer than the body and has a central spermiduct. There is a protruding, straight, body spermiduct.

Distribution
Cochlostoma (Clessiniella) anomphale inhabits the Karawanken in south-eastern Austria and northern Slovenia.

Habitat preference
The animals can be found under and on stones and boulders in shady habitats in forests.

Remarks
This species was initially described as a subspecies of Cochlostoma (Cochlostoma) septemspirale. Gittenberger (1971) considered it a separate species because of the sympatry with Co. (Co.) semptemspirale heydenianum and the lack of conchologically intermediate specimens. This is an epitome of quite common Cochlostoma taxonomical issues: based on shell morphology it was initially described as subspecies of a taxon which is only distantly related.

Diagnosis
Slender shell with 8½-10½ rather widely ribbed whorls. Spots always visible. Poorly developed lip with FF or AF external lobe. Visceral oviduct simplified and seminal receptacle sometimes oval.

Etymology
The epithet 'steluca' is an acronym noun formed from the initial syllables of the names of the wife and two daughters (Stefania, Ludovica and Camilla Zallot) of the senior author.

Description
Shell. The shell has prominent spots and strong and rounded ribs, which are regularly and widely spaced and coloured like the background. The ribs become less prominent on the body whorl. The body whorl slightly and gradually enlarges while approaching the aperture (Fig. 4A: FF, AF). In comparison with the other species of the subgenus, the whorls increase more slowly in size so that, given the same shell height, there is a higher number of whorls (Fig. 9).
Female genitalia. The visceral oviduct has only 1-2 superficial loops, what makes it one of the simplest in the genus Cochlostoma. The seminal receptacle is thin and may be oval, thus not always club-shaped as in the other species. male genitalia. The penis is longer than the body and inflated, although less so than in the other species of Clessiniella. The penial spermiduct is gently twisted. The body spermiduct is straight. There is a long groove, ending in a well-formed sperm pocket, delimited at the frontal side by an ascending sperm funnel.

Distribution
The species inhabits a restricted area in the eastern Southern Pre-Alps. Our sampling localities are located between Val Tramontina to the west and Val di Torre to the east (Friuli region, Italy).

Habitat preference
Like in most species of Cochlostoma, these are obligate rock-dwelling snails but, rather than on the surface of limestone cliffs, their main habitat is on smaller surfaces of stones and boulders which are normally found in large numbers at the foot of the cliffs.

Remarks
Cochlostoma (Clessiniella) stelucarum sp. nov. was introduced as Cochlostoma sp. by Zallot (2002: 98). At most of the localities, it lives syntopically with either Co. (Eupomatias) philippianum (Gredler, 1853) or Co. (Eupomatias) henricae (Strobel, 1851). Near the village of Anduins it has been found a few centimeters apart from both Co. (Eu.) henricae and Co. (Cochlostoma) septemspirale on stones and boulders. This is one of the rare cases where 3 species of Cochlostoma, belonging to different subgenera, live syntopically. The range of the species is situated in between the range of Co. (Cl.) villae in the west and that of the other species of Clessiniella in the east.

Remarks
The original indication of the type locality is rather vague, since only a city in the Friuli-Venezia Giulia region, in the easternmost part of Italy is indicated. The closest to Trieste where we found the species is at "Val Rosandra", 8 km north-east of the city. The fresh specimens collected there have shell features similar to those of the syntypes. We therefore consider them as topotypical.

Description
Shell. The size of the shells is extremely variable (height 6.1-9.6 mm in the females) as well as their general morphology (see Fig. 28). Several populations are spotless whereas others have clear spots on the teleoconch. Depending on the population, the lip can be either poorly or even hugely developed. In lateral view, the body whorl, while approaching the aperture, may widen in various ways, depending on the population (Fig. 4A: FF, AF, II). The H/W ratio is very variable and the slenderness of the shells in different populations varies accordingly.
Female genitalia. There is a long, club-shaped seminal receptacle. In some cases there is a change of colour at its base, presumably indicating the starting point of the rather long distal oviduct. As shown in Table 5, there is a considerable variability in the part of the seminal receptacle that is curved down and in the number of loops. male genitalia. The inflated penis is longer than the body. The penial spermiduct is confined to the central part of the penis and gently twisted. The body spermiduct is straight and well defined.

Distribution
This species inhabits the eastern side of the province of Trieste in Italy, the southwestern part of Slovenia and the Istrian peninsula. It has also been found in several Croatian localities in the surroundings of Bakar and, quite far from its main range, on the Villacher Alps, in Austria (see Mildner 1997). The species has been erroneously reported for Albania (see Fehér & Erőss 2009).

Habitat preference
It is an obligate rock-dwelling species. Co. (Cl.) tergestinum is found in a variety of habitats, from open, exposed limestone cliffs to shady spots in deciduous and evergreen forests.
For the time being, we here synonymize these nominal taxa because the conchological features which supposedly characterize them, like measures, density and prominence of the ribs, and the structure of the lip, are population features without an obvious geographical pattern. There is no significant correlation between latitude and longitude of the locality of the sample and the character states that are mentioned in the original description of the so-called subspecies. Moreover, in the surroundings of Trieste, the type locality of the nominate subspecies, only populations with some 'tortivum' shell features, like the thin lip, can be found (De Mattia & Prodan 2006). Some of the populations have a peculiar shell morphology (see for instance Fig. 28E-F); their taxonomical status should be investigated more thoroughly especially since the available molecular H3 analysis indicates that population "EZ0813" from Rasko Polje, corresponding to the specimen in Fig. 28E, stands apart from the others. (Wagner,

Description
Shell. The shell is more or less light corneous brown and spotless. The size of the aperture varies considerably among the populations; shells with a large aperture and a correspondingly large lip occur next to shells with a poorly developed lip and a relatively small aperture. The lip is gently curved backwards on the columellar side.

Distribution
The species inhabits the north-western and south-eastern part of Slovenia and an adjacent area in Croatia. It is reported from Austria for the Karawanken and the Gailtaler Alpen (Edlinger & Mildner 1979). Its range largely surrounds that of Co. (Cl.) tergestinum (see Fig. 1).

Habitat preference
The snails are found on rocky cliffs as well as on boulders in woody areas.

Remarks
To assign a given population to Co. (Cl.) waldemari rather than to Co. (Cl.) tergestinum on the basis of the morphology of the shell and the female genitalia alone could be challenging if not impossible (see . Only the structure of the male genitalia allows a convincing identification of the species. The H3 analysis (Fig. 7) confirms that these sibling species belong to separate clades within Clessiniella. Boeckel (1939) considered Co. (Cl.) waldemari a subspecies of Co. (Cl.) tergestinum. This view was contradicted by Bole (1994). In some areas, like on Mount Snežnik in southern Slovenia, the two species live not far from each other and here they can be distinguished also conchologically while differing in the presence or absence of the spots on the teleoconch (with Co. (Cl.) waldemari having a spotless shell) and in the shape of the body whorl approaching the aperture, in lateral view (Fig. 36).

Discussion
The lack of clear diagnostic features in shell morphology, with intraspecific sometimes overlapping interspecific variability as in Pyrenean Cochlostomatidae (Gofas 2001), is one of the reasons of the problematic taxonomy at the species level within the genus Cochlostoma. Based on only shell morphology, several populations of 4 of the 5 species of Clessiniella cannot be identified with certainty. The lack of clear distinguishing shell features may explain why a species like Co. (Cl.) stelucarum sp. nov. went unnoticed in an area that may be considered well explored. The multivariate analyses of   (Westerlund, 1878), Jakačici, Croatia (WdM6699). the shell measurements also fail to unequivocally diagnose the species. The shell features that cannot be quantified may be helpful for identification, but are hardly decisive, except for the identification of Co. (Cl.) anomphale. There is no general, always satisfying, single morphological dataset that enables indisputable identifications. The analysis of the female genital morphology sets apart Co. (Cl.) anomphale and Co. (Cl.) stelucarum sp. nov. from the other species but it does not clarify everything in Cochlostoma taxonomy. Similarly, the investigation of the male genitalia reveals a taxonomical relevant feature for the diagnosis of Co. (Cl.) waldemari but not of the other species.
A combined analysis of shell and genitalia is needed to clarify the taxonomic relationships within the genus in a satisfactory way. It should not be overlooked, however, that the results of our approach do not  (Westerlund, 1878), Val Rosandra, Italy (EZ0840). fundamentally differ from the views of our predecessors except for the phylogenetic relations amongst taxa. Three species of Cochlostoma (Clessiniella) got their names in the 19 th century, one in the 20 th century, and one species was discovered only in modern times.