Tandonia bolensis n . sp . , a new slug ( Gastropoda : Pulmonata : Milacidae ) from the island of Bra č ( Dalmatia , Croatia )

The genus Tandonia Lessona & Pollonera, 1882 is represented along the Western Balkans by about 13 taxa, most of which are regional endemics. Recent collecting on the island of Brač (Croatia) has provided a single, fully grown specimen of a totally black slug. A careful investigation of the genital anatomy and body traits, reveal characteristic and unique morphological features which until now have never been observed in other representatives of the genus. Such results have led to the description of new species. Tandonia bolensis n. sp. is easily distinguishable by virtue of its genital features, the dummy-like penial-atrial papilla, and the long and partially spiral-convoluted, thorny spermatophore.

Along the Western Balkans (from Istria southward, via Dalmatia as far as the Montenegrin coast) up to 13 taxa, belonging to Tandonia, are present and most of them are regional endemics (see Tab. 1).The most important diagnostic features are the morphology of the genitalia and the spermatophore, while the body (shape, coloration and size), the shell and other features such as the jaw or radula have been proved to be of poor diagnostic significance.For a full review of Tandonia's taxonomy see Wiktor 1987a: 165.Past authors who have dealt with the Western Balkan Tandonia, described new Dalmatian endemic species but provided only unclear or scant descriptions, without iconography and also often including errors and artefacts (cfr.Wiktor, 1996: 45): the description of T. reuleauxi by Clessin (1887: 46) was mainly based upon shell features; T. montenegrina was unclearly described by Simroth (1900: 107) as also stated by Wiktor (1996: 38).Simroth (1900: 106) also provided a brief description of the genitalia and the body of T. dalmatina, but it's status still remains unclear (Wiktor 1996: 31).Nevertheless Simroth's description of T. cavicola (1916: 4) was more complete, including both genitalia and body features and drawings, this iconography turns out to be too synthetic and lacking important taxonomic details.Wagner (1929: 333) Research Article described T. croatica only by its exterior appearance.In the same paper (Wagner 1929: 331) he also provided a full genital description for T. fejervaryi, allowing an easier systematics overview for this latter species.T. jablanacensis (Wagner, 1930) was fully described with both genitalia and body features, although never found again even after further field surveys (Wiktor 1987a(Wiktor , 1996)).Once more, the description of T. lagostana (Wagner 1940: 138) was incomplete and scant, probably based upon juvenile specimens.As a consequence many species classified within the genus Tandonia still have an uncertain systematic position (Wiktor 1987a(Wiktor , 1996)).
Only in more recent times has the description of a new endemic Tandonia species fully matched all the taxonomics requirements for an effective systematic evaluation.Wiktor (1996: 39) introduced T. rara from mid-Dalmatia, providing accurate descriptions and drawings, although he overlooked some remarkable taxonomical feature (De Mattia, personal unpublished data).
Recent collecting undertaken on the island of Brač (Splitsko-Dalmatinska županija, Croatia) has provided a single, fully grown specimen of a totally black slug, belonging to the genus Tandonia.A careful investigation evaluating the genital anatomy and body traits, revealed a set of characteristic and unique taxonomic features which have never been previously observed in other representatives of the genus.By virtue of such results a new species, Tandonia bolensis, has been described.

Material and Methods
The slug was drowned in water and then fixed in 80% ethanol.The shell was removed from its pocket beneath the mantle and the body was dissected under a Leica stereoscope.Anatomical details were photographed using a digital camera and later, freehand drawings were created from the photos.The shell and the body (including the jaw) was depicted and measured.The holotype is deposited in the Malacological Collection of the Museum and Art Gallery of the Northern Territory (NTM), Darwin, Australia.
The following abbreviations are used for the anatomical parts: A atrium; AG albumen gland; AAG accessory atrial glands; BC bursa copulatrix; DBC duct of bursa copulatrix; DRC double ringed collar; E epiphallus; FD fleshy disk; FO free oviduct; G gonad; NLP nipple-like papilla; OS ovispermiduct: P penis; PP penial papilla; T talon; V vagina; VD vas deferens.Figures 3-10.Genitalia and shell of Tandonia bolensis new species from Bol, Brač island, Croatia. 3 whole genitalia, gonad included; 4 distal genitalia; 5 inner structure of penial complex; 6 spermatophore; 7 magnification of the head of spermatophore; 8 magnification of the coiled central section of spermatophore; 9 shell; 10 talon.Scale bars: 1 mm.Shell (Fig. 9).The shell is whitish, sturdy and thick in the midpoint, overall hyaline, and peripherally thin and transparent.It is located in a small pocket beneath the posterior part of the mantle (clipeus).It is slightly elongated, dorsally convex and only slightly concave ventrally.From its rear embryonic part, many irregularly spaced growth lines develop frontward, almost concentrically.These growth lines are much more visible on the dorsal side of the shell.The ventral side is characterized by the presence of a fine granulated pattern.

Dimensions of shells.
The shell is 4.5 mm long and 2.3 mm wide.
Body (Figs 1-2).The animal is slim with a poorly developed keel, clearly visible along only the posterior third of the slug.The body is completely slate black, both dorsally and along the sides.A paler, creamy portion is visible under the mantle, appearing mainly after preservation and only partly visible in the live specimen.The head is also slate black.The skin grooves are equally black throughout the enitre body.No other patterns, such as dots or reticulations, are visible.The sole is clearly tripartite, with bands of muscles arranged in a chevron pattern, uniformly cream in colour.The slate black mantle makes up approximately one fourth of the total body length, and is finely granulated.The pneumostome is rounded, with a groove extending downward and reaching the mantle border.The mucus is colourless and milky when disturbed.The jaw is of oxygnathous type, chitinous and uniformly ochre/yellow.

Dimensions of body.
After preservation the body is 37.1 mm long and 8.1 mm wide.The mantle is 13.3 mm long and 8.4 mm wide.The pneumostoma is 0.8 mm in diameter.
Genital anatomy 10).The multilobed gonad is composed of hundreds of spherical acini and it is creamy-whitish in colour.It is located in the tail of the slug.A very long, non convoluted first hermaphrodite duct arises from it and ends laterally with a well developed, cylindrical talon (Fig. 10) which has a blunt apex.The albumen gland is relatively small and slim and is connected to a wide, lobated ovispermiduct consisting of prostatic and uterine portions.The prostatic portion continues turning in to the vas deferens.The uterine portion eventually leads into a long free oviduct, turning into vagina at the level of the bursa copulatrix duct.The free oviduct is about three times as long as the vagina, while the latter is much wider in diameter.The inner walls of the vagina have moderately elevated pleats running longitudinally.The small atrial accessory glands are four in number, gathered in two tufts with two glands each, placed at the opposite sides of the vaginal-atrial border.The duct of the bursa is wide and long, ending in a large oval, almost pointed bursa copulatrix.The inner walls of the duct have very fine longitudinal plicae while the inner walls of the bursa are smooth.Inside the duct-bursa copulatrix and its duct a well developed spermatophore was found (Figs 6-8).This spermatophore is 31.6 mm long and is corneous, hyaline and light yellowish in colour.The first third is tubular, smooth, moderately wide in diameter and thornless, gradually narrowing.The upper head is hook shaped (Fig. 7).The middle third is strictly coiled, with a dense thorny comb running along the entire section (Fig. 8).The last third of the spermatophore loses its coiled and thorny appearance, acquiring a small-chain-like structure, gradually widening and smoothing toward the "tail" part.The extremity abruptly ends with a blunt apex.The vas deferens opens apically into the epiphallus.The penial complex is cylindrical.The epiphallus is half the diameter and twice as long as the penis.The border between the epiphallus and the penis is evident externally while no retractor muscle is present (or is too thin to be detected).The inner structure of the epiphallus consists of a spongy architecture ending distally with a simple, round papilla leading to the penis.The inner walls of the penis have a faint radial pleat, mostly irregularly arranged A second, very unique and unusual papilla is present between the penis and atrium.This papilla is somewhat dummy-like shaped (Fig. 5), with an upper, double ringed collar ending in a fleshy disk that obliterates the whole section of the distal penis.A nipple-like papilla, with a thin base and a large head, arises from the middle of this disk and ends in the atrium.The atrium is simple, structureless, with very faint folds originating from the genital pore and running upwards.
Etymology.Named after the village of Bol, located on the southern coast of the Dalmatian island of Brač (also known as Brazza), which is the nearest town from the type locality of this new species.
Ecology.Tandonia bolensis sp.n. was found crawling on cretacean limestone of coastal cliffs during a heavy summer rainfall.Most probably the slug is usually hidden in deep rock crevices and cracks.The new species occurs in an extremely xeric and steep environment with coastal sclerophyll maquis and low scrubland.
As regards T. albanica, the vas deferens inserts asymmetrically on the epiphallus.The proximal portion of epiphallus shows two bumps and the penis has a strong, large retractor muscle.The many atrial accessory glands are big, somehow fused together at their base and distributed around the distal end of the vagina.The spermatophore is short and not spiral in form at any (Wiktor 1987a: 223-224;Wiktor 1987b: 99).In the case of T. cretica, apart from the genital morphology the major differences are found as regard the spermatophore, which is never spiral and is covered throughout by single, bifid or fringed spines (Wiktor 1987a: 238-240;Wiktor 1987b: 92, 97).
T. ehrmanni, apart from being biogeographically and ecologically distinct, shows a unique atrial ctenoid structure, between the openings of penis and vagina (Wiktor and Milani 1995;Nardi 2011).
T. fejervaryi, which is a northern Dalmatian endemic, has many long atrial accessory glands and the inner walls of the penial complex are covered throughout with dense, sharp pointed papillae.Moreover, the vagina seems to be much longer with a bursa copulatrix without a duct.Its spermatophore also shows considerable differences, being with long anchoring spines in thin section and heving completely smooth walls of container (Wiktor 1987a: 248-250;Wiktor 1987b: 89).
T. jablanacensis is a poorly known taxon, nevertheless the information so far recorded for this species allow us to easily distinguish it from T. bolensis sp.n..The bursa copulatrix is very long, almost twice as long as the ovispermiduct and wider, gradually widening posteriorly and ending with an oval distension; its spermatophore is unknown (Wiktor 1987a: 252).Even so, this species has been recorded only by the original author on the mainland (Wagner 1930: 46) far from Brač island.T. macedonica shows a very unique penial papilla, in the shape of a mushroom.The spermatophore is strongly elongated anteriorly, laterally covered by small, adhering hooks.Posteriorly the spermatophore is covered with several thick, branched, large hook-like spines (Wiktor 1987a: 263-264;Wiktor 1987b: 94).
T. nigra has a duct that is much shorter than the bursa copulatrix itself and the inner walls of the vagina have dense large conical pointed papillae (Wiktor 1987a: 270).Moreover it distribution is largely restricted to a relatively small area of the Central Alps (Lombardy in Italy and Ticino in Switzerland) (Manganelli et al. 2000;Boschi 2011).T. piriniana shows a peculiar pocket-like appendix between the vas deferens and the epiphallus, as well as a strong and large retractor muscle and very large atrial accessory glands.T. rara has a remarkably developed atrium with a set of spiny longitudinal crests, which are exposed during copulation, and a small and relatively thin penial complex.The penial complex is small and relatively thin (Wiktor 1996: 39-41).T. reuleauxi, distributed along southern Dalmatia and Montenegro, shows a different spermatophore structure and a different sculpture of the inner penial complex walls and penial papilla (Wiktor 1987a: 280-281;1996: 42).
Tandonia bolensis sp.n. is not the first species described from a Dalmatian island.Wagner (1940: 138) described Tandonia lagostana from the island of Lastovo (Italian: Lagosta), Dubrovačka-neretvanska županija, Croatia.Wagner provided a very poor iconography which does not allow for proper taxonomical evaluation.Nevertheless the description given by Wagner, both in regard to the body and genitalia, does not fit at all what we have provided for Tandonia bolensis sp.n.The body is presented as reddish-ochrous, brownish-violet on the back with a well defined reticulated pattern due to the pigment accumulation while the genitalia's description is too general, aspecific and lacking of many important taxonomical features (i.e.atrial accessory gland and spermatophore).For a review about this taxon see Wiktor (1996: 35).Simroth (1900: 106) described Amalia dalmatina from Dubrovnik (Dubrovačka-neretvanska županija, Croatia).Again the taxonomic description provided by the author is too scant to clearly identify it, but the brief description given by Simroth does not fit with Tandonia bolensis sp.n.(see also Wiktor 1996: 31).Similarly, the same principles can be applied to what Wagner (1929: 333) described as Milax croaticus and thus not even this name can be considered available for the species here described from the island of Brač.
Some of the remaining known Tandonia species show a livery with a combination of two or more hues (e.g.ochre, green, reddish or brown), usually darker on the back and gradually lightening along the sides, with a reticular, darker pattern following the skin grooves.Other species of Tandonia presents a more or less light and uniform creamy-brownish ground colour.In both basic liveries different patterns can be present: spots, blots or smears.However, genital features make Tandonia bolensis sp.n. easily distinguishable from other Tandonia species.Moreover, a fundamental and essential taxonomical feature seems to be represented by the overall spermatophore architecture.The upper part, cylindrical and without any accessory spines could resemble species such as T. fejervaryi, T. rara and T. robici but the lower section, shows the unique coiled morphology, with a dense thorny comb.It is also worth noting that some rare or cryptic Tandonia species have previously been described upon a single specimen (Wagner 1930;Wiktor 1987a) as in the present instance.
In the wake of these preceding papers, Tandonia bolensis sp.n. is unlikely to be abnormal or mutant by virtue of its peculiar anatomy which shows not a single, but many different peculiar traits.Defining whether or not Tandonia bolensis is an insular endemism will require further collecting activities throughout the whole area.Up to the end of the Würm/Weichsel glaciation (approximately 10.000-8.000years ago), the island of Brač was completely connected to the mainland thus its malacofauna probably has a common origin with the whole Southern Dalmatia.
Status and conservation.Its (until now) limited distribution makes it "vulnerable", according to the Categories and Criteria of the IUCN Red List of Threatened Species (www.iucnredlist.org,Version 3.1).

Table 1 .
Distribution of the Tandonia species living along the Western Balkans with references to the literature's iconography.