New marine gastropod records for the Hellenic waters

The Hellenic Seas are influenced by on-going environmental changes and the introduction of alien species, which are expected to have an impact on their biodiversity. This study contributes to the knowledge of the Hellenic marine gastropod biodiversity, expanding data over the entire Greek territory, during the period from October 2008 to March 2017. This work presents 45 species of gastropods not previously reported from Greece or reported only once, belonging to 19 families. From those species, one (Horologica sp.) is, most probably, an undescribed species, 17 are new for the Eastern Mediterranean Sea and 40 are new for the Hellenic fauna. Main taxonomic characteristics and ecological information such as habitat, distribution and origin, are given and discussed. By this report, the Hellenic gastropod biodiversity is enriched by 40 new records, out of which, 17 are new for the Eastern Mediterranean Sea, 4 are Lessepsian migrants previously reported for the Mediterranean Sea and 1 is probably a new species.


Background
The Mediterranean Sea is rich in biodiversity. Almost two decades ago, about 8500 species have been estimated to occur [1]; this number doubled recently to more than 17,000 species [2]. This impressive increase is attributed to (a) the rising number of relevant studies in new areas and at different depths and biotopes, (b) the introduction of alien species, which reached almost 1000 species by 2012 [2], and (c) the increasing rate of modification of the Mediterranean Sea ecosystems [3]. The environmental changes that have influenced the biodiversity during the last three decades, the reasons for the occurrence of aliens, the frequency of the records, the vectors and the distribution pathways have been extensively discussed [2,[4][5][6][7][8][9][10][11][12].
The Hellenic Seas, as a part of the Eastern Mediterranean Sea, have been influenced by the on-going environmental changes, and Aegean Sea, in particular, after a long and slow cooling period from the late sixties to the early nineties, started to warm rapidly [13]. As a result, dense waters from the Adriatic Sea shifted to the Aegean Sea, a phenomenon known as the "Eastern Mediterranean Transient" [14,15]. These environmental changes combined with the effects of navigation and sea currents, were expected to have an impact on the biodiversity. Indeed, recent publications revealed new species for the Hellenic Seas [16][17][18][19][20][21][22]. The aim of this study was to further contribute to the knowledge of the Gastropod fauna of the Hellenic Seas.

The documented records for the Hellenic Seas
All 1st and 2nd documented records from the Hellenic Seas are presented bellow per family; a brief description is given whenever additional information is supportive to their identification.

Chilodontidae Wenz, 1938
Danilia costellata (O.G. Costa, 1861) ( Fig. 5a-c). One live specimen and 13 shells (2.65-10.00 mm high, 2.55-6.55 mm wide), biogenic material and maerl, 80-120 m, stations 23, 24 and 25. Protoconch paucispiral of one smooth whorl followed by half a rather smooth whorl with axial ribs. Next whorls are decorated with distinct wavy spiral cords. The body whorl bears 14-15 axial prosocline (15 o -20 o ) ribs crossed by spiral cords and forming shallow cavities. Columellar tooth variable from small to big in size and from pointed to square in form. 14-15 main teeth on the outer lip and several secondary. Unicolor of moccasin color in general. Very similar to D. tinei (Calcara, 1839) ( Fig. 5d) but the latter bears 17-18 axial prosocline cords (30 o -35 o ) while the cavities of the decussated areas are deeper. Its outer lip bears less main teeth (10)(11)(12) and the shell is whitish with about 8 brown zigzag zones of flames (not as spots) [35]. Danilia costellata was referred from the Mediterranean Sea by Palazzi and Villari [45], Crocetta and Spanu [46] and Cossignani et al. [47] as certain, by Vilvens and Heros [48] as doubtful and Herbert [49] characterized that of Crocetta and Spanu [46] as tentative. In contrast, D. tinei was referred from the Mediterranean Sea by many more researchers including Gofas et al. [35], Ghisotti and Steinmann [50], Cachia et al. [51], Navaro and Capdevila [52], Scaperrotta et al. [53] and Öztürk et al. [54]. Based on the available bibliography, the two Mediterranean Danilia species have overlapping taxonomical characteristics including the shell outline, the ratio height/width, the shape and size of the collumellar tooth and the depth distribution range. Moreover, as the juvenile shells exhibit no  [55], a further study of these two congeneric species seems to be necessary.
Another specimen present as C. cf. scalaris Locard, 1891 (Fig. 13d, not in Table 1) was collected, mixed bottom, − 10 m, station 5. It is lighter in color (honeyyellow) with the adapical cord light brown instead of unicolor and dark chestnut-brown as referred by Gofas et al. [35], and with a cylindroconical protoconch of 4.5 whorls instead of 3.5-4.0 of C. scalaris [77,78]. Oliver et al. [79] pointed out that the teleoconch of C. scalaris may vary from cylindrical to cyrtocylindrical, that members of the genus Cerithiopsis with the same type of protoconch differ in their teleoconch and that cryptic species possibly exist within the C. scalaris complex.
Horologica sp. (Fig. 14a). One live specimen (2.55 mm high, 0.90 mm wide), Aplysina aerophoba (Nardo, 1833) sponge host, − 6 m, rocky bottom, station 16. Shell small, h/w ratio 2.8, fusiform, glossy, slightly scalaroid and moderately slender. Protoconch 400 μm high and 220 μm wide, conical, smooth, milky-white with light brown nucleus, translucent, consisting of about 4 slightly convex whorls less inflated adapically and exhibiting minute and dense dentition at the suture. Limit to the teleoconch sigmoidal. Teleoconch composed of 5.5 flat whorls with a broad base. Spiral sculpture of two wellseparated cords starting as two from the onset of the teleoconch, the adapical one being weaker in the first three whorls but becoming first equal and finally stronger in the last whorl. Two additional wide and strongly tuberculated spiral cords-peripheral and basal-and a swelling before the lower end. Axial sculpture of 14 strong, prosocline and axially aligned ribs on the body whorl, which at their intersections with the spiral cords form ovate, equidistant, conspicuous nodules with quadrangular interspaces. The nodules are elongated radially in the first four whorls and axially in the last one. Suture deep and evident separating clearly the whorls. Color walnut-brown with lighter nodules and the two basal cords darker brown. Aperture pear-shaped, wide, with white and simple outer lip. Columellar callus conspicuous, peristome thin, anal canal short and broad, siphonal canal short, open and stubby. Aperture showing by transparency the pattern of the spire. Anterior part of the animal grayish-white. The shell looks similar to Cerithiopsis minima (Brusina, 1865) in its outlook, the size and the white planktotrophic larval shell (only lacking the minute denticles in C. minima), but it bears two spiral cords as in the genus Dizoniopsis Sacco, 1895 and Horologica Laseron, 1956 [80]. It also looks similar to Dizoniopsis aspicienda Bouchet, Gofas & Warén, 2010, but it differs mainly in the type of protoconch and the color. As members of the genus Dizoniopsis bear a stiliform or globose larval shell decorated with axial ribs or spiral cords the specimen most probably belongs to the genus Horologica Laseron, 1956, the members of which bear a smooth multispiral conical/cylindrical protoconch and 2 spiral cords per whorl [80,81]. Oliver et al. [82] under the name Dizoniopsis sp. (Figure 77 in [82]) show a quite similar to our shell, with missing protoconch, from the Chafarinas Islands, Alboran Sea. The latter authors refer that their specimen differs from the known Dizoniopsis species from the Atlantic Ocean and the Mediterranean Sea.
Obesula marisnostri Bouchet, 1985 (Fig. 16a). One live mature specimen of dark chestnut-red shell (7.55 mm high, 1.90 mm wide) and lighter apex, in whitish sponge on maerl, − 100 m, station 23. The easily recognizable shell has been well described [21,35,85] but the animal coloration was lacking until now where one live specimen has been collected. The external parts are uniformly translucent-white with cream-white spots and black eyes. The species has been referred from Greece [21] and other areas of the Mediterranean Sea [35,77].

Eulimidae Philippi, 1853
Acrochalix cf. callosa Bouchet & Warén, 1986 (Fig. 18a  protoconch. The later has a bluntly rounded initial whorl, a visible limit with the teleoconch, consists of 2.5 evenly convex whorls, is 260 μm high and 185 μm wide. The teleoconch bears 6 perfectly smooth and nearly flat whorls of gradually increasing diameter and an evenly curved axis. Suture very indistinct while the incremental growth scars of all 6 whorls are strictly aligned, forming thus a perfect series oriented towards the upper right of the shell. The right side of the shell is almost straight. The shell is flattened dorso-ventrally with the diameter from the outer lip to the opposite side of the shell measuring 578 μm and that at a right angle to this measuring 522 μm. The relation is 0.90. The last whorl occupies nearly 50% of the shell's length. Aperture high and pyriform with its long axis towards the right, outer lip orthocline joining the suture at a right angle but slightly bending to the right at its high most end and slightly projecting at its lower part and at 2/5 of its height. Inner lip reflected both on the columella and the parietal wall forming a continuous callus while the columella is straight in its upper part, is curved in its lower part and continuous with the parietal wall. Outer lip sinuous at its vicinity with the suture. Acrochalix callosa is reported as a NE Atlantic species and differs from other Mediterranean minute and curved species by having a slender aperture, a straighter columella-parietal wall line and a well-developed, solid and continuous inner lip [86]. As the species is very rare and its variability unknown, we refer to our specimens as A. cf. callosa. Campylorhaphion cf. famelicum (Watson, 1883) (Fig. 19a). One young live specimen (1.50 mm high, 0.60 mm wide), on Holothuria (Holothuria) tubulosa Gmelin, 1791 host, − 35 m, mixed bottom, station 4. The hyaline, white, club-shaped shell has a paucispiral protoconch of 2 whorls (excluding the nucleus) and a diameter of approximately 200 μm. The suture of the protoconch whorls exhibits a weak and dense crenulation. The teleoconch is slightly curved, of 5 shiny whorls that are nearly smooth though with very fine axial sculpture approximately 30 μm apart that is more prominent close to the incremental scars (Fig. 19a). The body whorl occupies 48% of the shell's length and forms a rounded base, while the aperture is nearly twice as high as wide and occupies 34% of the shell's length. Its Vitreolina type incremental scars are not strictly aligned, are facing the lateral side of the shell over the outer lip with the two last scars (above the outer lip) in a slowly retreating series, nearly aligned with the outer lip and the two adapical (older ones) in advancing series, while the oldest one is more conspicuous. The inner lip is straight by the columella and projected over its lower part, while the outer lip is arquated and orthocline. Animal of light pink-purple color. We did not manage to find in the literature a description of a specimen of the same developmental stage as ours, therefore, we present the later as C. cf. famelicum. The specimen resembles Melanella spiridioni (Dautzenberg & H. Fisher, 1896) (Fig. 19b) from which it differs in having a cylindrical protoconch instead of a conical of M. spiridioni and a more fragile teleoconch, in its aperture which is, at least, twice as high as wide and in the color of the animal which is lemon-yellow with red speckles in M. spiridioni. It differs from the Vitreolina species in having a cylindrical protoconch with almost 2½ whorls. Campylorhaphion famelicum is uncommon in the Central Mediterranean Sea [27] and in the North Atlantic [86].

Discussion
Out of the 45 presented species, 40 are reported for the first time for the Hellenic fauna, raising its gastropod biodiversity from 651 [21] to 691 species, and 17 species are new records for the East Mediterranean Sea. Two families and 19 genera are also new records for the Hellenic fauna (see Table 1). Interestingly, the vast majority of the presented species is of minute size (nearly 70%) being collected from a variety of substrates and depths (Fig. 22).
The occurrence of only shells and among them juvenile ones of Rhinoclavis kochi accumulated in shallow water stations, could be explained by an initial rapid increase of the population density followed by a decline due to the sea temperature change [5] and subsequent accumulation of the shells by the sea currents. This may also mean a temporal establishment. A future occurrence of live specimens could not be excluded provided the environmental conditions are favorable and the corridors are open.
The almost simultaneous findings of Cerithiopsis buzzurroi, C. ladae, C. scalaris, Narrimania concinna, Nanobalcis nana and Sticteulima jeffreysiana from different areas of Greece indicates that they are more widely distributed in the Hellenic Seas and the Eastern Mediterranean Sea, and that targeted researches from the same type of substrates and direct sampling might bring to light more species. Field observations and the gross anatomy of their alimentary systems indicate that ptenoglossans are parasites [99]. Among them, cerithiopsids are usually associated with sponges while eulimids have a strict preference for certain classes of echinoderms [99]. At the same time the identification of cerithiopsids, in particular, necessitates the presence of intact protoconches and/or the observation of the color of the living animal and thus, the collection of live specimens. In the present study we have met those prerequisites by means of brushing their hosts and by keeping the animals alive until they were photographed.
After an extended survey of the literature on Eritrean and Indo-Pacific Triphorids and the lack, up to now, of intermediate records of M. amicitiae from the East Mediterranean Sea [83] almost rules out an Eritrean or Indo-Pacific origin of the species. The present new records from Greece, although cannot challenge that hypothesis, are indicative of a wider range of distribution.
The assignment of our specimen Horologica sp. to the genus Horologica Lasero, 1956 was based primarily upon the combination of its smooth protoconch (characteristic for the genera Cerithiopsis sp., Horologica sp. and Joculator sp.) and its spiral sculpture of 2 cords (genera Dizoniopsis sp., Mentax sp. and Prolixodens sp.). Cerithiopsis sp. and Joculator sp. were excluded as they bear 3 spiral cords while Dizoniopsis sp., Mentax sp. and Prolixodens sp. were excluded on the bases of their ribbed protoconches [80]. We hope that further and persistent efforts will bring to light more specimens to study the variability of the species and perhaps describing a new taxon.
The occurrence of Heliacus jeffreysianus in the Hellenic waters, besides the record from South Crete [97] considerably enlarges its distribution, which, apart from its extreme rarity, is indicative of a rather broad range of occurrence. The large hyperstrophic protoconch of about 1 mm, fitting the size range of those species with a planktotrophic development, supports the hypothesis of a potentially wide geographical distribution [100]. To our knowledge, the scattered occurrence and extreme rarity of H. jeffreysianus may be attributed to its cryptic life-style which may be intimately linked to a deep water zoanthid host, typical of some offshore bottoms [96]. 45 species, the majority of which is of minute size, belonging to 19 families were identified. Among those species, 17 are new for the Eastern Mediterranean Sea and 40 are new for the Hellenic fauna. The high number of new findings is attributed to the sampling methods applied, the under-or unsearched marine environments investigated, as well as the different types of substrates and depths covered, and the multilateral co-operation.

Methods
The sampling and handling of the specimens were conducted according to Manousis and Galinou-Mitsoudi [21] from October 2008 to March 2017 in certain locations throughout the Hellenic Seas (Fig. 1). In addition, we have applied: (a) brushing with a soft brush on holothurians, sea urchins and sponges either in situ or on live material brought temporarily to the surface in several localities and from various habitats, (b) sampling vertical substrates (e.g. poles, embankments) by means of a metal pole scraper connected to a 120 μm mesh nylon net, (c) keeping until examined and sorting out benthic material in cool sea water until photography of live specimens took place. Cooling of biological material in the sea water was achieved by means of a gel freeze ice pack under the examination vessel.
The protoconch whorls were counted according to Verduin [101], the protoconch's visible height was measured parallel to its axis, from the tip to the intersection of the larval scar and the teleoconch suture, while for the shell's slenderness (h/w) the outer lip of the aperture was included in the shell's width.
The species recognition was based on: (a) systematic guides and atlases (e.g. [5,27,29,35]); (b) faunistic and review articles [76,102], (c) studies on the Mollusca fauna in the Hellenic seas [18,21,103]. Information from specific web sites was also taken into account (31 March 2017). More specifically, for the species nomenclature update, besides the Marine Biodiversity and Ecosystem Functioning EU Network of Excellence (MarBEF) [58] and the World Register of Marine Species (WoRMS) [104] the Taxonomic on-line Database on European Marine Mollusca (CLEMAM) [34] was used. In addition, the Ellenic Network on Aquatic Invasive Species (ELNAIS) [61] and the Marine Mediterranean Invasive Alien Species database (MAMIAS) [105] were used for the alien species status in the Hellenic and Mediterranean Seas. Records were compared with the checklist of Koukouras [106]. For the molluscan life habits, the Todd databases [107] were taken into account. The specimens are deposited in the premises of the Alexander Technological Educational Institute of Thessaloniki and those of Dr. T. Manousis, C. Kontadakis, G. Polyzoulis and G. Mbazios. Scientists are welcome to have access to the biological material at will and by appointment.
Authors' contributions TM conceived the idea of the study, collected sea-bottom material by diving, searched for mollusks, participated in the identification of the species, processed the images and participated in the study's design and coordination and helped to draft the manuscript. CK collected sea-bottom material by diving, searched for mollusks, participated in the identification of the species, processed the images and participated in the study's design and coordination. GP collected sea-bottom material by diving, searched for mollusks, participated in the identification of the species, processed the images and participated in the study's design and coordination. GM collected sea-bottom material by diving, searched for mollusks, participated in the identification of the species, processed the images and participated in the study's design and coordination. SG-M collected biogenic sea-bottom material, searched for mollusks, participated in the identification of the species, designed figures and participated in the study's design and coordination and helped to draft the manuscript, supported with the bibliography sources and is the corresponding author. All authors read and approved the final manuscript.