Last snails standing since the Early Pleistocene, a tale of Calliostomatidae (Gastropoda) living in deep-water coral habitats in the north-eastern Atlantic

Three species in the gastropod genus Calliostoma are confirmed as living in Deep-Water Coral (DWC) habitats in the NE Atlantic Ocean: Calliostoma bullatum (Philippi, 1844), C. maurolici (Seguenza, 1876) and C. leptophyma Dautzenberg & Fischer, 1896. Up to now, C. bullatum was only known as fossil from Early to Mid-Pleistocene outcrops in DWC-related habitats in southern Italy; our study confirmed its living presence in DWC off Mauritania. A discussion is provided on the distribution of DWC-related calliostomatids in the NE Atlantic and the Mediterranean Sea from the Pleistocene to the present.


Introduction
The Senckenberg Institute and the Royal Netherlands Institute for Sea Research (NIOZ) investigate the geophysical, geological and biological characteristics of scleractinian-dominated Deep-Water Coral (DWC) habitats in the world. The north-eastern Atlantic Ocean has been a focus area and the current article is a spin-off of the biodiversity investigations in this region.
DWCs are commonly found along the continental slopes of all oceans and they occur from less than 100 m depth in colder regions to more than 1000 m in more moderate temperature regions (Rogers 1999) and shallowing up to 400-660 m off north-western Africa (Westphal et al. 2014;Ramos et al. 2017). These DWC habitats can develop into three-dimensional topographically well-defined reef structures, which influence the bottom current regime. Prerequisites for the presence of thriving DWC are a suitable range of water temperature, dissolved oxygen level, access to suspended food, a substrate and adequate sedimentation rate. Once a DWC habitat is established, they often quickly monopolise the seabed and eventually form self-sustaining reefs over time. If several fossil reefal episodes are stacked in one place over time, geologists speak of coral mounds (Roberts et al. 2006). Larger mound complexes measure 100-350 m in height and several tens to hundreds of kilometres in lateral extension (Rogers 1999;Roberts et al. 2006;Wheeler et al. 2007;Ramos et al. 2017).
Live and dead DWC framework provides a broad suite of microhabitats for other organisms. This habitat heterogeneity is a major attractor for a species and often biomass-rich community (Buhl-Mortensen et al. 2010). In an ongoing census of the DWC-associated fauna, more than 10.000 species have been reported from several hundred taxonomic publications starting from the famous H.M.S. Challenger expeditions to the present (Freiwald, unpublished). Our own research is focused on the shelly molluscs in the temperate and tropical North Atlantic, which has yielded about 1.600 mollusc species from approximately 280 stations from DWC settings held by NIOZ and Senckenberg Institutions. The geographic spread of these stations ranges from northern Norway (71°N) as far south as to Mauritania (17°N) with major study areas in the Oslofjord-Kosterfjord, Mingulay, Hatton Bank, Rockall Bank, Porcupine Bank, Celtic margin, Galicia Bank, Atlantic Morocco and Mauritania.
This study is based on collection campaigns during cruises held since 1997 (Table 1). These campaigns include geophysical mapping of the sea bottom and its subsurface to high-grade potential areas where DWC-habitats could be expected. When feasible, video data were gathered either by a towed camera, a lander or by Remotely Operated Vehicle (ROV), often complemented with actual sampling by a ROV or a video-guided box corer. Records from ROV stations are listed herein with coordinates and depth at starting point of dive. In addition, bottom samples were taken by using a ROV manipulator, van Veen grab, box corer or dredge. The live material collected by a ROV or using other sampling methods was preserved, mostly in ethanol. The remaining sediment with biological remains was rinsed with fresh water, dried, and retained for further analysis. The alcohol collection and sediment samples were studied as part of the biodiversity inventory efforts for the Mollusca. We selected three species representing the genus Calliostoma (Gastropoda) for this publication because the use of combined ROV footage, selected species sampling and bulk sediment sampling has led to unique insights about these species; the adult shells of the three species are large enough to be identified on video material especially from underwater high-definition cameras.
The Mollusca wet-collection (in ethanol, 96% plus 1% methyl ethyl ketone) and part of the dry material are stored at Senckenberg am Meer in Wilhelmshaven, Germany. Dried specimens are also stored at Naturalis in Leiden, the Netherlands.

Systematics
The family Calliostomatidae Thiele, 1924 (Trochoidea, Vetigastropoda, Gastropoda) has been reconfirmed as a distinct family by molecular studies (Williams et al. 2008(Williams et al. , 2010Williams 2012). Even though the family currently comprises about 29 extant genera, the genus Calliostoma takes the lion's share in species; all calliostomatids in the NE Atlantic are placed in Calliostoma. A generic classification was carried out for calliostomatids from New Zealand (Marshall 1995a) and New Caledonia, the Loyalty Islands, and the northern Lord Howe Rise (Marshall 1995b). The historic generic subdivision of NE Atlantic and Mediterranean calliostomatids was largely based on shell morphology; it was abandoned in the 20 th century; all genera are currently lumped under Calliostoma. A generic revision of these species is required but this effort is beyond the scope of this study. In the left map, empty shells of Calliostoma bullatum as red diagonal crosses, live observations as yellow triangles, empty shells of Calliostoma leptophyma as red normal crosses, live observations in this study as green triangles, live observations from literature as green diamonds. In the right map, empty shells of Calliostoma maurolici as red normal crosses, live observations as yellow triangles, live observations from literature as green diamonds. Bathymetry data source: GEBCO, depth contour intervals 500 m.
First teleoconch whorl convex with five rounded spiral ribs above periphery; shoulder area flattened with weaker spiral ribs (Figs 2-4). Base of first teleoconch whorl with nine, broad-flattened ribs and narrow, open umbilicus. Aperture angular, columella convex, inner shell nacreous. Outer lip of a juvenile blunt, prosocline at about 25° with shell axis. After 2 teleoconch whorls, outline more flattened; whorls with 6-14 spiral ribs which are finer, more numerous and evenly distributed; additional spiral ribs emerge as thin threads between two stronger ones. Spiral ribs stronger near the periphery on whorls six and seven; fine beads frequently present on the first and second spiral rib below the suture in the first whorls. Body whorl slightly more convex with blunt keel at periphery. Some specimens show slightly more convex whorls and a more regular pattern of fine spiral ribs on the whorl face. Base flattened with spiral ribs that are strongly developed near periphery and near columella. Umbilicus closed in adult specimens. Aperture angular and inside of the shell smooth and nacreous, columella convexly curved and its callus clearly demarcates the transition to the external shell. Lip blunt, strongly prosocline under 45° with shell axis. Colour cream white with a nacreous shine; periostracum absent (Figs 6-8).
Head white, centrally swollen, snout wide oblong with thin necklobes; necklobe rectangular at the front; lobes pinkish posteriorly (Fig. 9). Two long pointed cephalic tentacles, greyish at tips. Eyes black, situated on short ommatophores behind base of cephalic tentacles. Foot muscular with remarkable ability to adapt to rugose and variable substrates (Fig. 11). Back of the foot with wide dorsal groove (Fig. 10). At least four epipodial sensory tentacles on either side in dorsal position, emerging between shell and foot (Figs 9-12). Base of foot with wide edge corrugated up front (Fig. 9). Operculum circular, spirally developed, brown translucent, thin, chitinous.
Distribution. Calliostoma bullatum is commonly found as fossil in Early to Mid-Pleistocene formations that were deposited in bathyal depths in association with remains of the colonial Lophelia pertusa (Linnaeus, 1758) and Madrepora oculata Linneaus, 1758 (Barrier et al. 1996;La Perna & D'Abramo 2010). The fossil record suggests that this species has been living for about 2.5 million years.
Recent including living specimens have only been from off Mauritania in DWC habitats, dead at 414-588 m, alive at 450-642 m (Fig. 1). The species is unknown from Morocco's Atlantic Margin or from the Lusitanian islands or seamounts. It is possible that the species is living further south along the African shelf as DWC habitats along this coast are poorly sampled.
Remarks. Our findings are the first living records of this species that up to now was only known as fossil. They resemble Philippi's (1844) figure very well (Fig. 8) especially on size, outline, the incremental convexity of the body whorl, shape of the aperture, development of the columellar callus and the sculpture on the base of the body whorl, particularly that near the columella. Philippi (1844) mentioned about six spiral grooves. Fossil specimens from Sicily have 6-10 grooves or ribs on the body whorl. Specimens from Mauritania show typically 10-14 ribs. We consider this difference variability within the species. Variability of spiral rib sculpture is common in calliostomatids, for example in the European species Calliostoma zizyphinum (Cossignani & Ardovini 2011) and in several species in Maurea from New Zealand (Marshall 1995a).
Calliostoma bullatum is the largest representative of the genus in the NE Atlantic and the Mediterranean Sea. Calliostoma bullatum Coen 1933(non Philippi, 1844) is a synonym of Calliostoma conulus. The deep-water species Calliostoma maurolici (G. Seguenza, 1876) will be discussed in the following; it is smaller, with a stronger spiral ornament, more convex whorls, a wider top angle and an open umbilicus when adult. Calliostoma cleopatra (Locard, 1898) is another deep-water species described from off Western Sahara at 1035-1056 m; it is smaller, with more convex whorls, stronger spiral sculpture and stronger beads on the top spiral ribs. Calliostoma caroli Dautzenberg 1927 was described from the Azores at 1250 m; it has a more angular outline with a stronger spiral structure, and it is smaller than our species. Calliostoma leptophyma Dautzenberg & H. Fischer, 1896 will be discussed below; it is similar to Calliostoma cleopatra but it lacks the strong carinae on the ribs and it has a more compressed outline. Calliostoma normani (Dautzenberg & H. Fischer, 1897) is described from the Azores at 1600 m; it is similar to C. caroli but has finer spiral ribs, inflated whorls and lacks a blunt keel. We also considered other Western and Eastern Atlantic calliostomatids but we did not find a match with our specimens; all have either a stronger spiral (beaded) structure, are smaller or show a stronger keel. Interestingly, live and dead scleractinian corals were found in all above cited dredge hauls together with the calliostomatids.
The protoconch is similar to that of C. bullatum; 0.8 globular whorls ending with a rounded varix. The sculpture is hexagonally reticulated on a smooth background (Fig. 24). The hexagonal cells are arranged in about 15 spiral rows with a height of about 0.016 mm each.
The animal has an oblong snout with a swollen head with two long and pointed cephalic tentacles. Small black eyes on short ommatophores are present behind the base of the cephalic tentacles. A set of epipodial sensory tentacles is visible on the dorsal side of the foot below the shell. The living animal is pinkish white. The operculum is brown, translucent, thin, chitinous, spirally developed.
Remarks. Calliostoma maurolici is common in the NE Atlantic. The differences between the various Calliostoma species are given in the discussion of C. bullatum.

Calliostoma leptophyma
The protoconch is similar to that of C. bullatum, comprising 0.8 globular whorls with a rounded varix. Sculpture hexagonally reticulated on a smooth background (Fig. 37). Hexagonal cells loosely arranged in about 11 spiral rows and about 30 axial rows with a diameter of about 0.04 mm each; central pits round with a flat base.
Distribution. Calliostoma leptophyma is found from the Hatton, Rockall and Porcupine Banks in the north, the Azorean Seamounts in the west to the continental slope off Western Sahara in the south. Within this study, shells were found in the bathymetrical range of 557-1091 m and one live specimen at 876 m.   Beck et al. (2003) reported shells from the Seine and Gettysburg Seamounts. Rolán & Suárez (2007) mentioned three live specimens from the Galicia Bank at 769-1000 m and one live specimen off Cedeira, NW Spain at 600 m. Le Duff (2015) mentioned two live specimens from the Bay of Biscay at 519-650 m. Locard (1898) reported specimens from off Western Sahara as Zizyphinus laquatus (Talisman1883/DR71, 640 m) and Zizyphinus oppansus (Talisman1883/DR63, 640 m).
Remarks. The frequent occurrence of Calliostoma leptophyma in coral rubble sampled at all DWC locations and one live specimen from the Little Meteor Seamount suggest a close association with DWC habitats. It displays a considerable morphological variability. A syntype of the synonymous Zizyphinus laquatus Locard 1898 from off Western Sahara (MNHN-IM-2000-31194) is very similar to the holotype but it has fewer spiral ribs with beads. A syntype of the synonymous Zizyphinus oppansus Locard, 1898 from off Western Sahara (MNHN-IM-2000-31252) has a nearly smooth and flat base with a strong keel on the body whorl. In contrast, the holotype has a more rounded outline and flattened spiral ribs on the base of the body whorl.
The differences between the various Calliostoma species have been discussed above (C. bullatum). Two syntypes of C. cleopatra have been described from off Western Sahara at 1435 m (MNHN-IM-2000-31095); this species is more slender and has regularly spaced spiral ribs on the upper whorl face, on the wellrounded periphery and on the base. Moreover, it has more pronounced beads on the spiral ribs. Future physiological and molecular studies may prove this species to be synonymous with C. leptophyma. Specimens reported under the name C. cleopatra (Locard, 1898) by Hoffman et al. (2011) are considered to represent C. leptophyma herein.

Discussion
Calliostoma bullatum and Calliostoma maurolici were found alive on Lophelia pertusa and Madrepora oculata, partly with visual grazing traces on the frameworks. The oldest records of these calliostomatids are from the Early Pleistocene, co-occurring with DWCs in the Mediterranean (Barrier et al. 1996;La Perna & Abramo 2010).
Calliostoma maurolici and C. leptophyma have a wide distribution range from the Rockall Bank in the north to off NW Africa in the south and the Azorean Seamounts in the west. They are unknown from the Mauritanian slope.
Calliostoma bullatum is the only species known to live in DWC habitats on the Mauritanian slope. The current live population is separated from its original distribution during the Early Pleistocene that included the Mediterranean Sea. Mollusc species living since the Pleistocene are not uncommon. However, C. bullatum is the second rediscovery of a living mollusc from off Mauritania that has been surviving since the Pleistocene; the other species is Fissurisepta papillosa G. Seguenza, 1863(Hoffman & Freiwald 2018. The deep-sea fauna of the Mediterranean Sea has been subject to massive changes during the Messinian Salinity Crisis (7.15 Ma) when the Gibraltar gap started closing and water influx was limited (e.g. Freiwald 2019). The fauna was largely wiped out during the subsequent evaporation phases (Pérès 1985;Vertino et al. 2014;Freiwald 2019). When the Gibraltar gap opened up again at 5.33 Ma the Mediterranean Sea was repopulated with Atlantic fauna within a short period of time (Di Geronimo et al. 2005;Vertino et al. 2014).
A period of global cooling developed towards the end of the Pliocene and Early Pleistocene. Deep-water corals and associated fauna including Calliostoma bullatum and C. maurolici are part of the Early Pleistocene fauna and the outcrops near Messina date from this period. It was followed by a sequence of ice ages with short intermediate periods of warming. During the last glaciation period (Würmian, 115 -11.7 Ka), the sea level fell by some 120 m causing the water exchange at Gibraltar to diminish. Boreal species settled in the upper shelf areas of the western Mediterranean Sea but the deep-water fauna was largely impoverished. The gap at Gibraltar is narrowing again since the Late Pleistocene and water circulation in the Mediterranean Sea is decreasing, causing homothermic conditions in deep water. Mean annual water temperature in the deep of the Mediterranean Sea has risen to more than 13°C at present which causes the Palaeo-Atlantic cold-water fauna to perish (see for example Pérès (1985) on various taxa, Di Geronimo & La Perna (1997) on Mollusca, and Vertino et al. (2014) on scleractinians). The shallow gap at Gibraltar also forms a barrier to deep-water larvae, which complicates repopulation of Mediterranean deep-water habitats from the Atlantic Ocean. Migration of the DWC fauna from the Atlantic to the Mediterranean is further restricted because the massive Atlantic DWC communities have moved north with its current-day culmination off western Norway (Frank et al. 2011). There are few thriving DWC communities on the NW African Slope, off the Iberian Peninsula and in the Mediterranean Sea. They are limited in size and scattered along the continental slopes. The DWC-associated calliostomatids are probably extinct in the Mediterranean Sea even though DWC colonies are still living locally (Vertino et al. 2014). The calliostomatids in the scattered DWC communities in the NE Atlantic south of the Porcupine, Rockall and Hatton Basins are considered survivors ("last snails standing") since the Early Pleistocene.