Offshore Neopycnodonte Oyster Reefs in the Mediterranean Sea

: Oysters are important ecosystem engineers best known to produce large bioconstructions at shallow depth, whilst offshore deep-subtidal oyster reefs are less widely known. Oyster reefs engineered by Neopycnodonte cochlear (family Gryphaeidae) occur at various sites in the Mediterranean Sea, between 40 and 130 m water depths. Remotely Operated Vehicle surveys provide new insights on this rather neglected reef types with respect to their shape, dimensions and associated biodiversity. We suggest that these little contemplated reefs should be taken in due consideration for protection.

Oyster reefs are of paramount importance worldwide (e.g., [54][55][56][57][58]), and are considered under several protection and management measures (e.g., Habitat Directive: Council Directive 92/43/EEC). According to the EUR28 interpretation manual of the Habitat Directive for the class "1170 Reefs", it can be defined as reef any biogenic (concretions, encrustations, and bivalve beds originating from dead or living animals) or geogenic (reefs originating on non-biogenic substrate such rocks, boulders, etc.) structure arising from the seafloor [59]. Oyster reefs can raise the height of the seafloor from 0.15 m up to 6 m (e.g., Crassostrea virginica reefs in the US coasts and Ostrea edulis reefs in the Black Sea, respectively [60,61]).
Here, we describe N. cochlear reefs identified by Remotely Operated Vehicle (ROV) surveys in the Adriatic and Ionian seas offshore the Apulian margin of Italy ( Figure 1).

Materials and Methods
Several cruises have been carried out in the study areas that evidenced the occurrence of Neopycnodonte cochlear reefs. Remote Operated Vehicle (ROV) dives surveyed the SW Adriatic and N Ionian oyster sites ( Figure 1, Table 1). We used an ROV Pollux III (Global Electric Italiana) equipped with a low-resolution CCD video camera for navigation and a high-resolution (2304 x 1296 pixels) video camera. Three laser beams, each 20 cm apart, provided the scale bar on the videos. The ROV was equipped with an underwater acoustic tracking system that gives position and depth every second. Still-photo footage, one frame every 10 s, was analysed by the VLC freeware software providing taxonomic information. Macro-(> 2 cm) and mega-benthic organisms were identified to the lower possible taxonomic rank. Taxa unidentifiable at species level from images alone were categorized as morphological categories (e.g., [62,63]). Taxonomic classification adheres to the World Register of Marine Species database [64]. Species densities were calculated using three photos selected at random, showing live Neopycnodonte cochlear reefs, from each transect. ROV exploration was primarily for scientific surveys to monitor the environmental status of marine ecosystems in Italian waters (Marine Strategy Framework Directive).
Other Mediterranean sites not surveyed using ROVs proved to host abundant N. cochlear valves on the sea-bottom, suggesting the existence of related reefs ( Figure 1, Table 1). Large volume Van Veen grabs and epibenthic hauls provided samples of oyster specimens from Kerkyra Island (SE Adriatic), Eivissa Island (Balearic Sea), and the Gulf of Izmit (Marmara Sea) [65].

General Features of Neopycnodonte cochlear Reefs
The Neopycnodonte cochlear reefs surveyed in this research represent various typologies in terms of shape and dimension (Figures 2 and 3). Since based upon ROV observations, their thickness could not be assessed with precision and larger reefs could, in fact, represent encrustations of the underlying substrate by a few generations of grypheid oysters (Figures 2-4).
i) At Bonaccia in the northern Adriatic Sea (Figure 1), N. cochlear bioconstructions (Figure 2a,b) cover 4-5 m in lateral extension (length and width) and reach ca. 2 m in height by likely encrusting over bedrock substrate, reaching maximum (underestimate) oyster densities of 500 ± 158 ind·m -2 . The occurrence of substantial oyster growth and related biostromal deposits, including valve embedding into hydrocarbon-imprinted limestone was noticed previously [66].
iii) At Monopoli and Santa Maria di Leuca sites (Figures 1 and 3), the latter in Ionian waters, it is difficult to estimate reef dimensions. In fact, at both sites N. cochlear grows on a rocky substrate and covers this primary substrate for several meters, growing thicker than 10-20 cm. However, oyster densities are comparable with the Adriatic sites mentioned above.

A Glimpse at Reefs' Associated Biodiversity
The overall biodiversity associated with the Neopycnodonte cochlear reef is noticeable (Figures 2-5 and Table 2). Sponges dominate the macro-and mega-benthic associated fauna; Spongia officinalis, S. lamella, Axinella polypoides, Tethya aurantium (All listed in Annex II of the Barcelona Convention; if protected, they are so by the regulations of the countries who signed the Convention) and Ulosa stuposa are a common occurrence at Bonaccia and Vieste sites. Undetermined
The Neopycnodonte cochlear reefs in the central and eastern Mediterranean represent also a hotspot of biodiversity, as well as are the transitional areas between Neopycnodonte reefs and surrounding mobile sediment bottom. Lastly, these reefs are home to protected species such as Centrostephanus longispinus, recorded by the ROV surveys.
Despite intense investigation on littoral oyster reefs (e.g., Ostrea edulis [54,56,57,60]), information on intermediate Neopycnodonte cochlear reefs is still exiguous. The development and improvement of non-invasive technologies (i.e., ROV), coupled with high-definition image acquisition, has paradoxically provided more knowledge on deep Neopycnodonte zibrowii oyster occurrences (> 200 m) than on Neopycnodonte cochlear reefs at intermediate depths (30-150 m). The monitoring program under the Marine Strategy Framework Directive does not consider Neopycnodonte cochlear reefs among target habitats. Further research is needed to better understand such reefs, to expand our knowledge about their spatial distribution, associated biodiversity, and goods and services they may provide.
The Neopycnodonte cochlear reefs are not considered among the top marine bioconstructions listed by [69]. Their ecosystemic importance, however, strongly advises for the enforcement of adequate management measures to ensure their survival, including fishery restrictions. In fact, the impact by trawling and longlining on oyster reefs cannot be a priori excluded in consideration of the fishery effort in this region (e.g., [70][71][72]). This approach would be in line with European recommendations for "H1170 Reefs" in the Annex I of the Habitats Directive 92/42/EEC on the conservation of natural habitats and of wild fauna and flora [73]. The inclusion in protected areas of the sites of Bonaccia, Vieste, Monopoli and Santa Maria di Leuca would guarantee the preservation of examples of grypheid reefs growing at different depths and substrates and encompassing slightly different biodiversity content.

Conclusions
Albeit relatively neglected thus far, oyster reefs engineered by the grypheid Neopycnodonte cochlear are important bioconstructions in the mesophotic zone of the Mediterranean Sea.
Remarkable examples of such reefs occur in the Adriatic and Ionian waters from 40 m down to 130 meters. ROV inspection of these Neopcynodonte cochlear reefs testifies to dense oyster growth and considerable biodiversity of the associated fauna calling for their protection.