A unique and threatened deep water coral-bivalve biotope new to the Mediterranean Sea offshore the Naples megalopolis

The Gulf of Naples is an example of the most beautiful and biodiverse marine regions of the Mediterranean Sea and of the most impacted areas in terms of industrial activities, large contaminated areas, resource exploitation, infrastructures at sea and maritime transportation. We conducted Remotely Operated Vehicle surveys in the Dohrn Canyon in the Tyrrhenian Sea at approximately 12 NM off Naples metropolitan area, and revealed a hotspot of deep-sea benthic biodiversity of sessile fauna at ca. 400 m depth. The hard bottoms are characterized by a high abundance of charismatic species, such as the habitat forming cold-water corals (CWC) Madrepora oculata, Lophelia pertusa, Desmophyllum dianthus in association with the large size bivalves Acesta excavata and Neopycnodonte zibrowii. This CWC-bivalve co-occurrence represents a novel biotope for the Mediterranean Sea, which coexists with the evidence of severe anthropogenic threats, such as illegal dumping and fishery malpractices that were visually documented during the survey. We recommend the adoption of specific protection measures to preserve these unique deep-sea assemblages showing the uncommon co-existence of such a number of deep-sea species in a single habitat.

www.nature.com/scientificreports www.nature.com/scientificreports/ sea-level fall during the last glaciation enhanced by local subsidence, cutting through Pleistocene sediments and the Campana Ignimbrite 14 . The water mass structure of the GoN is predominantly linked to the main circulation of the southern and mid-Tyrrhenian Sea, with influences from local factors, such as the wind stress and the river runoff. The two main water masses flowing in the GoN are the Modified Atlantic Water (MAW) that occupies the upper 50-100 m and the Levantine Intermediate Water (LIW) located below ~200-300 m, with salinity of 38.65, temperature of 14.2 °C and a potential density (σ θ ) of 29.0 kg m −3 [17][18][19] . Depending on the season, other water masses can be recognized within the GoN, such as the Tyrrhenian Intermediate Water (TIW) formed during winter mixing at depths down to ~150 m and the Tyrrhenian Surface Water (TSW) found above 75 m as the result of summer warming and freshening of the TIW 20 .
In 2016, the first ecological exploration of the deep-sea habitats of the Dohrn Canyon was launched by means of Remotely Operated Vehicle (ROV) focusing on the steep walls of the northern flank of its western branch (Fig. 1B,C). The survey unveiled for the first time the presence of charismatic benthic life in the canyon system, with biological traits previously unreported for the entire Mediterranean basin.

Results
The living community. An abundant and diverse sessile fauna was observed by ROV image analysis . Several macro-invertebrates species were concentrated on rocky bottoms at depths of 350-470 m (Fig. 5), corresponding to the depth of the LIW (Fig. 6). We report here the presence of the frame-building cold-water colonial scleractinian corals Lophelia pertusa and Madrepora oculata. These species are key ecosystem engineers in deep-sea habitats of the Atlantic Ocean and have been reported with a fragmented distribution in the Mediterranean Sea 21,22 but were never reported previously in the GoN. Differently from what observed so far in the Mediterranean Sea, where cold-water coral habitats are predominantly characterized by M. oculata, in the Dohrn Canyon L. pertusa is the most abundant species, especially on the northwestern flank where as many as 10 colonies of each species have been counted on a distance of 235 m between 375-455 m water depth. Our ROV images and videos indicate that both species occur as small interspersed colonies on the rocky substrate (Figs 2A,B,E and 3B,E), isolated or admixed (Fig. 2F), and with polyps fully expanded (Figs 2A,B,F and 3B,E). On the canyon walls, we also found conspicuous populations of solitary corals (Figs 2C-E and 3B,D,F), including Javania cailleti and Desmophyllum dianthus (a target species for paleoclimatically-oriented studies 23 ). Such solitary corals settle on bedrock overhangs forming dense aggregations of up to more than 700 ind. m −2 , with the tentacles facing down, and often unevenly rimming the layered substrate (Fig. 2D). The sea anemone Protanthea simplex is equally abundant (Fig. 2C), often associated with solitary corals or settled on the dead frames of M. oculata and L. pertusa. This piezotolerant species, spanning from infralittoral to bathyal depths in the northern Atlantic, has been previously reported only once in the Mediterranean, in the Strait of Sicily 24 . Another unique finding is the discovery of the presence of large bivalves in association with such CWC (Fig. 5; Table 1), namely the limid Acesta excavata (Figs 2E and 3A,D), and the giant deep-sea oyster Neopycnodonte zibrowii (Fig. 4A), www.nature.com/scientificreports www.nature.com/scientificreports/ which colonize the vertical cliff of the canyon. These large bivalves are frequently reported in the Atlantic Ocean, but seldom documented alive in the Mediterranean Sea [25][26][27] , where their presence has been so far documented only in the western basin or as Pleistocene fossil in the eastern basin. Individuals attain a size of ca. 20 cm that is comparable with the NE Atlantic populations 28 . Individuals of A. excavata have been observed byssate on the wall at 380 m water depth down to the basal part of the canyon flank at 415 m. The long-lived oyster N. zibrowii is recorded in all ROV transects along the Dohrn Canyon (Figs 3A and 4A) but with variable density, occurring as individuals or aggregations (strings) in a depth range of 375-415 m. Remarkably, clusters of dead and blackcoated N. zibrowii occur cemented at the canyon's wall, some of which fouled by living epifauna that includes CWC. Overall we counted more than 120 living N. zibrowii reaching a maximum density of ca. 20 ind. m −2 in the northwestern part of the canyon between 375-395 m (Fig. 4B). The canyon's wall is inhabited by a number of other mega-and macrobenthic taxa including further cnidarians, sponges, polychaetes, molluscs, bryozoans, echinoderms, crustaceans, adding to the total biodiversity (see species list in Table 2). Other cnidarians include rare specimens of the antipatharian Parantipathes larix (Fig. 3C), the sea fan anemone Amphianthus dohrnii (Fig. 2F) abundant on long lines entangled in the wall, and an unidentified species of Epizoanthus sp. (Fig. 3E). The sponge assemblages are dominated by the encrusting species Desmacella inornata and Hexadella sp., and by a few massive and tubular sponges (i.a., Spongosorites flavens). Serpulid polychaetes, encrusting and erect bryozoans, byssate (Asperarca nodulosa, Delectopecten vitreus) and cemented (Spondylus gussonii) bivalves contribute to the high biodiversity of hard substrates of the canyon. The investigated system hosts also some vagrant macro-invertebrate species, such as the pencil urchin Cidaris cidaris (Fig. 3A), Gracilechinus cf. acutus (Fig. 3B), the asteroid Astropecten sp., and the decapods Munida intermedia (Fig. 3F) and Anamathia rissoana (Fig. 2F). The www.nature.com/scientificreports www.nature.com/scientificreports/ colonial coral framework provides a cryptic habitat populated by a diversified fauna such as the pink fish Bellottia apoda and unidentified crustaceans.
Nekton diversity during the ROV survey revealed the presence of a limited number of species. Shoals of Pagellus bogaraveo were observed swimming next to the canyon wall, and the silver scabbardfish Lepidopus caudatus was abundant near the wall and the bottom of the canyon. ROV explorations were extended also on the adjacent muddy bottoms of the canyon. Here we observed the presence of sparse cerianthiids, unidentified Paguridae, other benthopelagic fishes and sponges (Fig. 4C). In particular, the Ceriantharia Pachycerianthus cf. dohrni reached here densities up to 5-6 ind. m −2 (Fig. 4D). Finally, the ROV images documented a high zooplankton biomass, dominated by Euphausiacea and Chaetognata.
Dating the Canyon occupancy by the coral-bivalve biotope. We have observed the common presence of subfossil oysters cemented to the wall (Fig. 4A,B). Some dead D. dianthus and Caryophyllia calveri appear also completely patinated by ferromanganese oxides, serving as attachment bases for living individuals of D. dianthus and J. cailleti, respectively (Fig. 7B,C). Noticeably, incipient oxide patination was observed on some live D. dianthus corals (Fig. 7D), although such polymetallic patina often testifies at a prolonged post-mortem exposure of the calcareous skeleton within the water column 29,30 . One dead valve of N. zibrowii coated by a thick black ferromanganese patina (Fig. 7A) was collected from the wall and radiocarbon dated at the Poznan Radiocarbon Laboratory in Poland, providing a calibrated age of 5496 yr cal BP (2σ range: 5329-5588) ( Table 3). We hypothesize that this age might represent the time of coral-bivalve settlement of the Dohrn Canyon. The date matches www.nature.com/scientificreports www.nature.com/scientificreports/ well with the timing of re-colonization that followed the collapse of CWC-growth in the eastern Mediterranean Sea due to oxygen deficiency in intermediate and deep waters between 10.8-6.1 kyr BP 31 .
Thus, to the best of our knowledge, the Dohrn Canyon became home of this peculiar biotope since the mid Holocene at least, along with the onset of the LIW at the end of the sapropel S1 event at 6.1 kyr BP. We have no       (Fig. 8A,C), as well as lost nets and longlines seen enveloping also epifaunal megabenthos. A high number of longlines was reported entangled on substrate asperities (Fig. 8B,D) often close to colony of cold-water corals and bivalves. Longlines served also as attachment substrate for cnidarians, including M. oculata and D. dianthus (Fig. 8B,D). Our data pointed out the presence on an impressive amount of illegal dumping of garbage bags, so intense to completely drape the sea-bottom at places (Fig. 8E). Marine litter has been classified following the MSFD guidance provided by 33 , with density indicated as items/100 m based on the total length of the ROV tracks (Fig. 5B) Table 4).

Discussion
Despite the fact that intensive biological investigations have been conducted in the GoN since the foundation of the Stazione Zoologica in 1872, information on deep-sea benthos has remained surprisingly very scant and limited to mobile bottoms [34][35][36] . The hard bottoms of the Dohrn Canyon reveal further elements of seafloor heterogeneity in the Gulf, which increase the incredibly rich topographic features of the coastal habitats, with all predictable ecological consequences 37 . It is surprising, however, that the presence of such a highly diverse habitat was so close to the shoreline and remained unnoticed until present. This finding further strengthens the evidence of the still insufficient knowledge of the deep-sea realm even in proximity of the coast 38,39 , and of the deep-sea Mediterranean biodiversity in particular 40,41 .
With respect to the cold-water coral habitats of the Mediterranean basin, the Dohrn Canyon is of particular oceanographic and ecological interest as it represents an element of potential connectivity with other deep-water coral populations of the basin. The GoN indeed is peripheral, but still on the track of the LIW (Fig. 6A), which is hypothesized to control the distribution of major deep-water coral grounds in the Mediterranean basin 42 .
The deep-sea assemblages revealed by this study indicate that the Dohrn Canyon is a highly diverse cold-water coral site of the eastern Tyrrhenian Sea, for which available information previously confirmed the occurrence of living specimens of M. oculata, L. pertusa and D. dianthus (pers. unpublished data), but never the contextual presence of N. zibrowii and A. excavata except in the case of subfossil Pleistocene records 30,43 .
A unique trait of the faunal assemblage of the Dohrn Canyon, however, is the tight co-existence of living specimens of the bivalves A. excavata and N. zibrowii with the cold-water corals, which has never been previously reported in the Mediterranean Basin. The assemblages of the Dohrn Canyon share several common traits with those inhabiting the Whittard Canyon of the eastern Atlantic Ocean [44][45][46] . Both canyons share, indeed, the presence of the same large bivalves of A. excavata and N. zibrowii, although with a much higher bivalve density in the Whittard than in the Dohrn Canyon. However, in both areas N. zibrowii appears to preferentially settle at slightly deeper depths than A. excavata. Conversely to what reported from the Whittard Canyon, solitary corals predominate in the Dohrn Canyon.
The new finding reported here from the Dohrn Canyon clearly indicates that the GoN is a hot spot of deep-sea benthic diversity coexisting with high levels of anthropogenic impact. The canyon head, indeed, is very close to the Naples megalopolis, and is subjected to a severe dumping and ghost-fishing impact, that were clearly documented by ROV images. Data on the number of large litter, and plastic debris, and of fishing nets and lines in the deep are still very scant 47,48 , so that remains difficult to provide sound comparisons. However, we did not find evidence of trawling and topographic elements of physical damage and habitat destruction (e.g., dredging). Data reported in the present study, thus suggest that hot spot of deep-sea benthic biodiversity can coexist with these impacts (litter, long lines) if the latter are not coupled with the habitat destruction. We call for the preservation of these unique deep-sea habitats by adopting protection measures from bottom contact fishing and other sources    www.nature.com/scientificreports www.nature.com/scientificreports/ Geographic Information System (distributed by ESRI). The tool "minifilms" was used to extract high-resolution still images from ROV footage every 10 seconds: 2048 photographs have been obtained with each image covering ca. 6 m 2 . A total length of 2703.36 m (more than 15,000 m 2 ) was surveyed during the four dives, of which 1895.58 m pertain to mobile substrates (such as mud) and 807.77 to hard substrates (e.g., rocky outcrops).
Taxa recognition was based on still HD images analysis and on a limited number of samples obtained by the ROV's robotic arm. Sampled organisms were first photographed, and then fixed on board in a buffered 4% formaldehyde solution or preserved in proof ethanol for genetic analyses. Taxonomic identification was conducted in the laboratory using the proper techniques of each taxonomic group, and conforms to the World Register of Marine Species 49 .
A first ROV exploration of the Dohrn Canyon habitat was conducted in June 2016 during the oceanographic cruise ANOMCITY16 onboard R/V Minerva Uno (station code DOHRN_ROV_1), followed by others explorations in July 2016 and July 2017 for the initial assessment and monitoring of the new site within the frame of the Italian Marine Strategy Framework Directive (station codes MS16_ and MS17_, respectively). In total, four ROV dives explored three distinct sectors of the canyon head's NW branch, along transects perpendicular to the axis of the channel and ending on the steep rocky margin of the channel. Dives DOHRN_01 and MS16_7 have been the first two exploratory dives in the canyon revealing high biodiversity level (Fig. 1B,C, and Table 2). Both dives explored the northwestern flank of the canyon at depth comprised between 375 and 455 m. Deeper parts, from 420 to 455 m are characterized by gentle topography and muddy substrate with no evident mega-and macrofauna. The two dives explored in detail the canyon wall from 375 to 410 m where rocky substrate outcrop. To the west of this sector (MS17_81), the area is characterized by a relatively gentle morphology between 360-450 m water depth, with muddy sediment prevailing over scattered outcropping bedrock occurrences at 380 and 450 m respectively. The easternmost ROV survey (MS17_102) explored the canyon's head between 345-470 m. This side of the canyon is characterized by gentle muddy topographies alternating with rocky outcrops and cliffs (vertical walls) between 440 and 460 m and 390 and 430 m respectively.  Table 4. Number of litter identified through ROV analysis. Density of litter is reported as number of records for a distance of 100 m as indicated by the Guidance on Monitoring of Marine Litter for the Mediterranean MSFD 33 . The total density refers to the density of the identified objects for the entire length of the four ROV under study; Density refers to the density of the objects identified in mobile sediments (*) and hard substrates (**).