Visual Imaging of Benthic Carbonate-Mixed Factories in the Ross Sea Region Marine Protected Area, Antarctica

: Marine biogenic skeletal production is the prevalent source of Ca-carbonate in today’s Antarctic seas. Most information, however, derives from the post-mortem legacy of calcifying organisms. Prior imagery and evaluation of Antarctic habitats hosting calcifying benthic organisms are poorly present in the literature, therefore, a Remotely Operated Vehicle survey was carried out in the Ross Sea region Marine Protected Area during the 2013–2014 austral summer. Two video surveys of the seafloor were conducted along transects between 30 and 120 m (Adelie Cove) and 230 and 260 m (Terra Nova Bay “Canyon”), respectively. We quantified the relative abundance of calcifiers vs non-calcifiers in the macro- and mega-epibenthos. Furthermore, we considered the typology of the carbonate polymorphs represented by the skeletonized organisms. The combined evidence from the two sites reveals the widespread existence of carbonate-mixed factories in the area, with an overwhelming abundance of both low-Mg and (especially) high-Mg calcite calcifiers. Echinoids, serpulids, bryozoans, pectinid bivalves and octocorals prove to be the most abundant animal producers in terms of abundance. The shallower Adelie Cove site also showed evidence of seabed coverage by coralline algae. Our results will help in refining paleoenvironmental analyses since many of the megabenthic calcifiers occur in the Quaternary record of Antarctica. We set a baseline to monitor the future response of these polar biota in a rapidly changing ocean. C.M., P.M. S.S.; resources, P.M. and SS.; data curation, S.S. and S.C.; writing—original draft preparation, G.C., S.S. and M.T.; writing—review and editing, G.C., M.T., S.S., L.A., C.M. and P.M.; funding acquisition, P.M. and S.S. authors


Study Area
The Ross Sea region Marine Protected Area (RSRMPA) was established in December 2017 under Conservation Measure 91-05 (2016) [39]. After several years of laborious negotiations that have resulted in a significant reduction in area for protection, the MPA finally reached a consensus in 2016. By now, the RSRMPA encompasses a surface of ca. 1.55 million km 2 , which represents the world's largest marine protected area established under an international agreement to date.
The Ross Sea is counted amongst the least human-impacted marine environments worldwide due largely to its remoteness, intense seasonality and extensive ice cover [24]. In the 19th and 20th centuries, commercial hunting of whales and seals was in force, resulting in the loss of thousands of individuals [40]. Between the 20th and 21st centuries, intensive fishing of toothfish resulted in over-exploitation and depletion of stocks [41,42] until 1996, when CCAMLR initiated a program to reduce the Antarctic toothfish biomass exploitation by fishing activities by 50% within 35 years [43].
Human activity in the area is strictly regulated after the establishment of the RSRMPA, encompassing a vast area (1.12 million km 2 ) under full protection (General Protection Zone, GPZ) where commercial fishing is prohibited, a special research zone (SRZ) where the krill and commercial toothfish research fishery is regulated and a zone where research for krill is regulated (krill research zone, KRZ, Figure 1) [44].
Terra Nova Bay is a coastal marine area of ca. 30 km 2 located between the Adélie Cove and Tethys Bay within the RSRMPA and is part of the no-take General Protection Zone (GPZ) of the RSRMPA (Figure 1). There, the Italian summer station "Mario Zucchelli" is located on a small rocky peninsula along the coast of northern Victoria Land between the tongues of the Campbell and Drygalski glaciers (74°42' S, 164°07' E, Figures  1 and 2).
Since 1986, the area has been the focus of a variety of biological studies on benthic, nektic and pelagic aspects of resident communities (Table 1). Systemic biological research in TNB resulted in the discovery of a variety of taxa new to science, including ampharetids, amphipods, Porifera and coralline algae (e.g., [37,38,[45][46][47][48][49]). Checklists of species from TNB are systematically published and updated by the Italian national Antarctic Museum (MNA, Section of Genoa) [49][50][51][52][53]. The evidence of high diversity at both species and community levels fuelled the establishment of the Antarctic Specially Protected Area (ASPA) No. 161 of Terra Nova Bay (a coastal marine area encompassing 29.4 km 2 between Adélie Cove and Tethys Bay immediately to the south of the Italian Mario Zucchelli Station, MZS) and ASPA No. 173, which encompassed Cape Washington and Silverfish Bay in the northern Terra Nova Bay (a reproduction site for the for Antarctic silverfish Pleuragramma antarctica Boulenger, 1902).
Rocky cliffs alternating with occasional beaches characterize the coastline of TNB. Offshore the Mario Zucchelli Station, the seafloor is mostly composed of granitic rocks, with patches of gravels, coarse sands and muddy sediments. A large incision ("TNB Canyon") following the shoreline at ca. 0.4 km from the coast characterizes the seafloor geomorphology ( Figure 2).

Benthic Visual Surveys
During the 2013-2014 austral summer, three ROV dives were performed in TNB in the frame of the XXIX Antarctic Italian expedition. The first one aborted due to bad meteomarine conditions, while the following two were successful. The visual benthic surveys explored the seafloor offshore the Adélie Cove up to 120 m depth and TNB "Canyon" (a depressed segment of the seafloor) between 220 and 300 m depth ( Table 2). The activities were performed onboard the "Malippo" and "Skua" motor vessels when the weather conditions were favorable.
Video footage and still photographs were acquired using a ROV Pollux III (max working depth 500m) equipped with an underwater acoustic tracking system (USBL, Linkquest, TrackLink 1500 MA) which was connected to a Trimble dual-antenna system providing position and heading depth every 1 s. Three laser beams spaced 10 cm apart provided the scale bar on the videos. The ROV was equipped with a digital camera (Canon EOS 550, Canon EF-S 10-22mm f/3.5-4.5 USM lens with double Speedlite 270EX flash, Canon, Tokyo, Japan) and a high-definition video camera (SONY HDR-HD7, Tokyo, Japan).

Taxonomical Identification and Habitat Characterization
High-resolution images were collected with a digital camera during the surveys and analyzed for the taxonomic composition of biological communities. A total of 169 images were examined for Dive 2 and 148 for Dive 3. When necessary, the images were coupled with low-definition video recording to improve taxonomic identification efficiency. Macrofauna and megafauna were identified to the lowest possible taxonomic rank by considering previous knowledge established in more than 35 years of research activities in the area and the large collection of museum vouchers curated by the Italian National Antarctic Museum (MNA, Section of Genoa; online database available at: https://steu.shinyapps.io/MNA-generale/ (accessed on 27 July 2021)). Organisms unidentifiable at the genus or species level were categorized as morpho-species or morphological categories. The abundances of taxa along the exploration tracks were calculated and mapped by counting the number of taxa in each frame. Information about the different substrates and habitat explored was reported as a percentage of bottom covering. These percentages were converted to aerial extensions by considering that each image displayed 3 m (width) × 2 m (height) of seabed on average, which corresponded to 6 m 2 .

Adélie Cove
Dive 3 explored ca. 2000 m of seafloor in length characterized by hard substrate between 30 m and 120 m depth. Along the entire transect, more than 7400 specimens belonging to 79 different taxa and 10 Phyla were classified (Table 3). Up to 100 m depth, the seafloor was characterized by a dense coverage from coralline algae of the order Hapalidiales ( Figure 3A-C) with an extent of 351.6 m 2 . Below 100 m, patches of hard substrate started to alternate with soft substrate. Among the most abundant taxa, we noticed that the regular echinoid Sterechinus neumayeri (Meissner, 1910) and the pectinid Adamussium colbecki (Smith, 1902) Table 3). Bryozoans were sporadic along the track with 74 individuals censused.
Beside S. neumayeri, echinoderms were abundant in the surveyed area. Holothurians were largely represented ( Figure 3C,D,F) and amounted to more than 1000 individuals; however, they cannot be confidently determined from images as their taxonomy largely depends on microscopic features, i.e., the shape of the calcareous ossicles. Ophiuroids and asteroids were also very frequent with more than 270 specimens identified.
Sources of biogenic carbonates were different at Adélie Cove site. Firstly, the seafloor was characterized by the coralline algae of the order Hapalidiales belonging to a new genus and new species (I. Moro, pers. comm. 2021) in course of description. Secondly, a noticeable portion of the benthic community included calcifiers, with 13 taxa and more than 4450 individuals counted, which corresponded to ca. 60% of the identified organisms. The major contributors were S. neumayeri, S. narconensis and A. colbecki, amounting to 4170 individuals. Other echinoderms such as ophiuroids, asteroids and crinoids also concurred to the calcifiers component, with 6 different taxa and 248 individuals. A smaller contribution was provided by sponge specimens of the class Calcarea (35 ind.) and by octocorals belonging to the family Isidiidae (3 ind.).

Terra Nova Bay "Canyon"
Dive 2 explored over 2300 m of seabed between 230 m and 260 m depth, transiting an area of hard substrate covered by a thin layer of soft sediment and sporadic segments of mobile substrate with patches of organic matter in degradation ( Figure 4).
In total, 10 Phyla, 86 different taxa and more than 4700 specimens were identified and mapped ( Table 3). The sessile megafauna was dominated by cnidarians and sponges that densely colonized the hard substrates. The octocorals of family Isididae (bamboo coral), such as Primnoisis (Delicatisis) delicatula (Hickson, 1907), dominate the assemblages in the investigated area, with over 850 individuals counted and mapped (Table 3). A total of 11 morpho-species of alcyonaceans were identified, corresponding to more than 1200 individuals. Among these, the most frequent taxa in the whole investigated area included the genus Thouarella (420 ind.), Arntzia gracilis (Molander, 1929) (81 ind.) and Alcyonium antarcticum (27 ind., Figure 4).
The echinoderms were less common when compared to the Adélie Cove site, with Ophiuroidea representing the most abundant taxa (360 ind.). Holothurians colonizing the substrate and epibionts on cnidarians were also frequently observed (Figure 4). The echinoid S. neumayeri was occasionally present, counting 57 individuals (Figure 4). Crinoids were also a consistent presence.

Adélie Cove.
The shallow situation observed in Adélie Cove reveals the occurrence of four main calcifiers in the order of relative abundance: Hapalidiales coralline algae, which predominates in the shallower part of the transect (30-100 m, Figure 5) (which belong to a new genus and a new species, currently under study); S. neumayeri, A. colbecki and S. narconensis. Algal thalli calcify by large in the polymorph high-Mg calcite. S. neumayeri is characterized by a high-Mg exoskeleton (mean 9.58 mol% MgCO3 [30]). S. narconensis is equally made up of high-Mg calcite [36]. The shell of Adamussium colbecki possesses low-Mg calcite [92], with minor presences of myostrocal aragonite [94,95].
Subordinate to such main skeletal carbonate producers, other mega-epibenthic components representing a minor contribution of post-mortem carbonates were observed in the ROV frames. For instance, high-Mg calcitic ossicles and spicules derive from other echinoderm groups such as asteroids, ophiuroids, crinoids and holothuroids (e.g., [30]); bryozoans produce particles of mixed mineralogy, but with a net prevalence of calcite at polar latitudes [6,95]; the calcareous sponges shed spicules (actines) composed of Mg-calcite [96]. Only one aragonite producer was identified by the ROV survey, i.e., two individuals of the gastropod Neobuccinum eatoni. This species was repeatedly documented in the Terra Nova Bay area between 15-100 m of depth, as documented by MNA vouchers (S. Schiaparelli, pers. comm.).

Terra Nova Bay "Canyon"
The carbonate-producing organisms were ca. 50% (>2300 ind., Figure 5) of the overall benthic community, with the bamboo coral P. delicatula and S. narconensis as main contributors (858 and 828 ind., respectively). Octocorals such as the Primnoisis contribute to the carbonate sediment by shedding calcified internodes post-mortem, which is made up of high-Mg calcite [97] together with the serpulids, as mentioned above.

Ross Sea Carbonate Factories: Traits, Legacy and Future
The ROV surveys disclosed the supremacy of calcitic megabenthos over other calcifiers inside the carbonate-mixed factories of shallow to relatively deep settings in the Ross Sea. The taphonomic resilience of calcite polymorphs finds confirmation in the paleontological legacy of Quaternary Antarctica. By referring only to megabenthic organisms, shallow-water deposits are often enriched by A. colbecki shells [7], while more distal and deeper situations document their richness in bryozoans, isidids, serpulids and echinoids [1,5,10,27,99].
The mega-epibethos carbonate-mixed factories only accounts for part of the total carbonate biogenic production in the study context. Therefore, these results are conservative and somewhat biased in terms of carbonate polymorphs. It does not account for any additional skeletal input derived by infauna, which could be relevant especially at shallow depths (such as the aragonitic bivalves Aequiyoldia eightsii (Jay, 1839) and Laternula elliptica (P. P. King, 1832)), relative to the holopelagic input (mainly aragonitic pteropods), the occasional aragonitc and vateritic otolith shed by fishes (e.g., [100]), but mostly the important contribution provided by macrobenthos (such as molluscs, which are predominantly aragonitic) and microbenthos (mainly calcitic foraminifers and subordinate ostracods and serpulids). Furthermore, our case studies deal with intermediate water situations in the range of 30-260 m, but do not account for other important factories in the Ross Sea, i.e., the very shallow ones [101] which are significantly represented in the fossil record [1,7] as well as the offshore banks [5,20]; all of these are meritable for exploration in the future.
The current structure of marine communities inhabiting the Ross Sea region Marine Protected Area as described here could well change some traits in the near future under the pressure of global climatic perturbations. Indeed, Antarctic organisms are exposed to increasing pressure from multiple stresses, including seawater warming (e.g., [102,103]) and freshening [104], changes in sea ice dynamics and productivity (e.g., [105,106]) and ocean acidification (e.g. [107]). In particular, ocean acidification and the decrease in seawater pH and carbonate ion concentration due to the absorption of large amounts of CO2 by the oceans are expected to be the most critical changes facing Antarctic waters (e.g., [108][109][110][111]). Antarctic calcifying organisms, which are already living close to aragonite and calcite undersaturation, may not be able to cope with the projected changes, resulting in potential cascading consequences that might ultimately affect food webs and higher trophic levels. However, the responses of Antarctic marine calcifiers to ocean acidification may vary among taxa depending on their ability to actively control seawater chemistry at the site of calcification, with some species being more vulnerable than others. Additional studies on the physiology of marine calcifiers living at high-latitudes in the Southern Ocean are required to understand their long-term ability to adapt to ocean acidification and other climate-related changes.

Conclusions
Our study recognized the relative abundance and typology of macro-and megaepibenthic calcifiers from two sectors of the Ross Sea of contrasting bathymetric setting. As expected from carbonate factories located at high latitudes, calcitic taxa (mainly high-Mg calcite) present an almost total dominance.
With the exception of the shallow depths of Adélie Cove where coralline algae strongly prevailed, calcifiers equalize and are, on occasion, quantitatively comparable to other non-calcifying megabenthic taxa. Many such calcifiers are among the more common taxa encountered in the Quaternary record of Antarctica. As shown by ROV imagery, the original living carbonate factories are far more diverse than resulting fossil assemblages. This suggests an obvious taphonomic loss of important ecological information with respect to the structure and diversity of the original communities.
The ROV transects represent an important in situ photographic documentation of the current situation of carbonate-mixed factories in the Ross Sea. They provide, therefore, a geo-referenced and replicable baseline that is useful for monitoring future effects of progressive ocean acidification and global warming, both in terms of the hypothesized decline of calcifying vs non-calcifiying mega and macrobenthos, and of a selective taxonbased resilience.