Sanctuary for vulnerable Arctic species at the Borealis Mud Volcano

Domain: earth_science
Published: 2025-01-27
Editorial outcome: minor_revision

Abstract
Borealis is a recently discovered submerged mud volcano in the Polar North Atlantic, differing from the numerous methane seepages previously identified in the region. Here we show in situ observations from a remotely operated vehicle (ROV), capturing the release of warm (11.5 °C) Neogene sediments and methane-rich fluids from a gryphon at Borealis. The surrounding seafloor within the mud volcano feature extensive carbonate deposits, indicating prolonged diffuse methane migration. Sampling and imagery reveal that Borealis supports unique habitats adapted to low-oxygen conditions near methane seeps. Additionally, the irregularly shaped carbonate structures serve as a natural shelter from bottom trawling and a substratum for sessile fauna and may function as nursery grounds for threatened fish species. This discovery underscores the ecological significance of cold seep ecosystems in the Polar North Atlantic, highlighting their role in biodiversity by serving as refuges for marine species and emphasizing the need for their conservation.

Introduction
Over the past ten years, marine surveys in the Polar North Atlantic continental shelf and slope have consistently identified new methane seeps on the seafloor 1 (Fig. 1). These sites are of great interest because of their potential impact on the marine ecosystem and global climate. Methane, a potent greenhouse gas, has increased atmospheric concentrations since the start of the Industrial Revolution, accelerating climate change 2. Estimates suggest that between 218 and 371 teragrams of methane per year (Tg CH4 yr−1) are emitted from natural sources within terrestrial and aquatic settings, as determined by top-down and bottom-up approaches, respectively 3. Methane’s influence on the environment extends beyond its well-known role as a greenhouse gas; it is also a critical component in forming complex ecosystems that emerge from the interactions between biological, geochemical and geological processes 4. In marine environments, methane cold seeps contribute to regional biodiversity by supporting specialized microbial and faunal communities adapted to harsh conditions 4–6. In seep-impacted sediments, the anaerobic oxidation of methane (AOM) supports high fluxes of dissolved sulfide (H2S) toward the seafloor, which is colonized by sulfur-oxidizing microbial mats and chemosymbiotic organisms, such as clams, mussels and tubeworms 6–8. Methane seepage sites are often associated with widespread carbonate deposits that precipitate in situ due to the increased local alkalinity induced by AOM 9. These deposits have a wide range of morphologies, from flat pavements to vertical pinnacle-like structures, and highly variable dimensions from a few mm-sized concretion to beds several hundreds of meters in lateral extent and several meters in thickness 10. Seep carbonates provide hard substrata for sessile organisms 4 and resources for other species in adjacent areas, contributing to a broader ecological network 6. They are also a record of geological processes of methane oxidation spanning millions of years and have been used in high-resolution paleo-reconstructions 11–13.

Despite numerous observations of methane emissions from the seafloor in Arctic regions, only five mud volcanoes have been discovered in the Canadian Beaufort Sea (Western Arctic) 14, and three in Alaska 15, and so far, the Håkon Mosby Mud Volcano (HMMV) was the only known structure in Norwegian waters 16. Mud volcanoes are surface manifestations of focused fluid flow in hydrocarbon-rich sedimentary basins along passive margins and at convergent plates characterized by high sedimentation rates 17. Mud volcanoes collectively play a significant role in the atmospheric methane budget 18,19 by releasing an estimated 60 Tg CH4 yr−1 19 sourced from several kilometres depth 17. These estimates have large uncertainties since the total number of mud volcanoes worldwide and their temporal variability concerning methane emission rates are not confidently constrained, especially when considering undiscovered offshore mud volcanoes. Therefore, our current understanding of their potential contribution to atmospheric methane emissions and impact on climate, ocean chemistry and ecosystems is still limited. Likewise, the mechanisms triggering their activity and their role in promoting the survival of specialised fauna thriving at these sites remain debated 5,6.

Here, we present a comprehensive study of a recently discovered mud volcano in the Polar North Atlantic named Borealis mud volcano (MV) located in Outer Bjørnøyrenna (Bear Island Trough, 72° 26.304´N, 17° 40.626´E, ~390 m water depth) in the Barents Sea (Fig. 1). This discovery marks the second mud volcano of this kind identified in the region, among numerous methane seeps previously detected 20, thus opening a new chapter in our understanding of Arctic geology and related fields of research. Borealis MV is characterized by a cluster of craters from ~70 m to ~400 m in width within a major depression of 500–600 m diameter and an active gryphon (~7 m in diameter and 2 meters high) expelling warm fluids, gas and oil. Our observations also show that Borealis MV acts as a sanctuary for fauna vulnerable to anthropogenic perturbations, specifically seabed trawling, which continues to have a significant impact on benthic ecosystems in the region 21,22.

Results
Subseafloor, seafloor and water column insights
Borealis MV is located in the eastern parts of the Sørvestsnaget Basin, where a thick sequence of Cretaceous and Cenozoic sedimentary rocks is covered by a wedge of Pliocene to Pleistocene sediments 23,24. In this area, industry 3D-seismic data revealed the presence of a 500–600 m wide seismic chimney that extends from a depth of 300 m below seafloor (mbsf) and terminates at the seafloor into a complex network of crater-like depressions (Supplementary Fig. 1). Shallow bright amplitude acoustic anomalies observed in the Quaternary sediments are interpreted as local free gas accumulations within a vertical chaotic zone. The roots of the acoustic chimney are located at the base of the Quaternary/URU (Upper Regional Unconformity) boundary (Fig. 1).

The detection of the chimney structure steered our hydroacoustic observations, which subsequently revealed persistent, concentrated and active gas flares originating from the Borealis MV. Within a confined area (~0.14 km2) around an active gryphon, we have identified a total of 26 gas flares (Fig. 1). These individual flares exhibit a typical height of 160 m on average, potentially ascending up to 355 m into the water column. In some instances, flares even approach the sea surface (Supplementary Fig. 2). CTD water samples collected from one of these plumes showed the presence of methane with concentrations that remain relatively high throughout the water column and preserve notable levels, up to 11.390 nmol L−1 close to the seafloor (Supplementary Data 2). This suggests an intense release of hydrocarbon-charged fluids. In comparison, background ambient values of methane concentrations are around 0.9 nmol L−1, making the observed values approximately 100 times higher than the ambient conditions. Additionally, the spatial scale survey conducted with the sensor SAGE (Sensor for Aqueous Gases in the Environment) across the main craters of the mud volcano revealed that the high methane emissions from Borealis significantly affect all the surrounding areas, demonstrating the extensive impact of the active hydrocarbon emissions. SAGE, which uses a deep-sea membrane inlet to extract dissolved gas from seawater, revealed methane background levels (212 nmol L−1) to be well above the typical background ambient levels. The highest values of methane (levels ≥10.000 nmol L−1; Fig. 1 and Supplementary Fig. 3; Supplementary Data 3–6), were measured within the southern craters, where several flares and the gryphon were observed.

The subsequent ROV seafloor observations confirmed the persistent and ongoing methane venting from four main craters, within a major depression (Fig. 1). The two northernmost craters display extensive carbonate deposits exposed at the seafloor as stacked slabs forming tens of meters wide large pavements. The thickness of single slabs is in the order of tens of cm, but the total thickness of the deposits can reach ~5 m along the steep wall of the craters. While carbonate crusts have been observed in other cold seeps in the Barents Sea 10,11,25, the carbonates at Borealis are remarkably larger and thick (Fig. 2), emphasizing the exceptional nature of this site. The gryphon, on the southern flank of the deepest crater of the Borealis MV, has a central conduit that serves as the epicentre from which warm fluids and sediments are actively expelled, as observed from gas bubbles and sediment flows extending over the seafloor.

Origin of fluids and expelled mud
We collected the expelled unconsolidated sediments from the gryphon using a push core manipulated by the ROV (Supplementary Fig. 3). The sediments consist of medium-sized sand (grains from 0.25 to 0.5 mm), with minor clayey content (particles <0.002 mm), and a few small sedimentary rock fragments and coal pebbles (typically <2 cm) 26. The expelled sediment contains planktonic foraminiferal fauna correlated with the micropaleontological zonation for the Neogene on the Vøring Plateau 27 while the benthic foraminiferal fauna correlated with the one for the Cenozoic of the North Sea 28. Nearly all the individuals are extinct species typically associated with Pleistocene deposits on the Norwegian Shelf from ca 29 700–1000 mbsf. The lack of benthic foraminifera typical for Holocene and recent sediments on the Norwegian continental shelf, including Trifarina angulosa and Uvigerina peregrina 30–32, and the almost complete lack of warm water-dwelling planktonic foraminifera indicate that no Holocene sediments are present in the erupted material.

Geochemistry performed on the sediment from the push core indicates that the fluid emitted from Borealis gryphon has lower levels of organic carbon and nitrogen compared to the average southwestern Barents Sea surface sediments (i.e. TOC >0.5 %; TN >0.05 %) 33 with values of 0.33 wt.% and 0.02 wt.%, respectively. The isotopic composition of bulk decarbonated material (δ13C = −27.11 ‰ and δ15N = 4.58 ‰), associated with the high C/N ratio (20.2), are consistent with a high contribution of ancient organic sources that underwent prolonged microbial degradation in the deep subsurface 34,35. Organic biomarkers revealed the presence of detectable amounts of crude oil associated with immature bitumens showing virtually no biodegradation (Supplementary Fig. 4). The source rock appears to be marine shale/lacustrine deposits of Jurassic age or younger. Recent studies reported the widespread and extensive methane and oil release from geological reservoirs to the Arctic Ocean 20 and, although evident oil seepage or oil slicks were not observed at Borealis MV, this site can be considered as the surface expression of a deeper seated active petroleum system.

The 30-minute ROV dive over the gryphon revealed frequent (every 5–10 minutes) eruptions of warm water-mud-gas mixture that rises along the seep conduit below (Fig. 1). The gas composition of the bubbles escaping from the sediment is primarily methane, characterized by a typical microbial isotopic signature 36 (Fig. 3). Additionally, elevated fractions of ethane and propane, measured in a gravity core taken from outside the craters, suggest the presence of a deeper thermogenic component. Such spatial and temporal variations in subsurface fluid migration are typical at mud volcanoes and other cold seep environments and could account for the observed variability in gas compositions. Within the craters, it is possible that the microbial gas masks the presence of the thermogenic component.

The temperature of the discharged fluid measured above the active gryphon by the CTD installed on the ROV was 11 °C, and that of the T-probe was 11.42 °C. This temperature is substantially higher than the reference ca. 4 °C measurements away from Borealis.

A unique oasis for faunal communities
Borealis MV hosts a diverse array of faunal communities, including seep-associated and background fauna, together with species that have commercial value. Within the MV´s craters, we observed in the ROV video dense and extensive patches of microbial mats and siboglinid tubeworm aggregations (Oligobrachia sp.; Fig. 2) akin to those documented at other Arctic cold seeps 8. Microbial mats and tubeworms seem to be the main characteristic of high-latitudes compared to lower-latitudes cold seeps, which host clams, mussels and vestimentiferan tubeworms 4. The microbial mats, extending several square meters, form the foundation for a variety of microorganisms, including a multitude of foraminifera species. Data obtained from environmental DNA (eDNA) analysis revealed the presence of distinct species of hard-shelled foraminifera (Epistominella. sp., Reophax dentaliniformis, Stainforthia sp.), monothalamids and soft-shelled foraminifera nestled within these microbial ecosystems. Although, until now, no endemic foraminifera species have been documented within cold seeps 37, our preliminary eDNA data from Borealis MV suggest the possibility of previously unidentified species potentially unique to these types of environments.

The megafauna diversity at Borealis MV, as observed from ROV imagery, is relatively low, and appears to be influenced by several environmental factors, including elevated methane concentrations, extensive carbonate crusts on the seafloor and the suspended sediment particles emitted by the gryphon that further contribute to the challenging conditions that may be impacting the larger faunal assemblages. The megafauna is dominated by clusters of anemones and serpulids anchored to carbonate substrates and hydrozoan colonies (Tubularia sp.) that flourish on the crater slopes, while the presence of cladorhizid sponges and sea stars is more sporadic. In addition, our sample collections from various habitats within the Borealis MV have yielded taxa not detectable via ROV imagery, such as annelids, amphipods, gastropods, polyplacophorans, nemerteans (Nipponemertes spp.), and ophiuroids (Supplementary Data 1, Supplementary Fig. 5). Morphological and molecular analyses of these samples are currently underway to further elucidate the composition of these faunal communities. The observed scarcity of megafauna taxa suggests that the immediate environmental conditions surrounding the Borealis MV may be inhospitable for a broader range of organisms. Remarkably high methane concentrations (11.390 nmol L−1 near the seafloor) or toxic sulfide levels are factors known to significantly influence invertebrate community structure at seep sites 38. These chemically-enriched habitats create a gradient of extreme environmental conditions that can be detrimental to many forms of marine life, often resulting in reduced biodiversity. Additionally, the high volume of suspended sediment particles emitted by the gryphon may impact the diversity and density of filter- and suspension-feeding organisms, potentially by clogging their feeding apparatus 39. Nevertheless, some taxa appear resilient to the presence of suspended sediment. Hydrozoans, for example, are abundant in the carbonate area covered with sediment particles, similar to what was also observed on the Koryak slope at upper bathyal depths (∼660 m) 40.

While some parts of the seafloor within Borealis MV seem to be inhospitable for many organisms, the extensive carbonates provide additional habitat and suitable hard substratum for epifauna including dense aggregations of several species of anemones, serpulids, demosponges, nudibranchs, and octocoral colonies (Primnoa resedaeformis). P. resedaeformis was exclusively found in the jagged carbonate area (Fig. 2), characterized by minimal or absent sediment deposition from the gryphon plumes (Supplementary Data 8 and Fig. 4). As observed earlier at other offshore seepage sites 4,41–43, carbonate deposits represent oases for numerous sessile organisms. Moreover, the carbonate structures may offer both shelter and feeding opportunities, thereby playing a role in sustaining the local fish populations. We observed large schools of saithe (Pollachius virens) and various demersal species such as spotted wolffish (Anarhichas minor), cod (Gadus morhua), four-bearded rockling (Enchelyopus cimbrius), and several species of redfish (Sebastes spp.) clustering around the jagged carbonate formations (Fig. 2).

There are three species of Sebastes common in the Barents Sea (S. norvegicus, S. mentella and S. viviparus) 44,45, and it was not possible to separate these three with certainty using the ROV footage. However, based on the presence/absence of a pronounced protrusion on the lower lip (beak), proportional size of the eye and presence/absence of a dark patch on the gill covers, we assume that S. norvegicus was the most commonly observed, although all three species appear to be present. S. norvegicus is listed as endangered on the Norwegian Red List for Species and is subject to a fishing moratorium 45,46. The redfish were particularly numerous and clustered close to the carbonate structures. In the ROV videos, we observed several redfish individuals with clearly distended abdomens (Fig. 2). The complex three-dimensional structures and the irregular morphology of the carbonates provide protective shelters, and the site’s elevated temperatures may enhance reproductive success by accelerating egg development 47. During our expedition, we encountered lost fishing gear (Fig. 2) from bottom trawling snagged on the jagged carbonate rock around the perimeter of the craters. These were colonised by sessile fauna (e.g. anemones and hydrozoans), suggesting that the gear was lost several years ago. The current cessation of fishing activities in the Borealis area is confirmed by the Vessel Monitoring System (VMS) 48 and the Automatic Identification System (AIS) data from the OSPAR/ICES database 49 and the Global Fishing Watch database 50 on bottom fishing intensity for the area. This information, in conjunction with the presence of red-listed species S. norvegicus and taxa indicative of Vulnerable Marine Ecosystems (VMEs) as defined by FAO/ICES 51, such as the octocorals, suggests that the Borealis MV is a de facto sanctuary for these endangered species. The absence of bottom trawling and the natural habitat’s protective qualities offer a refuge where these species can thrive despite significant seafloor impacts from fishing in the surrounding Barents Sea 21,22.

Methods
Multibeam echosounder Water Column Data
Borealis MV’s morphometric data were obtained from a 3 × 3 m resolution morphobathymetric map generated onboard and obtained by processing multibeam echosounder data (i.e., depth measurements and backscatter) acquired using the hull-mounted Kongsberg MBES EM710, from which water column data were also recorded, documenting evident flares. We received permission from NOD, The Norwegian Offshore Directorate, to collect data from the Borealis study site.

Gas seep mapping at the study site was done using the QPS FMMidwater software. Gas seeps were detected as gas flares in the water column data caused by backscatter from the gas bubble streams. The acquired data from EM302 multibeam systems in *.all and *.wcd file formats were converted with FMMidwater to the generic water column format (*.gwc). *.gwc files were visualised in fan view and stacked view. The fan view allows for narrowing the opening beams to select individual flares in the stacked view and export them in sd file for visualisation in Fledermaus. Only one flare was kept when multiple flares showed the same source in Fledermaus. Locations of individual gas flares were retrieved from the lowest point of the flare in the sd file. This identification is enabled by the significant differences in velocity and density between chains of gas bubbles and the water column, leading to pronounced contrasts evident in the acoustic signals. Flare locations were plotted on the maps and used during ROV-flying during the ROV dives to locate streams of gas bubbles.

CTD
Temperature and salinity at specific depths in the water were obtained from a CTD (Conductivity, Temperature, Depth) mounted on a rosette, which was lowered in the water column from the hull of the vessel. Twelve 10-litre Niskin bottles were also mounted on the rosette, which we closed at specific depths to estimate dissolved methane.

ROV and video analyses
The Aurora work-class ROV is a 6000 m depth-rated ROV with a tethered management system (TMS) called Borealis and provides unique opportunities for science and filmmaking. Four dives (ROV13, ROV14, ROV15, and ROV16) were conducted along exploratory track lines crossing four craters (for a total of 14 hours and 4 minutes of diving time). The SfM (Structure from Motion), photogrammetry technique 37 was applied on selected ROV video frames to develop 2D photorealistic orthomosaics of representative physical habitats and sedimentary facies (Fig. 2).

All the collected videos were analyzed for substrate characterization and the recording and counting of the main vulnerable megafauna observed in the surveyed areas. To ensure the reliability of our seafloor classification, the substrate was differentiated into carbonate crusts (CC), Microbial Mats (MM), which are the more typical and representative seafloor types for a cold seep and all other substrates not clearly associated with seeping phenomena. All detected megafauna occurrences were considered variables and classified as Redfish and Coral. The Redfish category comprises all encountered species of redfish such as redfish (Sebastes norvegicus, S. viviparus and S. mentella) (denoted as RedF). The Coral category includes all species of octocorals (such as Primnoa resedaeformis and others classified at the lowest possible taxa level through videos). Each ROV dive surveyed different areas, with some level of overlapping between ROV track lines crossing the Borealis MV main seeping sites (Supplementary Fig. 7). The surveyed area covered by each ROV dive was then estimated using a GIS-based tool and applying a 1 m−2 buffer area surrounding each ROV position to remain conservative in estimating the observed area by focusing on areas well-framed by videos and easy to interpret. Because of the differences in the extent of the areas surveyed by each video, the abundance of the detected megafauna (Redfish and Coral) is expressed as number (N) per 10 m−2 in Supplementary Data 8. Data are shown in Fig. 4.

Fluid analyses
Temperature measurements. The temperature (T) of the fluid emanating directly from the crater of the Borealis MV was measured using the CTD sensor and a temperature probe, the ISD400 Depth and Temperature Sensor from Impact Subsea (precision level of ±0.01% °C), both mounted on ROV Aurora.

Methane measurements. Water samples collected from the CTD Niskin bottles were transferred into 120 mL glass bottles containing 5 mL of 1 M NaOH. The samples were stored in the dark at 4 °C until analyses with a Gas Chromatographer-FID (Thermo Scientific Trace 1310). Before the analyses, we created a 5 mL headspace and let the samples equilibrate overnight. Measurement analyses for each sample collected at different water depths are reported in Supplementary Data 2.

The dissolved methane was also continuously measured during the ROV dives using SAGE (Sensor for Aqueous Gases in the Environment), a dissolved methane instrument designed and built in the Chemical Sensors Laboratory at the Woods Hole Oceanographic Institution (WHOI) 38,39. SAGE has a detection range of 5–10,000 ppm CH4. SAGE uses a deep-sea membrane inlet to extract dissolved gas from seawater. Inside the instrument, the extracted gas fills a hollow core optical fibre. Laser spectroscopy measures the methane inside the optical fibre by coupling the light from a laser to the fibre. The dissolved methane data are reported in Supplementary Data 3.

Geochemistry
Sediment geochemistry. We prepared 0.3 g of dry sediment from a surface sample collected at the seeping spot to measure its organic carbon, nitrogen content, and isotopic composition (δ13C, δ15N). The carbonate material was removed by acid addition using 6N HCl. Analyses on decarbonated material were conducted at the SIL (UiT) using a Thermo-Fisher MAT253 Isotope Ratio Mass Spectrometer (IRMS) coupled to a Flash HT Plus Elemental Analyzer. The δ13C and δ15N values were determined and normalised to Vienna Pee Dee Belemnite (VPDB) (δ13C) and Air-N2 (δ15N) using 3 in-house urea and peptide calibrators. The analysis of soil control samples yielded a measurement repeatability (1s; n = 6) of 0.01‰ on δ13C and 0.15‰ on δ15N. The C/N atomic ratio was calculated using the atomic mass weighted ratio of TOC (Total Organic Carbon) and TN (Total Nitrogen) as C/N = (TOC/12.011)/(TN/14.007).

Oil geochemistry. Sediment samples for oil geochemistry were collected from sediment slices, wrapped in aluminium foil and stored at −20 °C for freeze-drying. All oil preparation and analysis procedures followed NIGOGA (Norwegian Industry Guide to Organic Geochemical Analysis), 4th Edition, and were conducted at Applied Petroleum Technology (APT, Oslo). Extractions were performed with a Soxtec Tecator unit and dried before deasphaltering. A small amount of dichloromethane (3 times the amount of EOM, Extractable Organic Matter) is added. Pentane is added in excess (40 times the volume of EOM/oil and dichloromethane). The solution is stored for at least 12 hours in a dark place before centrifugation and the weight of the removal of asphaltenes. Quantifying saturates, aromatics A and polar (NSO-fraction) were done using two HPLC pumps, a sample injector, a sample collector and two packed columns. The pre-column is filled with Kieselgel 100 and heated at 600 °C for 2 hours to deactivate it. The main column, a LiChroprep Si60 column, is heated at 120 °C for 2 hours with a helium flow to make it water-free. Approximately 30 mg of deasphaltened oil or EOM diluted in 1 ml hexane is injected into a sample loop. The solvents used are hexane and dichloromethane. The stable carbon isotope composition of the different fractions was measured on a Delta V Plus Isotope Ratio Mass Spectrometer (IRMS) (Thermo Fisher Scientific) via Conflo IV. A standard (NGS NSO-1, topped oil) is analysed for each 12th sample. The δ13C value obtained for this standard is –28.6‰ vPDB. The variation in the isotopic values for NSO-1 by repeated analysis over one year is ± 0.09‰. Age-specific biomarkers were measured via GC-MS/MS using a Thermo Scientific TSQ Quantum instrument. The column used is a 60 m CP-Sil-5 CB-MS with an i.d. of 0.25 mm and a film thickness of 0.25 µm. d4-27ααR was used as an internal standard.

Gas geochemistry. Two samples of seep gas were collected during the AKMA366 and Extreme24 expeditions using a bubble catcher and stored in steel flasks. One headspace gas sample was also obtained from a gravity core: a slice of sediment (~200 mL) was sampled from the bottom of the core and placed into an IsoJar™ paint can (Isotech Laboratories and Humble Instruments, USA), to which we added 0.5 mL of 10% benzalkonium chloride as antimicrobial agent and tap water. All the gas samples were stored at 4 °C. Aliquots of the samples were injected into an Agilent 7890R GAGC equipped with Molsieve and Poraplot Q columns, a flame ionisation detector (FID) and 2 thermal conductivity detector (TCD). Hydrocarbons were measured by FID. The carbon isotopic composition of methane was determined via GC-C-IRMS. Aliquots were sampled with Triplus RSH autosampler and analysed on a Trace 1310 gas chromatograph (Thermo Fisher Scientific), equipped with a Poraplot Q column and PTV (Programable Temperature Vaporizing) injector. The GC is interfaced via GC-Isolink II and Conflo IV to Delta V Isotope Ratio Mass Spectrometer (IRMS) (Thermo Fisher Scientific). Repeated analyses of standards indicate that the measurement repeatability for δ13C is better than 1‰ vPDB (2s). The hydrogen isotopic composition of methane was determined by a GC-H-IRMS system. Aliquots were sampled with a Triplus RSH autosampler and analysed on a Trace 1310 gas chromatograph (Thermo Fisher Scientific) equipped with a Poraplot Q column and PTV (Programmable Temperature Vaporizing) injector. The GC is interfaced via GC-Isolink II and Conflo IV to Delta V Isotope Ratio Mass Spectrometer (IRMS) (Thermo Fisher Scientific). Repeated analyses of standards indicate that the measurement repeatability for δD is better than 10‰ vSMOW (2s).

Biological samples and observations
Megafauna. The description of the fauna present and distribution across the main micro-habitats of the Borealis MV was obtained through the cataloguing and annotation of all the ROV Aurora imagery collected during the expedition. Additionally, experts collected and identified physical samples of both specimens and sediments to support the correct identification of the specimens observed in the videos. A complete list of the samples observed is reported in Supplementary Data 1. Fauna attached to collected carbonates was carefully removed with forceps and a scalpel, and the carbonates were thereafter rinsed with filtered seawater to collect small-sized specimens. Sediment samples were carefully sieved through a stack of sieves between 2 mm – 0.5 mm to extract the megafauna and fixed on absolute ethanol to enable downstream DNA extraction. It is important to note that not all samples were analyzed by experts during this initial phase since a more detailed and comprehensive investigation of these samples is planned to further enhance the understanding of the Borealis ecosystem. Fauna samples will be deposited in the University Museum of Bergen collections.

Foraminifera (eDNA). The foraminiferal species list (Supplementary Data 7) was inferred from two sediment samples collected from a microbial mat (Supplementary Fig. 4). Briefly, after the DNA extraction using the DNeasy PowerLyzer PowerSoil kit (Qiagen, Germany), the specific foraminifera 37f hypervariable gene of 18S rRNA gene was amplified with foraminifera specific primers 14F1 (14F1 (5′-AAGGG CACCACAAGAACGC-3′) and s15 (5’-CCACCTATCACAYAATCATG -3’) primers. Per sample, a different combination of tagged primer was used, and three PCR replicates were performed. The PCR products were then verified on agarose gel, pooled and added to a library. The sequencing library was prepared using TruSeq® DNA PCR-Free Library Preparation Kit (Illumina) and quantified by qPCR using Kapa Library Quantification Kit for Illumina Platforms (Kapa Biosystems). The library was paired-end sequenced on a MiSeq instrument using the kit v2 (300 cycles). The raw data was demultiplexed with a DTD module from SLIM 67. We used then DADA2 68 to generate Amplicon Sequencing Variants (ASV). Only ASVs containing foraminiferal “GACAG” 69 pattern and with at least 100 reads were retained. Those ASVs were finally taxonomically assigned using VSEARCH 70 with 90% min. similarity against a foraminifera database.

Biostratigraphy and sedimentology
The top 3 cm of a sediment core collected from the rim of the active emitting fluid gryphon (AKMA3 ROV16 PusCC5-M05, Supplementary Fig. 5) was analysed for biostratigraphy to date the formation from which the emitting sediments originate and correlate them with known geological time periods. The undried, unconsolidated sample was soaked in water, wet sieved and dried. Then the dry sample material was fractionated. The fraction of 0.1–0.5 mm was gravity-separated in heavy liquid. The air in the foraminiferal chambers caused them to float up, and the tests could be collected for identification. The fraction less than 0.06 mm (silt and clay) was washed down the sink. The other fractions were used for a simplified grain distribution analysis. The fractions larger than 0.5 mm and less than 0.1 mm were also examined to investigate whether any important foraminifera were left in these fractions. All foraminifera and some other microfossil of importance from all fractions were investigated and recorded (more than 300 individuals) using a stereomicroscope. For planktonic foraminifera we used the zonation of Spiegler and Jansen (1989) 27 for the Neogene on the Vøring Plateau while the benthic foraminiferal fauna can be correlated with the micropaleontological zonation of King (1989) 28 for the Cenozoic of the North Sea. Nearly all the forms are extant species typically associated with Pleistocene deposits on the Norwegian Shelf. The sediment size composition was described following the Wentworth scale 26.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript entitled “The discovery of Borealis mud volcano: a natural sanctuary for threatened Arctic species” discusses the main characteristics of Borealis, a submerged mud volcano system located in Norwegian waters. It conducts a comprehensive characterization that covers geological, geochemical, and biological components. The study makes a novel and significant contribution, though it seems to primarily interest a specific and localized research community. It is important to note that mud volcanoes are prevalent across various global oceanic regions. A quick search on Web of Science using the terms 'mud volcano' and 'sea' yielded approximately 700 articles, underscoring the ecological significance of these structures. However, the discussions in the study focus mainly on other cold seeps in the Arctic region, leaving the potential influence or benefits of the results on the international scientific community uncertain.\nThe manuscript requires revision. The title promotes the mud volcano as a 'natural sanctuary for threatened Arctic species', yet among the species observed, only one (Sebastes norvegicus) is listed as endangered. The authors note the presence of species indicative of Vulnerable Marine Ecosystems, such as octocorals (P. resedaeformis), but report that colonies of P. resedaeformis are 'sparse' and located only in areas with irregular carbonate formations where there is little or no sediment deposition. Therefore, the use of 'threatened' in the title appears sensationalist and should be reconsidered.\nThe methods should be presented after the introduction to enhance the understanding of the results and discussions.\nThe study does not specify the criteria for selecting sampling areas or the number of samples collected. It also lacks details on the statistical sampling procedures that would allow for an adequate characterization of the studied area. Statements, such as those in lines L129-132, are emphatic and seem unsupported by the sampling effort, as the conclusions were based on just two samples. This raises concerns about whether the sampled area is representative of the mud volcano's influence area. Additionally, seasonal aspects that affect the occurrence and distribution of species should be considered.\nSpecifically, Figure 2 in lines L205-209 does not support the statement that 'large schools of saithe and various demersal species are clustering around the irregular carbonate formations.' The discussion concerning the fish population in the area, particularly in line L211, questions whether there is actual support for local fish populations, given that most of the identified species undergo seasonal migrations between coastal and offshore regions, suggesting that the mud volcano may simply be a transit zone rather than a vital habitat as suggested. Moreover, when discussing aspects of fish fauna and other benthic organisms, the study fails to present quantitative data on abundance or density, which is critical for a detailed understanding of these species' ecological impact in the studied area. This absence of quantitative data further compromises the robustness of the conclusions drawn about the ecological role of the mud volcano as a habitat.\nAnalyses of oil geochemistry fall outside the scope of my expertise, and I am therefore unable to fully assess this aspect of the study.\nOther specific comments (minor comments) are listed below:\n- In Figure 1a, include a map that more clearly identifies the region highlighted in the Polar North Atlantic.\n- L124-125: What does “medium-sized sand” mean, and what is implied by “minor clayey content” and “few small sedimentary rock fragments”? These terms seem very subjective and not specific enough.\n- L170: The referred table does not contain information associated with that result, i.e., species composition.\n- L173: Was any measure of diversity estimated, or are the authors referring to the number of species? How is it relatively low compared to what?\n- L220-221: Where is this VMS information?\n- L418-419: Were the images completely analyzed (i.e., 14h and 4 min of images) or were only fractions of the videos analyzed?\n- L419-421: More details are needed regarding these physical samples and analyses (e.g., number of samples, volume, sampled areas, sample processing, etc).\n- L424: Which supplement is referred to as “Supp.X”?\n- L434: The citation format is different from the rest of the text.\n- L442: Change “Fiog.1_SI” to “Fig.1_SI”\n- L462-472: The citation format is different from the rest of the text.", "reviewer_2": "The paper presents a broad overview of a new submerged mud volcano, Borealis. It is my impression that this general paper will be followed up by many specialized papers of each of the authors. This paper focuses on the geophysical and geochemistry description of the volcano. Paleomicrobiology ages the rising sediments as Pleistocene. The organic geochemistry of the sediments and gas emissions suggest ancient organic sources that were subjected to long periods of microbial degradation. The article presents a geological history that strongly supports a deglaciation trigger model or a hot fluids surge for the origin of the volcano.\nBorealis is the second mud volcano to be identified in the Barents Sea. The paper successfully conveys the importance of studying our seafloor and discovering features that can contribute to climate change or serve as vital ecosystem resources. The Borealis mud volcano does both. CTD measurements indicate that methane is reaching the sea surface, unlike many known marine methane features. This discovery causes one to wonder if there are other marine structures contributing methane to the atmosphere that have not found yet.\nThe high methane and sulfide concentrations appear to be limiting the macrofauna diversity at the site, however many types of anemones, serpulids, demosponges, nudibranchs, and octocoral colonies live on the carbonate slabs, creating a reef-like environment. This reef welcomes many fish species, including the endangered Sebastes norvegicus. The authors speculate that the environment’s warm temperature and carbonate slabs shelter and promote reproductive success. The authors suggest that the ecosystem may enhance the breeding of many species, claiming, the potential nursery site as an asset to the fishing grounds of the Barnet Sea and worth protecting.\nOverall, the article is well-written and will be an interest to many deep-sea geobiologists. Methods are appropriate and standard to those in our field. The finding of the mud volcanic system is significant considering the volume of methane it is releasing and the habitat it create.\nI have very few edits.\nAbstract: I don’t think it is necessary to start the abstract with “this paper presents”. This may be a personal preference, but I would start with “Borealis is a recently discovered submerged mud volcano system…”\nSection 5.2 suggests that there is a foraminiferal species list. It is labeled as X and no table was included.\nFigure 1. It is my interpretation of the legend that the diamonds mark where the CTD sampled the water column, and the color of the diamond represents CH4 concentrations. However, all the diamonds are the same color and don’t reflect the concentrations in Table SI 3. Please correct the figure or make the explanation of the diamonds clearer.\nFigure 2. I would note the colors of the comparison sites before the description of 2.a as the comparisons are made in several plots. Additionally, the Mosby Modu Volcano isn’t charted on 2.a, which is confusing when it is listed under 2.a.\nTable SI 3. The formatting of this table causes the header to be on the second page. It would be ideal if the entire table fit onto one page."}, "author_response": "We thank the reviewers for their thorough reading of our manuscript and their constructive comments.\nBelow we have copied each review in full (in black text), and highlighted (main) reviewer comments in\nblack bold text. We provide our response to them in Blue text.\nThanks to these requested comments and suggestions, we feel the manuscript has improved\nconsiderably and hope that our proposed revision will meet the criteria for publication in Nature\ncommunications.\nSincerely,\nGiuliana Panieri (on behalf of all authors)\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nReviewer: The manuscript entitled “The discovery of Borealis mud volcano: a natural sanctuary for\nthreatened Arctic species” discusses the main characteristics of Borealis, a submerged mud volcano\nsystem located in Norwegian waters. It conducts a comprehensive characterization that covers\ngeological, geochemical, and biological components. The study makes a novel and significant\ncontribution, though it seems to primarily interest a specific and localized research community. It is\nimportant to note that mud volcanoes are prevalent across various global oceanic regions. A quick\nsearch on Web of Science using the terms 'mud volcano' and 'sea' yielded approximately 700 articles,\nunderscoring the ecological significance of these structures. However, the discussions in the study\nfocus mainly on other cold seeps in the Arctic region, leaving the potential influence or benefits\nof the results on the international scientific community uncertain.\nResponse: We acknowledge the reviewer for the comments and the opportunity to clarify the broader\nimplications of our study on the Borealis mud volcano. We appreciate the recognition of the novelty and\nsignificance of our research within its specific field, and we have now improved out manuscript quite\nsubstantially. In response to the reviewer´s concerns regarding the focus of our study and its appeal\nto the international scientific community, we would like to clarify the followings:\n- while our manuscript does concentrate on the Borealis mud volcano in the Arctic, our findings\nhave broader implications that extend beyond this area in the Barents Sea. Mud volcanoes, as\nthe reviewer rightly pointed out, are widespread across various regions. However, there are not\nmany papers dealing with biological characteristics of these features. We reported remarkable\nobservations of the biological characteristics from this site and for this resubmission, we have\nalso added the abundance of the vulnerable main macrofauna species strengthening the\nhypothesis that those sites represent oasis for vulnerable species.\n- The geological, geochemical, and biological characteristics observed at Borealis can provide a\ncomparative dataset that increase the understanding of similar structures worldwide. It is\nimportant to acknowledge, as we briefly mentioned earlier, the scarcity of comprehensive data\non many mud volcanoes and their biology. As pointed out by the reviewer, it is true that a search\non Web of Science using the terms 'mud volcano' and 'sea' yielded approximately 700 articles,\nbut a limited number of them (ca. 20) report on their ecological significance, which is what we\naim with our work. It needs to be mentioned that the scarcity of comprehensive data (from\ngeophysics, geochemistry and biology), is largely due to the logistical challenges and significant\nresources required to conduct such studies. These environments are difficult to access and\nrequire research vessels and specialized equipment. This reality underscores the value and\nnecessity of our detailed study at Borealis, which contributes data to a field where\ncomprehensive information is still limited and might trigger international collaborations for the\ninvestigations of similar sites.\n1\n- The ecological role of Borealis in supporting threatened species is a critical aspect of our study\nthat has an environmental relevance that go beyond the site itself. As the Reviewer 2 pointed\nout “This discovery causes one to wonder if there are other marine structures contributing\nmethane to the atmosphere that have not found yet”. The comment is suggesting that similar\nmarine structures could play unrecognized roles in both biodiversity support and climate\ndynamics. We agree with the reviewer, and we are positive that the discovery of Borealis might\ntrigger other similar discovery in the Arctic in the future now that we are aware of this geological\nfeature.\nWe think that our arguments and the additions to the text will make the significance of our findings\nclearer to a broader audience and underscore the global importance of studying such unique ecological\nsystems.\nReviewer: The manuscript requires revision. The title promotes the mud volcano as a 'natural\nsanctuary for threatened Arctic species', yet among the species observed, only one (Sebastes\nnorvegicus) is listed as endangered. The authors note the presence of species indicative of Vulnerable\nMarine Ecosystems, such as octocorals (P. resedaeformis), but report that colonies of P. resedaeformis\nare 'sparse' and located only in areas with irregular carbonate formations where there is little or no\nsediment deposition. Therefore, the use of 'threatened' in the title appears sensationalist and\nshould be reconsidered.\nResponse: We acknowledge the reviewer for the comments. We understand the reviewer´s concerns\nabout the use of the term \"threatened\" in the title and the representation of the species observed at the\nBorealis mud volcano.The term \"threatened\" might convey an unintended sense of urgency or\nsensationalism, as the reviewer has pointed out. To address this concern and to more accurately reflect\nthe content of our study, we propose revising the title to: \"The Discovery of Borealis Mud Volcano: A\nRefuge for Vulnerable Arctic Marine Species\". We think that the revised title maintains the integrity and\nfocus of our research while addressing the concerns about the representation of the ecological status\nof the species observed. It also aligns with the data presented in the manuscript, emphasizing the role\nof Borealis as a habitat for species within Vulnerable Marine Ecosystems without implying a broader\nthreat status than is supported by our findings.\nReviewer: The methods should be presented after the introduction to enhance the understanding of\nthe results and discussions.\nResponse: We followed the template provided by Nature communications but we are willing to change\nthe order of the chapters if the editor ask to do to.\nReviewer: The study does not specify the criteria for selecting sampling areas or the number of\nsamples collected. It also lacks details on the statistical sampling procedures that would allow for an\nadequate characterization of the studied area. Statements, such as those in lines L129-132, are\nemphatic and seem unsupported by the sampling effort, as the conclusions were based on just\ntwo samples. This raises concerns about whether the sampled area is representative of the mud\nvolcano's influence area. Additionally, seasonal aspects that affect the occurrence and\ndistribution of species should be considered. Specifically, Figure 2 in lines L205-209 does not\nsupport the statement that 'large schools of saithe and various demersal species are clustering\naround the irregular carbonate formations.' The discussion concerning the fish population in the\narea, particularly in line L211, questions whether there is actual support for local fish\npopulations, given that most of the identified species undergo seasonal migrations between\ncoastal and offshore regions, suggesting that the mud volcano may simply be a transit zone\nrather than a vital habitat as suggested. Moreover, when discussing aspects of fish fauna and other\nbenthic organisms, the study fails to present quantitative data on abundance or density, which is\ncritical for a detailed understanding of these species' ecological impact in the studied area. This\n2\nabsence of quantitative data further compromises the robustness of the conclusions drawn about the\necological role of the mud volcano as a habitat.\nResponse: The association of saithe with deep-water reef habitat is well established off the\nNorwegian coast, albeit observations have mainly concerned occurrence of this species with Lophelia\npertusa reefs (e.g. Costello et al (2005) in Freiwald A, Roberts JM (eds) Cold-water Corals and\nEcosystems. Springer-Verlag Berlin Heidelberg, pp 771-805; Mortensen et al (1995) Sarsia 80: 145-\n158; see evidence for also shoaling around artificial reefs [rigs] Soldal et al (2002) ICES Journal of\nMarine Science, 59: S281–S287). It is therefore unsurprising that this species is associated with\nirregular seafloor structures associated with broken carbonate pavement around the Borealis mud\nvolcano. The referee is correct in that this species undergoes migrations between more inshore and\noffshore areas during spring (e.g. Olsen et al., (2009) ICES Journal of Marine Science, 67: 87–101),\nhowever, the use of such reef-like habitats during such migrations does confer shelter, perhaps\nforaging opportunities and protection from fishing for these species during their migratory\nmovements. Therefore, irrespective of whether use of the habitat is temporary or longer term,\nprotection during part of the life history is provided.\nReviewer: lines L129-132: The reviewer refers to those lines in the main text:\n“Nearly all the individuals are extinct species typically associated with Pleistocene\n129 deposits on the Norwegian Shelf from ca 700-1000 m bsf28,29. The lack of benthic foraminifera\n130 typical for Holocene and recent sediments on the Norwegian continental shelf, including Trifarina\n131 angulosa and Uvigerina peregrina30-32, and the almost complete lack of warm water-dwelling\n132 planktonic foraminifera indicate that no Holocene sediments are present in the rising sediment.”\nResponse: In the specified lines, our discussion focuses on the findings related to foraminifera and\nbiostratigraphic analyses. It is important to note that the nature of biostratigraphic research,\nparticularly when involving foraminifera, does not typically require extensive sampling to yield\nsignificant insights and often one sample is enough to establish the age of a sediment. Foraminiferal\nanalyses can provide valuable information on the environmental conditions and geological age, even\nfrom a limited number of samples. This is due to the fact that foraminifera are highly sensitive to their\nenvironments, and their presence, absence, or community structure can be highly indicative of specific\necological conditions or geological interval.\nFurthermore, to address the reviewer comment regarding the representativeness of our sample,\nwe\nwould like to clarify that the analyzed samples were directly collected from the mud expelled by the\nvolcano so it is highly indicative of the expulsion activity of material from below and provides essential\ndata needed to characterize Borealis as a mud volcano. To illustrate this, we have included a frame\nfrom the ROV video in Figure 4_SI, which clearly shows the precise location where the samples were\ntaken. We hope this explanation clarifies the methodology and rationale behind our sampling strategy\nand the conclusions drawn from our biostratigraphic analysis.\nReviewer: “seasonal aspects that affect the occurrence and distribution of species should be\nconsidered”\nResponse: Regarding the comment of the reviewer about seasonality, we must clarify that our current\ndataset is limited to observations made during the single expedition that discovered Borealis MV. As\nsuch, we do not have other observational data that allow us to evaluate seasonal variations in species\nbehaviour or distribution around Borealis. We must admit that we did not think about this earlier, but\ndefinitely Borealis presents an ideal location for establishing a permanent monitoring station to\ncontinuously measure methane emissions and other environmental parameters, in addition to observe\nfish migrations. In any case, we answered to this pint in the text above while commenting on the general\ncomment of the reviewer.\nfails to present quantitative data on abundance or density, which is critical for a detailed\nunderstanding of these species' ecological impact in the studied area. This absence of quantitative\ndata further compromises the robustness of the conclusions drawn about the ecological role of the\nmud volcano as a habitat.\nResponse: In response to the reviewer your concerns, we would like to clarify that the primary focus of\nour initial study was to establish a baseline presence and distribution of species associated with the\nmud volcano Borealis habitat, providing a data set (from geophysics through biostratigraphy and\ngeochemistry) that clarify the nature of this unique geological feature for the Polar North Atlantic. Given\nthe exploratory nature of this research, our initial approach regarding all the macrofauna from this site\nwas qualitative. However, we recognize the critical importance of quantitative assessments in\necological studies, as the reviewer pointed out, and agree that such data are essential for a more robust\nanalysis of the ecological roles of these species. To address this gap, we are planning more than one\nfollow-up studies that will specifically target quantitative measurements of species abundance and\ndensity. However, because we acknowledge the concern of the reviewer, we viewed all the ROV dives\n(ROV 13, ROV 14, ROV 15 and ROV 16) and annotated the presence of all the main vulnerable\nmacrofauna occurrences and produced a matrix as described in the Method section in the chapter 3.\nROV and video analyses and included in Table 8_SI (Table 8_SI: Abundance of the vulnerable main\nmacrofauna species observed at Borealis MV). We then produced histograms showing the abundance\nof main vulnerable macrofauna occurrences (RedF: Red Fishes; CC: Coral), identified through video\nanalyses and expressed as the number of occurrences/10 m2, per substrate (BG: Sedimented\nbackground; MM: Microbial Mat; CC: Carbonate Crust) and each video (ROV 13: ROV 14; ROV 15; ROV\n16) and an histogram showing the abundance of the main vulnerable macrofauna occurrences (Red\nFishes and Coral), identified through video analyses and expressed as the number of occurrences/10\nm2, considering the total visualized area per each substrate (MM: Microbial Mat; CC: Carbonate Crust;\nBG: Background). Now those histograms are included in the main text as Fig. 4. As we have now\nexplained in the text, we observed that red fish are consistently more abundant around the carbonate\ncrusts, exhibiting occurrence rates ranging from approximately 0.02 to 0.76 per 10 square meters.\nThese numbers, when compared with the background fish populations investigated around Borealis\nfrom sedimented background where the number are basically 0, clearly indicate that the fish\npreferentially inhabit areas around the carbonate crusts. Additionally, corals (which are also vulnerable\nspecies, as described in the main text) are exclusively found on the carbonate crusts, with occurrence\nrates varying from 0.09 to 0.03 per 10 square meters. Those type of corals do not live on sediment\nseafloor.\nReviewer: Analyses of oil geochemistry fall outside the scope of my expertise, and I am therefore\nunable to fully assess this aspect of the study.\nResponse: We cannot comment this point, but we are confident of our work.\nOther specific comments (minor comments) are listed below:\nReviewer:- In Figure 1a, include a map that more clearly identifies the region highlighted in the Polar\nNorth Atlantic.\nResponse: We acknowledge the comment of the reviewer, and the Figure 1 is now clearer and identify\nbetter the area of investigation in the Polar North Atlantic.\nReviewer: - L124-125: What does “medium-sized sand” mean, and what is implied by “minor clayey content”\nand “few small sedimentary rock fragments”? These terms seem very subjective and not\nspecific enough.\nResponse: To address your concerns about the terms \"medium-sized sand,\" \"minor clayey content,\"\nand \"few small sedimentary rock fragments,\" we provide clarifications that have been included in the\n4\nsection “Methods” in the subchapter 6. Biostratigraphy and sedimentology where we indicated the\nclassification used (Wentworth scale, also included in the reference list) for the definition of the term\nused to describe the sediment emitted from Borealis. In addition, we also added information of the\nBiostratigraphy, that was non originally included.\nReviewer: - L170: The referred table does not contain information associated with that result, i.e.,\nspecies composition.\nResponse: We appreciate the reviewer's attention in identifying this error. We now added the Table 7_SI\nwith the foraminifera list identified from eDNA analyses with the following detail: Table 7_SI: Amplicon\nSequencing Variants (ASV) of foraminiferal eDNA. For each ASV the foraminifera taxon identified on a\nnumber of reads > 100 are indicated.\nReviewer: - L173: Was any measure of diversity estimated, or are the authors referring to the number\nof species? How is it relatively low compared to what?\nResponse: In this respect, in the text we refer to the observed number of species based on visual\ninspections from ROV video footage, rather than a calculated measure of diversity such as Shannon or\nSimpson indices that are planned. The term \"relatively low\" was used to qualitatively describe the\nobserved species richness in comparison to the background area and other similar habitats. To clarify\nthis in our manuscript, we revised the text to specify that our reference to diversity is based solely on\nobserved species trend and that these observations are not backed by statistical analyses (that are\nplanned for future work).\nReviewer: - L220-221: Where is this VMS information?\nResponse: We now included in the text the reference to the Vessel Monitoring System (VMS) data that\nare reported in a well-known publication from the MOREANO project and the EU-based bottom fishing\nintensity data from the OSPAR/ICES database, that we used to extract GIS data to confirm that no\nrecent fishery has occurred in the area where Borealis is located. We also confirmed with Automatic\nIdentification System (AIS) data from the Global Fishing Watch database, to double check from the\n2020s onwards data, and the same output, no fishing\nthere:https://globalfishingwatch.org/map/index?latitude=72.99823215175326&longitude=12.510194\n070543434&zoom=3.7222661775128367&start=2024-03-09T00%3A00%3A00.000Z&end=2024-06-\n09T00%3A00%3A00.000Z. All these information have been added in the text.\nReviewer: - L418-419: Were the images completely analyzed (i.e., 14h and 4 min of images) or were\nonly fractions of the videos analyzed?\nResponse: We thank the reviewer for the comment, that gave us the possibility to clarify the work we\ndid on all the videos. In Table 1_SI (reporting the type of sample and analyses/measurement performed\non each) we indicated the entire duration of each ROV video (in the column “note”) and all the videos\nwere completely analysed not fractions of them.\nReviewer: - L419-421: More details are needed regarding these physical samples and analyses (e.g.,\nnumber of samples, volume, sampled areas, sample processing, etc).\nResponse: We thank the reviewer for the comment that allow us to clarify those aspect of our work,\nas we have also indicated in the main text in the sub chapter 5.1 Mega/macrofauna under\n5. Biological samples and observations. During this initial phase of our research, not all collected\nsamples were analyzed. Our primary goal with this paper was on those samples that would most likely\nprovide immediate clarification and support for the video-based identifications. A more detailed and\ncomprehensive investigation of all collected samples is planned for October as part of our ongoing\nresearch in a Workshop funded by Ocean Census. This future work will include a thorough analysis of\n5\nthe remaining samples, which will give us the possibility to write one (or more) paper with the details\nsuggested by the reviewer\nReviewer: - L424: Which supplement is referred to as “Supp.X”?\nResponse: We thank the reviewer for the comment. We now added the Table 7_SI with the foraminifera\nlist identified from eDNA analyses with the following detail: Table 7_SI: Amplicon Sequencing Variants\n(ASV) of foraminiferal eDNA. For each ASV the foraminifera taxon identified on a number of reads > 100\nare indicated.\nReviewer: - L434: The citation format is different from the rest of the text.\nResponse: We thank the reviewer for the comment. Now the citation is formatted following the Nature\ncomm guidelines.\nReviewer: - L442: Change “Fiog.1_SI” to “Fig.1_SI”\nResponse: Corrected as suggested.\nReviewer: - L462-472: The citation format is different from the rest of the text.\nResponse: We thank the reviewer for the comment. Now all the citations that were included in the\ncaption of Fig. 3 are formatted following the Nature comm guidelines.\n-----------------------------------------------------------------------------------------------------------------\nReviewer #2 (Remarks to the Author):\nThe paper presents a broad overview of a new submerged mud volcano, Borealis. It is my impression\nthat this general paper will be followed up by many specialized papers of each of the authors. This\npaper focuses on the geophysical and geochemistry description of the volcano. Paleomicrobiology\nages the rising sediments as Pleistocene. The organic geochemistry of the sediments and gas\nemissions suggest ancient organic sources that were subjected to long periods of microbial\ndegradation. The article presents a geological history that strongly supports a deglaciation trigger\nmodel or a hot fluids surge for the origin of the volcano.\nBorealis is the second mud volcano to be identified in the Barents Sea. The paper successfully\nconveys the importance of studying our seafloor and discovering features that can contribute to\nclimate change or serve as vital ecosystem resources. The Borealis mud volcano does both. CTD\nmeasurements indicate that methane is reaching the sea surface, unlike many known marine\nmethane features. This discovery causes one to wonder if there are other marine structures\ncontributing methane to the atmosphere that have not found yet.\nThe high methane and sulfide concentrations appear to be limiting the macrofauna diversity at the\nsite, however many types of anemones, serpulids, demosponges, nudibranchs, and octocoral\ncolonies live on the carbonate slabs, creating a reef-like environment. This reef welcomes many fish\nspecies, including the endangered Sebastes norvegicus. The authors speculate that the\nenvironment’s warm temperature and carbonate slabs shelter and promote reproductive success.\nThe authors suggest that the ecosystem may enhance the breeding of many species, claiming, the\npotential nursery site as an asset to the fishing grounds of the Barnet Sea and worth protecting.\nOverall, the article is well-written and will be an interest to many deep-sea geobiologists. Methods are\nappropriate and standard to those in our field. The finding of the mud volcanic system is significant\nconsidering the volume of methane it is releasing and the habitat it create.\nReviewer: Abstract: I don’t think it is necessary to start the abstract with “this paper presents”.\nThis may be a personal preference, but I would start with “Borealis is a recently discovered\nsubmerged mud volcano system…”\nResponse: We have followed the suggestions of the reviewer and changed the text as suggested. Now\nthe abstract start with “Borealis is a recently discovered submerged mud volcano system…..”\nReviewer: Section 5.2 suggests that there is a foraminiferal species list. It is labeled as X and no\ntable was included.\nResponse: We thank the reviewer for the comment. We now added a Supplementary Table 7 with the\nforaminifera list identified from eDNA analyses and the new table name is Supplementary Table 7:\nAmplicon Sequencing Variants (ASV) of foraminiferal eDNA. For each ASV the foraminifera taxon\nidentified on a number of reads > 100 are indicated.\nReviewer: It is my interpretation of the legend that the diamonds mark where the CTD sampled the\nwater column, and the color of the diamond represents CH4 concentrations. However, all the\ndiamonds are the same color and don’t reflect the concentrations in Table SI 3. Please correct the\nfigure or make the explanation of the diamonds clearer.\nResponse: We thank the reviewer for the comment. We modified Figure 1 according to the suggestions\nand made it better and more readable than before. We add the CTD and SAGE Methane concentration\ndata in a zoomed circle where the concentration can be easily seen and added also the number to better\nappreciate the results of the measurements.\nReviewer: Figure 2. I would note the colors of the comparison sites before the description of 2.a as\nthe comparisons are made in several plots. Additionally, the Mosby Modu Volcano isn’t charted on\n2.a, which is confusing when it is listed under 2.a.\nResponse: We think that the suggestions of the reviewer refers to Figure 3. As the reviewer\nsuggested, we moved the comparison to the colours characterizing the different sites in several plot in\nthe first part of the Figure description and adjusted the captions.\nReviewer: Table SI 3. The formatting of this table causes the header to be on the second page. It\nwould be ideal if the entire table fit onto one page.\nResponse: All the tables have been reformatted according to “Nature communication guide to\nformatting articles”. Now there are 4 Tables (Supplementary Table 3-6) instead of Table 3_SI reporting\nthe SAGE data.\n7"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I appreciate the comments and concerns of reviewer 1 and the changes that the writers have made to address all the comments in general. I still have a few suggestions.\nline 32. What is I-oxygen?\nLine 33. I agree with Reviewer #1, I don't think one expedition can ensure the reasons why fauna were found at this site. Maybe word the sentence \"carbonate structures may serve as\"\nline 60. Is there a period after organisms(4)? If so, please remove.\nFigure 1. I still do not understand the relevance of the diamond colors. I appreciate that the methane concentrations are written to the side. Maybe make the diamonds all one color and depend only on the written values. Additionally, what is the significance of the gas flare colors? Are the temperatures? If so, were is the legend for that?", "reviewer_3": "Dear authors, thank you for a great paper of a important discovery of a mud volcano system “Borealis” located in the North Atlantic (Bear Island Trend). I have only revied te faunistic part of this manuscript, and understand that a following up paper will be made for the fauna.\nFor this manuscript I would recommend to clarify the points I identified so that the reader will feel faunistic convinced at this preliminary stage, and should look forward for a more “in depth” paper that will come and will apply the evidence (lacking in this manus) for the described observations in this paper.\nThe review of the manuscript entitled \"The discovery of Borealis mud volcano: a natural sanctuary for vulnerable Arctic species\" (NCOMMS-24-20285A).\nGiuliana Panieri, Claudio Argentino, Alessandra Savini, Bénédicte Ferré, Rune Mattingsdal, Sofia Ramalho, Mari Eilertsen, Tor Eidvin, Sarah Youngs, Beckett Colson, Anna Michel, Jason Kapit, Fereshteh Hemmateenejad, Denise Swanborn, Alex Roger, Adriano Mazzini, Ines Barrenechea Angeles, Stephane Polteau, Dimitri Kalenitchenko, and Stefan Buenz\nI will only review the “biological” aspects of this work.\nThis manuscript describes a mud volcano system “Borealis” located in the North Atlantic (Bear Island Trend). Methane-\ndominated fluids are eruption of from a localized site within a  500 m diameter crater. The seafloor around comprises\nlaterally extensive carbonate deposits.\nSampling and seafloor images reveal that Borealis hosts unique habitats thriving in the environments around methane\nseeps. Additionally, the irregular shaped carbonate structures serve as a substratum for sessile fauna and function as\nnursing grounds for threatened fish species. These carbonate structures are also a natural refuge from bottom trawling.\nThis highlights the ecological value of cold seep ecosystems, which play a critical role in biodiversity by acting as\nsanctuaries for marine species, hence emphasizing the importance of their conservation.\n- What are the noteworthy results?\nA newly discovered mud volcano named Borealis mud volcano located in Outer Bjørnøyrenna at 390 m water depth in the\nBarents Sea.\nWithin the craters dense and extensive patches of microbial mats and tubeworm aggregations (Oligobrachia sp.) are\nobserved which seem to be the main characteristic of high-latitudes compared to lower-latitudes cold seeps. A variety of\nmicroorganisms/multitude of foraminifera species were obtained from environmental DNA (eDNA) analysis (hard-shelled\nforaminifera (Epistominella sp., Reophax dentaliniformis, Stainforthia sp.), monothalamids and soft-shelled foraminifera\nnestled within these microbial ecosystems.\nThese preliminary eDNA analyses suggest the possibility of previously unidentified species potentially unique to those type\nof environments.\nA observed scarcity of megafauna diversity suggests that the immediate environmental conditions surrounding Borealis may\nbe inhospitable for a broader range of organisms (elevated methane concentrations, extensive carbonate crusts on the\nseafloor and the suspended sediment particles emitted by the gryphon).\nROV imagery revealed that the megafauna is dominated by dense clusters of anemones and serpulids anchored to\ncarbonate substrates. Tubularia sp. colonies flourish on the crater slopes appearing resilient to the presence of suspended\nsediment. Cladorhizid sponges and sea stars is more sporadic.\nSample collections (possible taken by a push core, but not explained) from various habitats within the Borealis yielded taxa\nnot detectable via ROV imagery, such as annelids, amphipods, gastropods, polyplacophorans, nemerteans (Nipponemertes\nspp.), and ophiuroids. The results from these samples are not included in this manuscript but will be elucidated later.\nThe extensive carbonates provide additional habitat and suitable hard substratum for erect epifauna and dense\naggregations of several species of anemones, serpulids, demosponges, nudibranchs, and sparse octocoral colonies.\nIt was annotated by the ROV videos that the corals P. resedaeformis was only located in the jagged carbonate area with\nlower or no sediment deposition from the plumes of the gryphon was evident.\nLarge schools of saithe (Pollachius virens) and various demersal species such as spotted wolffish (Anarhichas minor), cod\n(Gadus morhua), four bearded rockling (Enchelyopus cimbrius), and redfish (mainly Sebastes norvegicus but a few\nindividuals tentatively identified as S. viviparus and S. mentella) clustering around the jagged carbonate formations suggest\nthat these structures serve as critical habitats, offering both shelter and abundant feeding opportunities, thereby playing a\npivotal role in sustaining the local fish populations.\nSeveral individuals of the red-listed S. norvegicus observed in the ROV videos appear to be pregnant.\nThe morphology of the carbonates providing a protective shelter, and the site's elevated temperatures are said to possible\nenhance reproductive success by accelerating egg development.\nLost fishing gear on the carbonate rock around the perimeter of the craters was observed by ROV imagery. The gear was\nheavily colonised by sessile fauna (e.g., anemones and hydrozoans), suggesting being old. No resent fishing activities\n(VMS and AIS, OSPAR/ICES) are recorded around the mud volcano system.\nThe presence of red-listed S. norvegicus and VME octocorals suggests that the Borealis is a sanctuary for endangered\nspecies.\nThe absence of bottom trawling and the natural habitat's protective qualities offer a refuge where these species can thrive\ndespite significant seafloor impacts from fishing in the surrounding Barents Sea.\n- Will the work be of significance to the field and related fields? How does it compare to the established literature? If the work\nis not original, please provide relevant references.\nDespite numerous observations of methane emissions from the seafloor in Arctic regions, only five mud volcanoes have\nbeen discovered in the Canadian Beaufort Sea (Western Arctic), and three in Alaska, and so far, the Håkon Mosby Mud\nVolcano was the only known structure in Norwegian waters.\nThis work is significant to the field because it is an extremely important new and seldom discovery of a mud volcano that will\nneed to be registered on sea-maps as soon as possible to avoid damaging this complex habitat and its associated fauna.\nComparison to established literature is good, but there are issues that need to be taken care of.\n- Does the work support the conclusions and claims, or is additional evidence needed?\nLines 215-218\nSaithe (Pollachius virens), spotted wolffish (Anarhichas minor), cod (Gadus morhua), bearded rockling (Enchelyopus\ncimbrius) can be identified on images. But splitting redfish into Sebastes norvegicus, S. viviparus and S. mentella is\nchallenging and would need more verification to become trustworthy (e.g., to be taken by physically by equipment and\nidentified by experts). This would need to be explained in the text.\nLines 223-225: Several individuals of the red-listed S. norvegicus observed in the ROV videos appear to be pregnant.\nThis is written without any references or further description. As above, this would need much more evidence since it is\nextremely difficult (impossible) to judge if a S. norvegicus is pregnant from a picture.\nLines (226-228) 287-290: The elevated temperatures around the seepage sites may create optimal conditions for\nreproductive processes, leading to more effective breeding cycles. This thermal advantage can enhance the survival and\ngrowth of fish populations in the deep sea, and the Barents Sea, one of the world's most productive fishing grounds,\nrepresents a significant ecological asset.\nIt is highly unlikely that a 500m diameter spot can have an effect on the “fish-population” in the Barents Sea. It may have an\nimpact on the local fish using this area for spanning. This is written without any references or further evidence and need to\nbe more carefully evaluated.\nLines 216-221: Large schools of fish are clustering around the jagged carbonate formations suggest that these structures\nserve as critical habitats, offering both shelter and abundant feeding opportunities, thereby playing a pivotal role in\nsustaining the local fish populations.\nThis is built on evidence from observations in Lophelia reefs and artificial reef-life structures – but I am missing evidence that\nthe pelagic prey that the mention fish species are eating are found at the volcano structures. Did you register these pelagic\nprey-species on your video-records?\nLines 169-204\nIt is very hard to understand what was observed via ROV imagery, what was sampled using a push core, and how sediment\nwas taken for DnA analyses. I question is it may be too premature to include the faunistic information in this paper since it\nanticipated that another and likely more describing paper will be made by the authors.\nLines 187-192: Anemones, annelids, amphipods, gastropods, polyplacophorans, and ophiuroids.\nThese large animal groups are found everywhere and does not really provide any information. I may suggest describing their\ntraits or morphological characteristic in this paper instant, and then in next paper provide more detailed taxonomy.\n- Are there any flaws in the data analysis, interpretation and conclusions? - Do these prohibit publication or require revision?\nSome sentences need to be strengthened and definitions clarified before publication:\nLines 352-374\nA photogrammetry technique (SfM) was applied and selected ROV video frames provided physical habitats and sedimentary\nfacies. But are these pictures also analyzed for fauna – please explain?\nThe substrate was differentiated in carbonate crusts (CC), Microbial Mats (MM) and all other substrate (denoted as BG in\nfigure 4, but not as such in the text, and this is confusing).\nMacrofauna occurrences were classified as Red Fishes (RedF) and Coral (CC). The Coral category are denoted ”CC” in line\n366 (but not in figure 4), -the same as carbonate crusts – and this is confusing.\nEach ROV dive surveyed different areas, with some level of overlapping between ROV tracklines crossing the Borealis MV\nmain seeping sites. – Does this represent a danger of counting and recording of observations more than ones?\nThe surveyed area covered was estimated by using GIS-based tool and applying a 1 m2 buffer area surrounding each ROV\nposition. Because of the differences in the extent of the areas surveyed by each video, the abundance of the detected\nmacrofauna (Red Fishes and Coral) are expressed as number per 10 square meter.\n- Is the methodology sound? Does the work meet the expected standards in your field?\nThe analyzing of fauna from video (and maybe images?) is an accepted and well used method. The abundance of observed\nfauna is standardized.\nBut the paper would be clearer and more convincing if explained and verified more deeply (see above). I guess that this will be\nthe case for the next paper wish I will look very much forward to.\n- Is there enough detail provided in the methods for the work to be reproduced?\nThere are not enough details provided for the fauna-work to be reproduced. I guess that this will be the case for the next\npaper wish I will look very much forward to."}, "author_response": "We thank the reviewers for their thorough reading of our manuscript and their constructive\ncomments. Below we have copied each review in full (in black text) and highlighted (main)\nreviewer comments in black bold text. We provide our response to them in blue text.\nThanks to these requested comments and suggestions, we feel the manuscript has\nimproved considerably from the previous version submitted and hope that our proposed\nrevision will meet the criteria for publication in Nature Communications\nSincerely,\nGiuliana Panieri (on behalf of all authors)\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nI appreciate the comments and concerns of reviewer 1 and the changes that the writers\nhave made to address all the comments in general. I still have a few suggestions.\nline 32. What is I-oxygen?\nWe corrected the text and changed it to low oxygen.\nLine 33. I agree with Reviewer #1, I don't think one expedition can ensure the reasons\nwhy fauna were found at this site. Maybe word the sentence \"carbonate structures\nmay serve as\"\nThe concern raised by Reviewer #1 has been better explained in the revised version of the\ntext in the chapter “A unique oasis for faunal communities” also following the suggestions\nof Reviewer #3. Regarding this comment from Reviewer #2, we changed the sentences\nsuggested and added the word “may” to the sentence.\nline 60. Is there a period after organisms(4)? If so, please remove.\nThe period has now been removed as suggested.\nFigure 1. I still do not understand the relevance of the diamond colors. I appreciate\n1\nthat the methane concentrations are written to the side. Maybe make the diamonds all\none color and depend only on the written values. Additionally, what is the significance\nof the gas flare colors? Are the temperatures? If so, were is the legend for that?\nWe appreciate the reviewer's comment, which prompted us to enhance Figure 1 and its\ncaption. The diamond symbol in the figure represents the CTD samples measured for\nmethane concentration. Since these samples reflect methane concentration\nmeasurements in water, similar to the SAGE data, we have used the same color scale for\nboth, to maintain consistency. Additionally, we have included a zoomed-in view of the\ndepression where methane measurements were conducted to improve clarity. To facilitate\nthe reader's appreciation of Figure 1, we labelled all figures using the a, b, c convention and\nprovided a more detailed explanation in the caption.\nRegarding the significance of the gas flare colors. The flares represent the backscattering\nintensity of the reflected acoustic signals (red indicating the highest values and light green\nthe lowest). However, the colour variations only help assess the distribution of the gas\nbubbles within the water column but is not indicative of methane concentration. This is a\nstandard way of visualising acoustic data to facilitate initial analysis and guide further\ndetailed investigations.\nThe revised version of the caption is the following:\nFigure 1. Borealis MV. a Map showing the location of Borealis mud volcano and other cold\nseeps in the area (for some of them, the origin of emitting methane is shown in Fig. 3). b\nActive gryphon emitting warm fluid, methane and Neogene sediments. c Compiled\nobservations, including seabed topography from high-resolution multibeam data (5 m grid\ncell), a seismic cross-section from 3D-seismic dataset SPE16M01 (the complete seismic\nsection is available in Supplementary Figure 1) and the multi-beam echosounder data (320\nkHz) tracing streams of gas bubbles (gas flares) in the water column with variations in the\ncolours of flares representing the backscattering intensity of the reflected acoustic signals\n(red indicating the highest values and light green the lowest). d Georeferenced detail of the\nconfined depression (~0.14 km2) around the active gryphon showing the methane water\nconcentration (represented with the same colour scale to maintain consistency) measured\nin CTD water samples (position of the water sampling indicated by the diamonds in the\nvertical line) and real-time SAGE measurements (data showed as ROV tracks)\n(Supplementary Tables 3-6). e Georeferenced ROV images show a carbonate pinnacle\ncolonised by Octocorallia (pinnacle high ca 120 cm) and f the red fish Sebastes norvegicus\n(ca 30 cm in length).\nDear authors, thank you for a great paper of a important discovery of a mud volcano system\n“Borealis” located in the North Atlantic (Bear Island Trend). I have only revied te faunistic\npart of this manuscript, and understand that a following up paper will be made for the\nfauna.\nFor this manuscript I would recommend to clarify the points I identified so that the reader\nwill feel faunistic convinced at this preliminary stage, and should look forward for a more\n“in depth” paper that will come and will apply the evidence (lacking in this manus) for the\ndescribed observations in this paper.\nThe review of the manuscript entitled \"The discovery of Borealis mud volcano: a natural\nsanctuary for vulnerable Arctic species\" (NCOMMS-24-20285A).\nGiuliana Panieri, Claudio Argentino, Alessandra Savini, Bénédicte Ferré, Rune Mattingsdal,\nSofia Ramalho, Mari Eilertsen, Tor Eidvin, Sarah Youngs, Beckett Colson, Anna Michel,\nJason Kapit, Fereshteh Hemmateenejad, Denise Swanborn, Alex Roger, Adriano Mazzini,\nInes Barrenechea Angeles, Stephane Polteau, Dimitri Kalenitchenko, and Stefan Buenz\nI will only review the “biological” aspects of this work.\nThis manuscript describes a mud volcano system “Borealis” located in the North Atlantic\n(Bear Island Trend). Methane-dominated fluids are eruption of from a localized site within a\n500 m diameter crater. The seafloor around comprises laterally extensive carbonate\ndeposits.\nSampling and seafloor images reveal that Borealis hosts unique habitats thriving in the\nenvironments around methane seeps. Additionally, the irregular shaped carbonate\nstructures serve as a substratum for sessile fauna and function as nursing grounds for\nthreatened fish species. These carbonate structures are also a natural refuge from bottom\ntrawling.\nThis highlights the ecological value of cold seep ecosystems, which play a critical role in\nbiodiversity by acting as sanctuaries for marine species, hence emphasizing the\nimportance of their conservation.\n- What are the noteworthy results?\nA newly discovered mud volcano named Borealis mud volcano located in Outer\nBjørnøyrenna at 390 m water depth in the Barents Sea.\nWithin the craters dense and extensive patches of microbial mats and tubeworm\naggregations (Oligobrachia sp.) are observed which seem to be the main characteristic of\n3\nhigh-latitudes compared to lower-latitudes cold seeps. A variety of\nmicroorganisms/multitude of foraminifera species were obtained from environmental DNA\n(eDNA) analysis (hard-shelled foraminifera (Epistominella sp., Reophax dentaliniformis,\nStainforthia sp.), monothalamids and soft-shelled foraminifera nestled within these\nmicrobial ecosystems.\nThese preliminary eDNA analyses suggest the possibility of previously unidentified species\npotentially unique to those type of environments.\nA observed scarcity of megafauna diversity suggests that the immediate environmental\nconditions surrounding Borealis may be inhospitable for a broader range of organisms\n(elevated methane concentrations, extensive carbonate crusts on the seafloor and the\nsuspended sediment particles emitted by the gryphon).\nROV imagery revealed that the megafauna is dominated by dense clusters of anemones\nand serpulids anchored to carbonate substrates. Tubularia sp. colonies flourish on the\ncrater slopes appearing resilient to the presence of suspended sediment. Cladorhizid\nsponges and sea stars is more sporadic.\nSample collections (possible taken by a push core, but not explained) from various\nhabitats within the Borealis yielded taxa not detectable via ROV imagery, such as\nannelids, amphipods, gastropods, polyplacophorans, nemerteans (Nipponemertes\nspp.), and ophiuroids. The results from these samples are not included in this\nmanuscript but will be elucidated later.\nWe appreciate the reviewer's comment. In response, we have enhanced the manuscript by\nincluding detailed information on the sample collection processes in Table 1 and Section\n5.1 on Mega/Macrofauna in the Methods part. This addition clarifies our work and provides\ntransparency regarding our data collection.\nThe extensive carbonates provide additional habitat and suitable hard substratum for erect\nepifauna and dense aggregations of several species of anemones, serpulids,\ndemosponges, nudibranchs, and sparse octocoral colonies.\nIt was annotated by the ROV videos that the corals P. resedaeformis was only located in the\njagged carbonate area with lower or no sediment deposition from the plumes of the\ngryphon was evident.\nLarge schools of saithe (Pollachius virens) and various demersal species such as spotted\nwolffish (Anarhichas minor), cod (Gadus morhua), four bearded rockling (Enchelyopus\ncimbrius), and redfish (mainly Sebastes norvegicus but a few individuals tentatively\nidentified as S. viviparus and S. mentella) clustering around the jagged carbonate\nformations suggest that these structures serve as critical habitats, offering both shelter\nand abundant feeding opportunities, thereby playing a pivotal role in sustaining the local\nfish populations.\npregnant.\nThe morphology of the carbonates providing a protective shelter, and the site's elevated\ntemperatures are said to possible enhance reproductive success by accelerating egg\ndevelopment.\nLost fishing gear on the carbonate rock around the perimeter of the craters was observed\nby ROV imagery. The gear was heavily colonised by sessile fauna (e.g., anemones and\nhydrozoans), suggesting being old. No resent fishing activities (VMS and AIS, OSPAR/ICES)\nare recorded around the mud volcano system.\nThe presence of red-listed S. norvegicus and VME octocorals suggests that the Borealis is a\nsanctuary for endangered species.\nThe absence of bottom trawling and the natural habitat's protective qualities offer a refuge\nwhere these species can thrive despite significant seafloor impacts from fishing in the\nsurrounding Barents Sea.\n- Will the work be of significance to the field and related fields? How does it compare to the\nestablished literature? If the work is not original, please provide relevant references.\nDespite numerous observations of methane emissions from the seafloor in Arctic regions,\nonly five mud volcanoes have been discovered in the Canadian Beaufort Sea (Western\nArctic), and three in Alaska, and so far, the Håkon Mosby Mud Volcano was the only known\nstructure in Norwegian waters.\nThis work is significant to the field because it is an extremely important new and seldom\ndiscovery of a mud volcano that will need to be registered on sea-maps as soon as possible\nto avoid damaging this complex habitat and its associated fauna. Comparison to\nestablished literature is good, but there are issues that need to be taken care of.\n- Does the work support the conclusions and claims, or is additional evidence needed?\nLines 215-218\nSaithe (Pollachius virens), spotted wolffish (Anarhichas minor), cod (Gadus morhua),\nbearded rockling (Enchelyopus cimbrius) can be identified on images. But splitting\nredfish into Sebastes norvegicus, S. viviparus and S. mentella is challenging and\nwould need more verification to become trustworthy (e.g., to be taken by physically by\nequipment and identified by experts). This would need to be explained in the text.\nWhile we acknowledge the reviewer's suggestion that collecting specimens would have\nbeen ideal for species identification and confirming pregnancy, capturing redfish using the\nROV was not feasible due to the size and mobility of the fish. Attempting to do so would\n5\nhave been cruel and could have caused unnecessary suffering to the animals and potential\ndamage to the ecosystem. Instead, we relied on the expertise of fish specialists from the\nUniversity Museum of Bergen to assist with identifying the fish from the ROV footage, as\nnoted in the acknowledgements section. We have revised the text to emphasize the\nuncertainty surrounding the identification of Sebastes spp. and have detailed the specific\ncharacteristics used for differentiation where possible.\nLines 223-225: Several individuals of the red-listed S. norvegicus observed in the ROV\nvideos appear to be pregnant.\nThis is written without any references or further description. As above, this would\nneed much more evidence since it is extremely difficult (impossible) to judge if a S.\nnorvegicus is pregnant from a picture.\nWe thank the reviewer for the comment that allowed us to explain this part better, which is\nvery important for our paper. We have expanded upon the assumption that S.norvegicus\nobserved in the ROV videos appears to be pregnant, explaining that it is based on the\nobservation of the fish's distended abdomens coupled with the timing of these\nobservations (early May), which aligns with the known spawning season for this species as\nsupported by publications indicated now in the main text. On this matter, our opinion\ndiverged from the reviewer's opinion, and as explained above, instead, we relied on the\nexpertise of fish specialists from the University Museum of Bergen to assist with identifying\nthe fish from the ROV footage.\nLines (226-228) 287-290: The elevated temperatures around the seepage sites may\ncreate optimal conditions for reproductive processes, leading to more effective\nbreeding cycles. This thermal advantage can enhance the survival and growth of fish\npopulations in the deep sea, and the Barents Sea, one of the world's most productive\nfishing grounds, represents a significant ecological asset.\nIt is highly unlikely that a 500m diameter spot can have an effect on the “fish-\npopulation” in the Barents Sea. It may have an impact on the local fish using this area\nfor spanning. This is written without any references or further evidence and need to\nbe more carefully evaluated.\nThere have now been several observations of animals in the deep sea aggregating and\nspawning around seeps and vents (e.g. Drazen et al., 2003; Hartwell et al., 2018; Barry et\nal., 2023). The direct causation of such aggregations is not known for sure, however, several\nexplanations have been offered. First, deep-sea organisms often exist at low population\ndensities and so aggregation around prominent topographic features can be advantageous\n6\nfor reproduction. The conditions around seeps provide both enhanced local productivity\nand higher than surrounding ambient temperatures. These offer enhanced feeding\nopportunities for both adult and juvenile stages of organisms. Elevated temperatures are\nlikely to enhance the development times of eggs and larvae of organisms in areas of\nseepage, with Drazen et al. (2003) estimating an increase in temperatures of 1.5oC at the\nGorda Ridge in the Pacific could reduce egg incubation time by 10% in benthic fish and\noctopus assuming a Q10 of 2. However, we recognize that the taxa observed at Borealis\n(e.g. the redfish) have a different reproductive ecology than the species in the referenced\npapers since they have a direct development releasing free-swimming larvae that might not\nbe as affected by the increased temperature as eggs incubated directly on the seafloor.\nStill, the environment around the Borealis MV may provide both elevated food,\ntemperature, an obvious “reef-like” effect, aggregating fish taking advantage of shelter and\nprotection from bottom trawling. Given the conservation status of the fish in the Barents\nSea, with some species threatened as a result of overexploitation, even a relatively small\nlocation like the Borealis MV and associated carbonates could be significant as a\npopulation refuge.\nLines 216-221: Large schools of fish are clustering around the jagged carbonate\nformations suggest that these structures serve as critical habitats, offering both\nshelter and abundant feeding opportunities, thereby playing a pivotal role in\nsustaining the local fish populations.\nThis is built on evidence from observations in Lophelia reefs and artificial reef-life\nstructures – but I am missing evidence that the pelagic prey that the mention fish\nspecies are eating are found at the volcano structures. Did you register these pelagic\nprey-species on your video-records.\nWe thank the reviewer for the comment. This comment is connected to the previous\ncomment, where we have explained the role of jagged carbonate formations, which provide\nhabitat and feeding opportunities for large schools of fish and draw parallels with\nobservations made in Lophelia reefs and artificial reef-like structures. However, we\nacknowledge that our manuscript did not explicitly document direct evidence of pelagic\nprey species associated with these carbonate formations at the Borealis MV. Additionally,\nit's important to consider that the feeding dynamics at these sites might involve a broader\nrange of prey than previously observed in the gut content of fishes captured in the pelagic\nzone. Sebastes spp. have quite a variable diet composed largely of small fish and\ncrustaceans, with relative contribution of specific food-items changing with size of the fish\n(Dolgov and Drevetnyak, 2011). The biologically enriched environment of Borealis MV\n7\ndriven by the seep activity and associated bacterial production could be supporting a more\ncomplex food web that benefits the resident fish populations.\nLines 169-204\nIt is very hard to understand what was observed via ROV imagery, what was sampled\nusing a push core, and how sediment was taken for DnA analyses. I question is it may\nbe too premature to include the faunistic information in this paper since it anticipated\nthat another and likely more describing paper will be made by the authors.\nWe thanked the reviewer and agreed that the text needed to be clarified regarding the\nanalysed samples and how they were collected. We have added additional information in\nseveral paragraphs throughout the text to clarify which observations are from ROV imagery\nversus samples and information in Supplementary Table 1.\nWe value the reviewer's comment regarding the potential prematurity of including faunistic\ndata in this paper. We are convinced that incorporating this faunistic information is crucial\nat this stage, as it establishes a baseline that highlights the unique ecological\ncharacteristics of this mud volcano in the Barents Sea. Given that this area is likely to be\nexploited in the future (in fact, it has been included in one of the drilling permits from the\nNorwegian Offshore Directorate), presenting this data now underscores the need for\ncareful management to mitigate potential risks associated with its development. To avoid\nincluding preliminary identifications of fauna that are likely to contain errors that have to be\ncorrected in the following papers, we have maintained a focus in this paper on the\nmegafauna that could be identified with relative certainty based on ROV imagery and kept\nidentifications of macrofauna to very broad categories. Still, we believe that the information\nprovided in the paper is sufficient at this stage to support our conclusions and hypotheses\nabout the importance of the Borealis faunal communities for local productivity and\nbiodiversity.\nLines 187-192: Anemones, annelids, amphipods, gastropods, polyplacophorans, and\nophiuroids.\nThese large animal groups are found everywhere and does not really provide any\ninformation. I may suggest describing their traits or morphological characteristic in\nthis paper instant, and then in next paper provide more detailed taxonomy.\nWhile we agree with the reviewer that the faunal groups identified is not very informative,\nmore detailed information on the taxonomy or traits of the collected fauna is not possible\nto supply at this point as the faunal samples are still under processing. As described\n8\nabove, we kept identifications at higher taxonomic levels at this stage to avoid including\npreliminary identifications with a likelihood of error. Identifications to lower taxonomic\nlevels (genus and species) were only included where we were quite confident they were\ncorrect.\n- Are there any flaws in the data analysis, interpretation and conclusions? - Do these\nprohibit publication or require revision?\nSome sentences need to be strengthened and definitions clarified before publication:\nLines 352-374\nA photogrammetry technique (SfM) was applied and selected ROV video frames\nprovided physical habitats and sedimentary facies. But are these pictures also\nanalyzed for fauna – please explain?\nWe thank the reviewers for the comments and recognizing that our initial explanation might\nnot have been sufficiently clear. In our study, we utilized the same video footage for\nmultiple purposes: to construct the mosaic presented in Figure 2 using the Structure from\nMotion (SfM) technique and to perform detailed video analyses for seafloor\ncharacterization and fauna annotation that gave us the possibility to estimate the\nabundance of redfish and corals as shown in Figure 4. These methodologies are now better\nexplained in Chapter 3 \"ROV and Video Analyses”.\nThe substrate was differentiated in carbonate crusts (CC), Microbial Mats (MM) and all\nother substrate (denoted as BG in figure 4, but not as such in the text, and this is\nconfusing).\nMacrofauna occurrences were classified as Red Fishes (RedF) and Coral (CC). The\nCoral category are denoted ”CC” in line 366 (but not in Figure 4) -the same as\ncarbonate crusts – and this is confusing.\nWe thank the reviewer for this comment, which allowed us to refine and clarify this part in\nour manuscript. We have now detailed the various substrates identified in the study area\nthrough video analysis in the section titled \"A Unique Oasis for Faunal Communities.\"\nAdditionally, we have ensured consistency in the terminology used for different substrate\nanalyses across the main text, the tables, and Figure 4.\nEach ROV dive surveyed different areas, with some level of overlapping between ROV\ntracklines crossing the Borealis MV main seeping sites. – Does this represent a danger of\ncounting and recording of observations more than ones?\nour study was to spatially visualise associations among observations rather than\nperforming statistical analyses on fauna abundance. However, the reviewer raises a valid\npoint regarding the potential issues in recording observations, especially for mobile\norganisms like fish. From our observations during the expedition and the following video\nanalyses, we noted that the redfish has minimal movement, often remaining stationary\naround the carbonate crusts. This behaviour reduces the probability of double-counting\nthese particular fish in our recordings. We also observed other types of fish, and those were\nmore mobile than the redfish. This is why we decided not to include them in our\nclassification. As for the corals, each coral observed was precisely pinpointed and\ngeoreferenced using the ROV's navigation system on the tracklines. These locations were\nmarked as waypoints on our maps during the dives, and corrected once we performed the\nobservations of the videos onshore. This method allowed us to maintain a reliable record of\ncoral positions, avoiding the danger of recording the exact coral multiple times.\nThe surveyed area covered was estimated by using GIS-based tool and applying a 1 m2\nbuffer area surrounding each ROV position. Because of the differences in the extent of the\nareas surveyed by each video, the abundance of the detected macrofauna (Red Fishes and\nCoral) are expressed as number per 10 square meter.\n- Is the methodology sound? Does the work meet the expected standards in your field?\nThe analyzing of fauna from video (and maybe images?) is an accepted and well used\nmethod. The abundance of observed fauna is standardized.\nBut the paper would be clearer and more convincing if explained and verified more\ndeeply (see above). I guess that this will be the case for the next paper wish I will look\nvery much forward to.\nWe have provided a more precise and better explanation of the methods used to analyse\nour data and samples as better explained above.\n- Is there enough detail provided in the methods for the work to be reproduced?\nThere are not enough details provided for the fauna-work to be reproduced. I guess\nthat this will be the case for the next paper wish I will look very much forward to.\nthe methodology section, and further information about the fauna samples collected has\nbeen added to Table 1 in the supplementary material.\nOn behalf of the co-authors\nGiuliana Panieri\nNovember 18, 2024\n11"}]

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