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The Antarctic scallop Adamussium colbecki is one of the few species of benthic organisms that we routinely observed in the icy upper reaches of the shallow anchor-ice zone in Explorer’s Cove, in western McMurdo Sound, Antarctica (Supplementary Movie 1). They inhabit both the deep, ice-free zones and the shallowest, iciest areas27 where they can even be found sitting atop the thick, growing anchor-ice blanket (Fig. 1c–e). The external surfaces of this benthic bivalve (hereafter referred to as “shell”) appear to remain ice-free, while rocks and other inanimate materials in the scallops’ vicinity are completely covered with ice (Supplementary Movie 1). In possessing an inanimate calcitic shell, this species may provide a unique example of a marine invertebrate that avoids cryofouling by truly passive means.Similar to the well-studied locales in southeastern McMurdo Sound17,26, diver observations in Explorer’s Cove (2015, 2017, 2018, by PAC) revealed that the prevalence and thickness of the anchor-ice formations are highest in the shallowest locales and decreased with increasing depth of the seabed. From the underside of the surface sea ice (~2–3 m) to about 6 m depth, anchor ice formed a 60-cm-thick mat, covering greater than 90% of the seabed (Zone 1, Fig. 1c). From ~6 to 20 m depth, the anchor-ice cover was patchier, appearing to cover only 30–50% of the seabed (Zone 2; Fig. 1d). Anchor ice was never observed at depths below ~20 m (Zone 3; Fig. 1e). This depth may demarcate the maximum depth of supercooling occurrence in the study area (Fig. 1b). Live scallops were found to be distributed in all three zones. At the shallowest depths, aggregations of scallops were common even atop the growing anchor-ice blanket (Fig. 1c and Supplementary Movie 1). Interestingly, the shells of live scallops were never observed to be cryofouled during ~30 research dives completed during the peak anchor-ice growth periods in Austral spring (October to December of 2015, 2017, and 2018), nor did any scallops appear to be frozen to the actively growing anchor-ice matrix on which they sat.Demonstrating the potential dangers of cryofouling for Antarctic scallops is non-trivial because the scallops themselves appear to naturally avoid cryofouling. However, on numerous occasions, PAC documented cryofouling on bush sponges (Homaxinella balfourensis) that had colonized the external shell surfaces of Antarctic scallops. In these observations, the growth of ice24 on the epizoic sponges caused involuntary flotation of the host scallops due to the buoyancy28 of the ice attached to the sponge (Supplementary Movie 2). This resulted in the transport of both species to the underside of the growing sea-ice cover above, where they presumably froze in and died (Fig. 2a–f). Strikingly, the shells of cryofouled sponge-colonized scallops themselves, as well as uncolonized scallops in their immediate vicinity, appeared to remain devoid of ice. These observations suggest that the scallops themselves must be cryofouling-avoidant compared to the sponge, as well as to non-biogenic minerals in their vicinity (rocks, sand, and fine sediment) (Supplementary Movie 2).Fig. 2: Sea sponge-colonized Antarctic scallops demonstrate the potential dangers for organisms that do not avoid cryofouling.a–c Unless colonized by the cryofouling-susceptible bush sponge (H. balfourensis), Antarctic scallops appear to be unaffected by cryofouling, even in areas where underwater ice growth is prevalent (Zones 1 and 2). d–h Photographic images depicting the progression of cryofouling-induced uplift (by buoyant ice) of sponge-colonized scallops in Zone 2. The negative consequence of cryofouling for scallops becomes apparent only when its surfaces have been colonized by the bush sponge H. balfourensis, which readily accrete ice. g When a sufficient volume of ice has accreted on the sponge, both species are rafted to the underside of the sea ice by buoyant flotation, where both appear to freeze in and die. Scallop is freezing into the underside of the sea ice. Scallop shell exteriors appear to be free of icing even after arriving at the underside of the sea ice. Note: Scallops arriving and freezing into the underside of the sea ice will eventually be enveloped by further sea ice growth. Scale bars indicated are approximates.Full size imageCryofouling of Antarctic scallops could potentially be deleterious in multiple ways beyond that resulting in buoyant uplift and relatively rapid death. For example, ice adhered to a scallop’s shell could impact their swimming and escape behaviors or occlude water flow paths necessary for filter-feeding. In this case, if the Antarctic scallops do not possess anti-cryofouling capabilities, a larger proportion of the population may likely suffer from the same hazards posed by icing to epizoic sponges: ice growth, involuntary flotation, and likely death when merged into the sea ice. Given that no observations of a cryofouled live scallop exist, the icing process on scallops may be different, posing lower risks for accumulation and flotation. The negative consequences on both feeding and motility functionalities (Fig. 2c–h) associated with cryofouling suggest that for organisms inhabiting areas of supercooled seawater, the selection pressure to evolve passive or active anti-icing strategies could be relatively strong.The ability of surfaces to avoid in-air icing is influenced by properties like micro-structuring, surface roughness, hydrophobicity, chemical composition, or specific topological patterns4,5. Figure 3a–e shows the morphological characterization of the shells of the Antarctic scallop. In contrast to two non-Antarctic control species (bay scallop and sea scallop), the shells of the Antarctic scallop are macroscopically smooth, with only minute, elevated concentric growth rings and gently undulated primary ridges apparent to the naked eye. Scanning electron microscopy (SEM) images of the Antarctic scallop shells revealed a highly structured surface29 with concentric growth rings that separate micro-ridges and micro-valleys, forming a distinctive, regular hierarchical texture (Fig. 3f–h). That is, the areas between growth rings are defined by a series of relatively uniform radial micro-ridges. The shell surfaces of the two control species did not exhibit any ordered or repeating microstructures (Supplementary Figs. 2 and 3). For the Antarctic scallop, the spacing between concentric growth rings and radial micro-ridges along a line from the umbo to the margin were determined to be ~250 and ~10 µm wide, respectively. These features are not strictly uniform over the shell and may vary up to an order of magnitude, with the spacing of features increasing as a function of distance from the umbo. The microscopic surface features of larger (older) shells were slightly abraded, with surface wear appearing to increase towards the umbo–the oldest portion of the shell.Fig. 3: Surface characteristics of the Antarctic scallop’s shell features.a–c Terminology and schematics of features on the shell surface, a, b in-plane, and c section view. d, e Macroscopic images in visible light of the Antarctic scallop’s shell surface without magnification. The shell is covered by a thin, proteinaceous covering (periostracum), under which the calcitic material lies throughout the thickness of the shell. Radial rounded, primary ridges, and concentric growth rings are visible. f–h Scanning electron micrographs showing increasing magnification. f Concentric growth rings separating repeating series of g radial micro-ridges (c. 20 µm peak-to-peak). h Small protuberances are irregularly dispersed throughout the radial micro-valleys (Supplementary Fig. 3). Shells are oriented with the umbo at the top in all panels, except c. Shells of temperate control species are substantially rougher and less ordered than those of the Antarctic scallop (Supplementary Fig. 2).Full size imageNext, we determined the elemental composition and roughness at different positions on the shell surface. Energy-dispersive X-ray spectroscopy revealed that the shell surface predominately consists of oxygen, carbon, calcium, traces of silicon, sodium, aluminum, and magnesium (Supplementary Fig. 4). These findings are consistent with a typical calcitic shell (CaCO3) laminated with proteinaceous periostracum. Surface composition was found to be identical at different locations of the analyses, including along concentric growth rings, micro-ridges, or the micro-valleys in between. The surface roughness was determined using atomic force microscopy (AFM) and revealed that the peaks of the concentric growth rings were the roughest (RMS roughness of 135 ± 57 nm), followed by the radial micro-ridges (RMS roughness of 96 ± 3 nm). In contrast, valley floors between these micro-ridges were relatively smooth (RMS roughness of 38 ± 9 nm), interrupted only by small protuberances (nano-grains, diameter of 83 ± 26 nm) that were irregularly dispersed throughout the radial micro-valleys.In nature, unwanted ice accretion on surfaces can occur either by the accumulation of suspended adhesive frazil ice particles or by in situ initiation of ice growth (heterogeneous ice nucleation) on a surface18,25. To test the possibility of preferential ice nucleation, the scallops were subjected to ice nucleation measurements and an in-air frosting assay. Figure 4a, d shows the results of in-air frosting experiments performed using a custom-built apparatus30,31 in an air-filled climate-controlled (20 °C) chamber at controlled humidity (60%). Compared to the temperate control species, ice nucleation on the Antarctic scallop shell occurred later and subsequent ice growth was directed toward specific surface features (Fig. 4a, b and Supplementary Movie 3). Ice appears to accumulate only on the growth rings, leaving ice-free micro-ridges. As the experiment progressed, ice that had nucleated on the growth rings continued to grow vertically, thereby preventing further imaging of the ice-shell interface (Supplementary Movie 3). We observed no inter-ring bridging of ice and nucleated ice crystals grew preferentially upwards while enlarging in size. In contrast, neither control species showed any obvious signs of directed ice nucleation and appeared to frost uniformly over the surface (Fig. 4d, e). These results indicate that the Antarctic scallop may possess an ability to control ice nucleation and directed ice growth on its shell surfaces.Fig. 4: In-air frosting of scallop shells.The progression of ice accumulation was observed for shells placed atop a cold source in a temperature- and humidity-controlled chamber (20 °C, 60% relative humidity). a The Antarctic scallop preferentially directed ice nucleation and subsequent growth to the shell’s growth rings, termed directed frosting. b This patterned ice accumulation may also apply to ice growth in underwater environments, where it could reduce overall ice-shell contact area (c). d Directed frosting was not observed in control species (e.g., Bay scallop), where ice accreted in a patchy or uniform fashion over the entire surface (e). Likewise, such random but homogenously distributed nucleation-growth behaviors are likely to apply for ice growth in underwater environments, with continuous ice mats over the surface of the shell establishing higher overall contact adhesion force (f).Full size imageFor the Antarctic scallop, the mechanism of directed frosting occurs over two sequential steps: (1) initial condensation-frosting on growth rings, followed by (2) continuous frost growth. (1) The presence of sharp/rough edges (such as those on the growth rings), is known to enhance liquid nucleation by reducing the energy barrier32. Since more liquid water resides at the growth rings, here the nucleation of microscopic ice is more likely33. (2) Upon the formation of these microscopic ice domains as ice stripes (on growth rings), the vapor field around the ridges becomes compressed. The compressed vapor field generates locally steeper vapor gradients and thus stronger vapor fluxes towards the ice on the ridges (Fig. 4b). As a consequence, ice growth on the stripes is enhanced compared to the valleys. The vapor transport in the air during frosting is analogous to the heat transport underwater during icing. At the ridges, the latent heat generated is removed more quickly, facilitating accelerated growth34,35. Furthermore, since the vapor pressure of ice is lower than that of water36, the condensed water in the micro-valleys would be redirected to the ridges. This process would result in the micro-valleys being left comparatively free of ice, per Fig. 4a.A caveat remains, as the shell surface temperature during frosting assays (in-air) is significantly lower (c. −10 to −15 °C) than in nature. In its natural habitat, the lowest temperatures experienced by surfaces immersed in seawater (Fig. 4c, f) are rarely much below the equilibrium freezing point (~−1.9 °C), thus making heterogeneous ice nucleation on shells unlikely. However, the ability to direct ice growth may influence the probability of frazil ice adhering and anchoring strongly to the shell surface, thus facilitating easier removal through mechanical or passive environmental forces.We performed ice-adhesion measurements to test whether the apparent cryofouling avoidance of the Antarctic scallop’s shell in nature could arise from reduced adhesion forces between adhered ice and the shell’s surface. If adhesion is sufficiently low, ice could detach and float away under behavioral (movements, locomotion37) and/or environmentally induced forces (physical interactions, water currents, buoyancy of ice), or a combination of the above. This is particularly relevant once a sufficiently high ratio of ice volume to attachment surface area has been achieved. To provide complementary insight, both in-air and underwater ice-adhesion measurements were performed.The in-air ice-adhesion strength was determined30 by laterally shearing off drops of frozen freshwater from target scallop shells (Supplementary Movie 4). The recorded force curves show that ice adheres less strongly to the Antarctic scallop compared to the control species, the sea and bay scallops, as shown in Fig. 5a. For the Antarctic scallop, the in-air ice-adhesion strength was 145 ± 24 kPa (mean ± SE), ~2–3 times lower than both control species, with adhesion strengths of 335 ± 23 and 405 ± 27 kPa for the sea scallop and bay scallop respectively (Fig. 5a). We further observed that when the frozen drops sheared off the Antarctic scallop, the ice-shell fracture interface exhibited distinct growth ring-patterned fracture lines. In contrast, frozen drops detached from the control species appeared to have a uniform fracture interface (Supplementary Fig. 5 and Supplementary Movie 5). This observation could be explained by the incomplete contact adhesion of the frozen drops to the Antarctic scallop’s shell due to the spacing of small repetitive structural elements on the shell’s surface. As such, the overall area of ice-shell adhesion is reduced, thereby lowering the overall effective adhesion force.Fig. 5: Ice-adhesion measurements for Antarctic, Sea, and Bay scallops.a Displacement (lateral) of ice drops (10 µL) from the surface of shells in a humidity-controlled chamber. Shell temperature: −10 to −15 °C (thickness-dependent). Peak force was achieved immediately before the complete detachment of the drop from the surface. b Displacement (normal) of accreted ice grown in simulated seawater at its freezing point (35 g/L NaCl, c. −2 °C). Mean peak recorded force (dashed lines) ±1 SE (shaded areas) for three repetitions of each experiment are shown. Experimental details are available in the Supplementary Methods and Materials.Full size imageThe strength of ice adhesion (N/m2 or Pa) to shells underwater was determined using a custom-built apparatus. The apparatus determined the forces required for ice detachment when adhered ice was pulled perpendicular to and away from the shell surface. Underwater ice-adhesion experiments were performed in saltwater (35 g/L NaCl), simulating the seawater conditions in Antarctica. A cold source beneath a small section of the shell induced ice growth up to 6 mm in height above the shell surface, (Supplementary Movie 6), thereby encasing a perforated titanium plate (1 cm square, 4 × 2 mm-diameter holes) which was in turn connected to the force probe. The probe was then drawn upwards (normal to the shell, at 30 µm/s, Supplementary Movie 7). As for experiments in air, the recorded force curves in underwater experiments reveal that ice adheres weakly to the Antarctic scallop shell as compared to those of the two control species (Fig. 5b and Supplementary Movie 8). The Antarctic scallop experiences up to 3 times lower ice adhesion (37 ± 7 kPa, mean ± SE) than the sea scallop (112 ± 14 kPa) and more than 6 times lower adhesion than the bay scallop (243 ± 14 kPa) (Fig. 5b). At 37 ± 7 kPa, the Antarctic scallop’s shell shows underwater ice adhesion (normal to the surface) comparable to industrially-developed in-air anti-icing surfaces (1–50 kPa)4. However, if attachment forces in nature approach this value in a static environment (i.e., no water currents or scallop swimming behaviors), ≥3.7 cm3 of ice would need to be attached per mm2 of ice-shell contact area for the passive removal of ice under its own buoyant forces. Therefore, the “passive” removal of ice from Antarctic scallops must result from a combination of surface-to-environment factors. That is, the removal of ice crystals with small contact areas, such as platelet ice with its fine dendritic structure, may be facilitated by drag forces arising in natural water currents or induced by the rapid opening and closing of the scallop’s valves during swimming behaviors37. In these cases, adherent ice on the microstructures of the shell would likely experience the initiation of cracks that would subsequently facilitate ice removal given water movements or other physical disturbances.Whether the cryofouling-avoidant surfaces of the Antarctic scallop’s shell arose under evolutionary selection pressure remains unclear. Based on fossil records, the exclusively Antarctic scallop genus Adamussium first appeared in the early Oligocene, some 33 million years ago (mya)38,39. This time period roughly coincides with the onset of the major glaciation of Antarctica (c. 35 mya). The Antarctic scallop, Adamussium colbecki, appears to have arisen more recently, in the late Pliocene ( More

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Our results show that differences in environmental conditions between inland and coastal sinkholes, caused mainly by the inflow of seawater in the latter, influence the microbial community structure of their sediments. Furthermore, the microbial community structure also varied within the sinkholes and according to the sediment zone sampled, suggesting that a connection between the atmosphere in the outermost location of the sediments and sunlight creates an environment distinct from that found in deeper caves. Together with the different environmental factors that were measured (in situ physicochemical composition of water and sediment) these characteristics could drive niche-specific microbial community structures associated with the sediment zones. Additionally, beta-diversity analysis showed separate clustering of the sediment microbial communities from the coastal and inland sinkholes, and of the WM zone from cavern and cave zones at both sinkholes. Microbial community structure associated with karst environments have shown to be significantly influenced by environmental factors as seen in the Bahamian blue holes19, a coastal sinkhole13, a Floridan anchialine sinkhole20, and sediments from Chinese karst caves21.Microbial communities from karst sediments can be limited by nutrients such as carbon, phosphorus, and nitrogen21, therefore, influencing their structure. Differences in the microbial community composition associated with multiple environmental factors (moisture, type of niche, nitrogen) were also reported in karst cave sediments from China21. Previous studies had shown there was no effect on the alpha diversity of water column assemblages in the Yucatan groundwater associated with the type of sinkhole (inland or coastal)6. However, this observation may be limited due to the low sample number used in the study6. For other karst sinkholes, the microbial community dynamics differ between the water column and the sediments6,22,23,24.The karst caves and sinkholes of the underground river in Yucatan are characterized by low phosphorus concentrations and high levels of nitrate, mostly related to Anthropocentric activities (urban developments, farms and agriculture)3. The inland sinkhole at Noh Mozón showed the highest concentration of nitrate detected in the study and, not surprisingly, the area is surrounded by agricultural fields. The presence of organic matter in the sinkholes from the Yucatan peninsula are highly dependent on the connection between the cave systems, on the levels of exposure to light, and on their morphology3. High concentrations of organic carbon (661 ± 132 μM) and methane (6466 ± 659 nM) have been reported in the top layer of the water masses in coastal sinkholes before13. In this study, the highest concentration of organic carbon was observed in the sediments from the coastal sinkhole, likely originating from the surrounding vegetation and from seawater intrusion. We hypothesize that the differences in nutrients found at these two types of sinkholes influence the structure of their microbial communities.Other environmental factors such as pH and dissolved oxygen (DO), may also contribute significantly to the composition and structure of microbial communities, as seen in freshwater lake sediments22. Davis and Garey20 reported distinct microbial communities with unique functions for each water layer from an anchialine sinkhole from the Florida karst aquifer and suggested that this occurred as a result of the influence of the hydrochemistry, including differences in the concentration carbon and other nutrients from the environment20. Analyses of the sinkhole caves from the Yucatan underwater river support observations that physical and chemical parameters create distinct ecological niches which host unique microbes, as a high abundance of exclusive (not shared) ASVs were observed in the three sediment zones at both locations.The taxonomic diversity from the coastal and inland sinkholes included Chloroflexi, Crenarchaeota, Desulfobacterota, Proteobacteria, Nitrospirota, Bacteroidota, and Firmicutes as the most abundant phyla in the sediment samples, however, there were differences in the relative abundance associated with the type of sinkhole and sediment zone. Some of these phyla (Chloroflexi, Proteobacteria, and Bacteroidetes) have been reported in sediments from freshwater karst sinkholes from Lake Huron25 in water and sediments from other sinkholes in the Yucatan peninsula6, and in the karst caves bacteriome from southwest China21. A study that included coastal marine sediments from two sites in the Yucatan peninsula, showed high abundances of Spirochaeta, Desulfococcus, Clostridium, Psychrobacter26, four genera that were abundant in the coastal sinkhole. However, Desulfococcus, Synechococcus were also abundant in the inland sinkhole. Of the most abundant genera reported for sediments from different marine environments in the Yucatan coast23, Acinetobacter, Desulfotignum, Desulfovibrio, Pseudomonas, Sedimenticola, and Sulfurimonas were also present in the coastal sinkhole while only Pseudomonas and Sedimenticola were also present in the inland sinkhole23. The high number of families shared between the coastal sinkhole and marine sediments from the Yucatan coast, together with the salinity levels registered at the bottom layer of the water column in the coastal sinkhole, suggest an interconnection between these two environments which shapes the microbial communities present in the sediments of caverns and caves of this sinkhole. The genus Nitrospira was abundant in the WM from the coastal sinkhole and in all sediment zones from the inland sinkhole. This genus has been reported as one of the most abundant in the surface of speleothems from El Zapote coastal sinkhole2, and is considered a complete ammonia oxidizer (comammox), meaning it converts ammonia to nitrate through nitrite. A negative correlation between abundance of this genus and salinity has been reported before, which could explain the low concentration of Nitrospira in the cavern and cave from the coastal sinkhole, where the highest salinity was observed27. Connectivity between coastal sinkholes and the ocean, as well as the terrestrial input of soil organic matter (OM) has been reported for the underground karst aquifer in the Yucatan peninsula13. As in other sediments, degradation of OM is carried out by several MFGs including acetogenic bacteria, methanogens, and sulfate reducers13,20,28. When these MFGs were analyzed in coastal and inland sinkholes, differences in their relative abundances were clearly marked by the type of sinkhole and by the sediment zone analyzed, supporting the hypothesis that environmental differences drive microbial community distributions in these niches. The high abundance of sulfate-reducing bacteria (SRB) in the three sediment zones from the coastal sinkhole suggests that sulfate reduction is a predominant function. SRB degrade organic matter using sulfate with sulfide as waste or end-product19,30, originating hydrogen sulfide (H2S)29, which could explain the low concentration of sulfate the hydrogen sulfide (H2S) cloud observed and previously reported in the WM zone of El Zapote coastal sinkhole30. In this study, high levels of sulfate (SO−4) were measured in the water samples from the cavern and cave zones from El Zapote sinkhole, which could be associated with sulfate-rich deposits, such as gypsum beds, which have been reported in other sinkholes from the Yucatan peninsula (up to 2400 mg/L of sulfate concentration)3. However, we do not disregard other possible sources of sulfate, associated with seawater intrusion or as a product of sulfide or sulfur oxidation29,31 by sulfur-oxidizing bacteria detected in this study.The inland sinkhole had a low concentration of sulfate and low abundances of SRB. The high abundance of methanogenic bacteria in the WM zone from the coastal sinkhole detected in the MFG analysis supports the previous hypothesis of acetoclastic methanogenesis due to high inputs of organic matter13. Methylotrophic bacteria were most abundant at the inland sinkhole in the WM zone, suggesting the presence of methyl compounds, such as methane or methanol which can be used as a source of carbon and energy32. High methane concentrations have been quantified in shallow water masses from the Yucatan aquifer system13, consistent with observations from this study. ‘Candidatus Methylomirabilis’ was identified in the sediment of the WM zone from Noh-Mozón and has been previously described as being able to perform nitrite-dependent anaerobic methane oxidation, using methane as electron donor and nitrate and nitrite as electron acceptors33, which would be possible in these sediments considering the low levels of oxygen (average of 2.3 mg/L) detected in the water column above them and assuming this would lead to lower levels of oxygen in the sediments. We hypothesize that bacteria from this genus could be using the nitrite produced by ammonia oxidizing bacteria and archaea observed in this zone (Nitrosomonadaceae, Nitrospiraceae, and Nitrosococcaceae). The low abundance of methanotrophic microbes in the coastal sinkhole (mainly the cavern and cave zones) could be derived from the high concentrations of hydrogen sulfide previously reported at this location, which have been suggested to be toxic to methane-oxidizing microbes34. Therefore, a decrease in the anaerobic oxidation of methane, and a poor methane removal capacity is hypothesized in the sediments from this coastal sinkhole. Further research could focus on the influence of the saline intrusion on methanotrophic microbes and methane levels in El Zapote sinkhole sediments. As expected, photosynthetic bacterial abundances differed with the type of sinkhole and sediment zone. Both sinkholes are so the presence of daylight can start the photosynthetic process which would occur most in the WM zone. However, only the inland sinkhole showed a high abundance of photosynthetic bacteria within its WM zone. The coastal sinkhole water column and sediments would be deprived of photosynthetic bacteria since the water source is the underground aquifer, lacks photosynthesizers. Ammonia oxidizing bacteria (AOB) and archaea (AOA), and nitrite-oxidizing bacteria (NOB) were relatively abundant in the three sediment zones from the inland sinkhole and in the WM from the coastal sinkhole, these observation at the WM from El Zapote agree with previous observations2. Anaerobic ammonium oxidation (anammox) uses nitrite (as a product of nitrate reduction), as electron acceptor35. The high levels of nitrate concentration in the water column from the three sediment zones at the inland sinkhole and at the WM from the coastal sinkhole may influence the abundance of AOB, AOA and NOB in the sediments from these zones, while NH4+ and nitrite values were below detection limit ( More

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