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Calcium carbonate (CaCO3) biomineralizing organisms have played major roles in the history of life and the global carbon cycle during the past 541 Ma. Both marine diversification and mass extinctions reflect physiological responses to environmental changes through time. An integrated understanding of carbonate biomineralization is necessary to illuminate this evolutionary record and to understand how modern organisms will respond to 21st century global change. Biomineralization evolved independently but convergently across phyla, suggesting a unity of mechanism that transcends biological differences. In this review, we combine CaCO3 skeleton formation mechanisms with constraints from evolutionary history, omics, and a meta-analysis of isotopic data to develop a plausible model for CaCO3 biomineralization applicable to all phyla. The model provides a framework for understanding the environmental sensitivity of marine calcifiers, past mass extinctions, and resilience in 21st century acidifying oceans. Thus, it frames questions about the past, present, and future of CaCO3 biomineralizing organisms.

The mature skeletons of hard corals, termed stony or scleractinian corals, are made of aragonite (CaCO3). During their formation, particles attaching to the skeleton's growing surface are calcium carbonate, transiently amorphous. Here we show that amorphous particles are observed frequently and reproducibly just outside the skeleton, where a calicoblastic cell layer envelops and deposits the forming skeleton. The observation of particles in these locations, therefore, is consistent with nucleation and growth of particles in intracellular vesicles. The observed extraskeletal particles range in size between 0.2 and 1.0 μm and contain more of the amorphous precursor phases than the skeleton surface or bulk, where they gradually crystallize to aragonite. This observation was repeated in three diverse genera of corals, Acropora sp., Stylophora pistillata─differently sensitive to ocean acidification (OA)─and Turbinaria peltata, demonstrating that intracellular particles are a major source of material during the additive manufacturing of coral skeletons. Thus, particles are formed away from seawater, in a presumed intracellular calcifying fluid (ICF) in closed vesicles and not, as previously assumed, in the extracellular calcifying fluid (ECF), which, unlike ICF, is partly open to seawater. After particle attachment, the growing skeleton surface remains exposed to ECF, and, remarkably, its crystallization rate varies significantly across genera. The skeleton surface layers containing amorphous pixels vary in thickness across genera: ∼2.1 μm in Acropora, 1.1 μm in Stylophora, and 0.9 μm in Turbinaria. Thus, the slow-crystallizing Acropora skeleton surface remains amorphous and soluble longer, including overnight, when the pH in the ECF drops. Increased skeleton surface solubility is consistent with Acropora's vulnerability to OA, whereas the Stylophora skeleton surface layer crystallizes faster, consistent with Stylophora's resilience to OA. Turbinaria, whose response to OA has not yet been tested, is expected to be even more resilient than Stylophora, based on the present data.

Soft corals (Cnidaria, Anthozoa, Octocorallia, Alcyonacea) produce internal sclerites of calcium carbonate previously shown to be composed of calcite, the most stable calcium carbonate polymorph. Here we apply multiple imaging and physical chemistry analyses to extracted and in-vivo sclerites of the abundant Red Sea soft coral, Ovabunda macrospiculata, to detail their mineralogy. We show that this species' sclerites are comprised predominantly of the less stable calcium carbonate polymorph vaterite (> 95%), with much smaller components of aragonite and calcite. Use of this mineral, which is typically considered to be metastable, by these soft corals has implications for how it is formed as well as how it will persist during the anticipated anthropogenic climate change in the coming decades. This first documentation of vaterite dominating the mineral composition of O. macrospiculata sclerites is likely just the beginning of establishing its presence in other soft corals. STATEMENT OF SIGNIFICANCE: Vaterite is typically considered to be a metastable polymorph of calcium carbonate. While calcium carbonate structures formed within the tissues of octocorals (phylum Cnidaria), have previously been reported to be composed of the more stable polymorphs aragonite and calcite, we observed that vaterite dominates the mineralogy of sclerites of Ovabunda macrospiculata from the Red Sea. Based on electron microscopy, Raman spectroscopy, and X-ray diffraction analysis, vaterite appears to be the dominant polymorph in sclerites both in the tissue and after extraction and preservation. Although this is the first documentation of vaterite in soft coral sclerites, it likely will be found in sclerites of other related taxa as well.

Spherulites are radial distributions of acicular crystals, common in biogenic, geologic, and synthetic systems, yet exactly how spherulitic crystals nucleate and grow is still poorly understood. To investigate these processes in more detail, we chose scleractinian corals as a model system, because they are well known to form their skeletons from aragonite (CaCO3) spherulites, and because a comparative study of crystal structures across coral species has not been performed previously. We observed that all 12 diverse coral species analyzed here exhibit plumose spherulites in their skeletons, with well-defined centers of calcification (CoCs), and crystalline fibers radiating from them. In 7 of the 12 species, we observed a skeletal structural motif not observed previously: randomly oriented, equant crystals, which we termed "sprinkles". In Acropora pharaonis, these sprinkles are localized at the CoCs, while in 6 other species, sprinkles are either layered at the growth front (GF) of the spherulites, or randomly distributed. At the nano- and micro-scale, coral skeletons fill space as much as single crystals of aragonite. Based on these observations, we tentatively propose a spherulite formation mechanism in which growth front nucleation (GFN) of randomly oriented sprinkles, competition for space, and coarsening produce spherulites, rather than the previously assumed slightly misoriented nucleations termed "non-crystallographic branching". Phase-field simulations support this mechanism, and, using a minimal set of thermodynamic parameters, are able to reproduce all of the microstructural variation observed experimentally in all of the investigated coral skeletons. Beyond coral skeletons, other spherulitic systems, from aspirin to semicrystalline polymers and chocolate, may also form according to the mechanism for spherulite formation proposed here. STATEMENT OF SIGNIFICANCE: Understanding the fundamental mechanisms of spherulite nucleation and growth has broad ranging applications in the fields of metallurgy, polymers, food science, and pharmaceutical production. Using the skeletons of reef-building corals as a model system for investigating these processes, we propose a new spherulite growth mechanism that can not only explain the micro-structural diversity observed in distantly related coral species, but may point to a universal growth mechanism in a wide range of biologically and technologically relevant spherulitic materials systems.

Reef-building corals and their aragonite (CaCO3) skeletons support entire reef ecosystems, yet their formation mechanism is poorly understood. Here we used synchrotron spectromicroscopy to observe the nanoscale mineralogy of fresh, forming skeletons from six species spanning all reef-forming coral morphologies: Branching, encrusting, massive, and table. In all species, hydrated and anhydrous amorphous calcium carbonate nanoparticles were precursors for skeletal growth, as previously observed in a single species. The amorphous precursors here were observed in tissue, between tissue and skeleton, and at growth fronts of the skeleton, within a low-density nano- or microporous layer varying in thickness from 7 to 20 µm. Brunauer-Emmett-Teller measurements, however, indicated that the mature skeletons at the microscale were space-filling, comparable to single crystals of geologic aragonite. Nanoparticles alone can never fill space completely, thus ion-by-ion filling must be invoked to fill interstitial pores. Such ion-by-ion diffusion and attachment may occur from the supersaturated calcifying fluid known to exist in corals, or from a dense liquid precursor, observed in synthetic systems but never in biogenic ones. Concomitant particle attachment and ion-by-ion filling was previously observed in synthetic calcite rhombohedra, but never in aragonite pseudohexagonal prisms, synthetic or biogenic, as observed here. Models for biomineral growth, isotope incorporation, and coral skeletons' resilience to ocean warming and acidification must take into account the dual formation mechanism, including particle attachment and ion-by-ion space filling.

Corals and other biomineralizing organisms use proteins and other molecules to form different crystalline polymorphs and biomineral structures. In corals, it's been suggested that proteins such as Coral Acid Rich Proteins (CARPs) play a major role in the polymorph selection of their calcium carbonate (CaCO3) aragonite exoskeleton. To date, four CARPs (1-4) have been characterized: each with a different amino acid composition and different temporal and spatial expression patterns during coral developmental stages. Interestingly, CARP3 is able to alter crystallization pathways in vitro, yet its function in this process remains enigmatic. To better understand the CARP3 function, we performed two independent in vitro CaCO3 polymorph selection experiments using purified recombinant CARP3 at different concentrations and at low or zero Mg2+ concentration. Our results show that, in the absence of Mg2+, CARP3 selects for the vaterite polymorph and inhibits calcite. However, in the presence of a low concentration of Mg2+ and CARP3 both Mg-calcite and vaterite are formed, with the relative amount of Mg-calcite increasing with CARP3 concentration. In all conditions, CARP3 did not select for the aragonite polymorph, which is the polymorph associated to CARP3 in vivo, even in the presence of Mg2+ (Mg:Ca molar ratio equal to 1). These results further emphasize the importance of Mg:Ca molar ratios similar to that in seawater (Mg:Ca equal to 5) and the activity of the biological system in a aragonite polymorph selection in coral skeleton formation.

Do corals form their skeletons by precipitation from solution or by attachment of amorphous precursor particles as observed in other minerals and biominerals? The classical model assumes precipitation in contrast with observed "vital effects," that is, deviations from elemental and isotopic compositions at thermodynamic equilibrium. Here, we show direct spectromicroscopy evidence in Stylophora pistillata corals that two amorphous precursors exist, one hydrated and one anhydrous amorphous calcium carbonate (ACC); that these are formed in the tissue as 400-nm particles; and that they attach to the surface of coral skeletons, remain amorphous for hours, and finally, crystallize into aragonite (CaCO3). We show in both coral and synthetic aragonite spherulites that crystal growth by attachment of ACC particles is more than 100 times faster than ion-by-ion growth from solution. Fast growth provides a distinct physiological advantage to corals in the rigors of the reef, a crowded and fiercely competitive ecosystem. Corals are affected by warming-induced bleaching and postmortem dissolution, but the finding here that ACC particles are formed inside tissue may make coral skeleton formation less susceptible to ocean acidification than previously assumed. If this is how other corals form their skeletons, perhaps this is how a few corals survived past CO2 increases, such as the Paleocene-Eocene Thermal Maximum that occurred 56 Mya.

Coral skeletons were long assumed to have a spherulitic structure, that is, a radial distribution of acicular aragonite (CaCO3) crystals with their c-axes radiating from series of points, termed centers of calcification (CoCs). This assumption was based on morphology alone, not on crystallography. Here we measure the orientation of crystals and nanocrystals and confirm that corals grow their skeletons in bundles of aragonite crystals, with their c-axes and long axes oriented radially and at an angle from the CoCs, thus precisely as expected for feather-like or "plumose" spherulites. Furthermore, we find that in both synthetic and coral aragonite spherulites at the nanoscale adjacent crystals have similar but not identical orientations, thus demonstrating by direct observation that even at nanoscale the mechanism of spherulite formation is non-crystallographic branching (NCB), as predicted by theory. Finally, synthetic aragonite spherulites and coral skeletons have similar angle spreads, and angular distances of adjacent crystals, further confirming that coral skeletons are spherulites. This is important because aragonite grows anisotropically, 10 times faster along the c-axis than along the a-axis direction, and spherulites fill space with crystals growing almost exclusively along the c-axis, thus they can fill space faster than any other aragonite growth geometry, and create isotropic materials from anisotropic crystals. Greater space filling rate and isotropic mechanical behavior are key to the skeleton's supporting function and therefore to its evolutionary success. In this sense, spherulitic growth is Nature's 3D printing.

Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years.

Ocean acidification caused by shifts in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations is threatening many calcifying organisms, including corals. Here we assessed autotrophy vs heterotrophy shifts in the Mediterranean zooxanthellate scleractinian coral Balanophyllia europaea acclimatized to low pH/high pCO2 conditions at a CO2 vent off Panarea Island (Italy). Dinoflagellate endosymbiont densities were higher at lowest pH Sites where changes in the distribution of distinct haplotypes of a host-specific symbiont species, Philozoon balanophyllum, were observed. An increase in symbiont C/N ratios was observed at low pH, likely as a result of increased C fixation by higher symbiont cell densities. δ13C values of the symbionts and host tissue reached similar values at the lowest pH Site, suggesting an increased influence of autotrophy with increasing acidification. Host tissue δ15N values of 0‰ strongly suggest that diazotroph N2 fixation is occurring within the coral tissue/mucus at the low pH Sites, likely explaining the decrease in host tissue C/N ratios with acidification. Overall, our findings show an acclimatization of this coral-dinoflagellate mutualism through trophic adjustment and symbiont haplotype differences with increasing acidification, highlighting that some corals are capable of acclimatizing to ocean acidification predicted under end-of-century scenarios. Low seawater pH impacts trophic dynamics in corals, increasing symbiont density and autotrophic/heterotrophic ratios and varying dinoflagellate haplotypes under ocean acidification. [ABSTRACT FROM AUTHOR]