Amorphous calcium carbonate particles form coral skeletons

Abstract

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.

Keywords: PEEM; calcification crisis; mesocrystal; ocean acidification; vital effects.

Fig. 1.

Component spectra and component maps of tissue and fresh, forming coral skeleton in a spine that appears separate from the rest of the skeleton, as it is curved and incompletely exposed in this polished surface. These data were acquired 25 h postmortem. (A) The reference Ca spectra used as components in all component maps in this work. (B) Component map of forming coral at the tissue–skeleton interface in S. pistillata. In a component map, colors indicate mineral phases, as distinguished by their spectra in A, and are correspondingly colored. In A–E, blue pixels are mostly or completely aragonite; ACC-H₂O, ACC, and pAra are displayed in red, green, and yellow, respectively. (C–E) Magnified component maps from the correspondingly labeled boxes in B. C and D show three particles (arrows) surrounded by tissue, which clearly contain more ACC-H₂O, ACC, and pAra pixels than any other region in this work. In the forming coral skeleton, instead, there are only ACC-H₂O and aragonite, very little ACC, and no pAra at all, as shown in E. The spectra in A are normalized to have the same area under the curve between 345 and 355 eV. They are vertically displaced here for clarity, not during component mapping. The maps in B–E were obtained from a stack of PEEM images, fitting every pixel with a linear combination of the four component spectra in A and displaying in each pixel the proportion of each spectrum that best fits the spectrum of that pixel in red, green, blue, and yellow colors. Since yellow never occurs as a mixture of red and green, displaying pAra as yellow is unambiguous (Figs. S8 and S9). Important additional data for this region are in Figs. S1–S3, S8, and S9, and repeat component maps are in Fig. S3. Quantitative analysis for the abundance of each mineral phase is presented in Table 1.

Fig. 2.

CoCs in a more mature part of the coral skeleton. (A) Visible light microscopy image obtained with crossed polarizers. In A and B, the magenta boxes indicate the region magnified in C and D. (B) Zoomed in cross-polarizer micrograph showing crystals that radiate out of the CoCs. (C) Zoomed in component map showing abundant ACC-H₂O, ACC, and pAra in the CoCs, indicated by the white arrows. The repeat component map for this region is in Fig. S3. (D) PIC map of the same CoCs shown in B, with white arrows in precisely the same positions. Notice that, where the amorphous precursors are localized in C, this PIC map shows no crystallinity; that is, no polarization dependence, displayed as black pixels.

Fig. 3.

Amorphous precursor phases in a forming spine in a 2-wk-old spat (that is, a coral larva right after it settled and started forming a skeleton). These data were acquired 28 h postmortem. ACC-H₂O is abundant, ACC is sparse, and pAra is absent from all skeleton regions (spat here, adult in Figs. 1 and 4), except for the CoCs (Fig. 2). (A and B) Cross-polarizer micrographs of a spat spine, with magenta arrows and boxes indicating the area magnified in C and D. (C) Component map obtained with the same four reference spectra as in all other data in this work, although here, we omitted the pAra label to stress that we found no pAra. C, Inset shows a zoomed in map for the white box region. (D) Average of 121 stacked PEEM images (termed movie), from which the component map in C was obtained. The repeat component map for this region is in Fig. S3, and quantitative phase analysis is in Table 1.

Fig. 4.

Amorphous precursors in a growing coral septum. These data were acquired 31 h postmortem. (A and B) Cross-polarizer micrographs, with magenta arrows and boxes showing the region magnified in C and D. (A) The whole coral branch, termed nubbin. (B) A flower-shaped holey structure termed corallite, with four fully formed and two forming septa. (C) Component map of a forming septum, showing that, in this adult coral, the forming skeleton has a lot of ACC-H₂O, very little ACC, and almost no pAra. (D) PIC map showing aragonite crystals in the septum, distinguished by different colors, which quantitatively identify their crystal orientations. By the time that the PIC map was acquired (32 h postmortem), all of the amorphous precursor phases had already crystallized to aragonite because of time delay and radiation damage. The repeat component map for this region, acquired immediately after the first and before PIC mapping, is in Fig. S3, and quantitative phase analysis is in Table 1.

Fig. S1.

Fresh, forming coral at the tissue–skeleton interface. (A) Cross-polarizer micrograph showing mature coral, corallite holes, and the growing coral front. (B) Zoomed in micrograph where the coral skeleton and the calicoblastic tissue (green arrows) depositing the mineral are visible. Magenta arrows and boxes in A and B indicate the area magnified in C–E. (C) Average of 121 PEEM images acquired across the Ca L₂₃ edge. The boxes indicate the areas magnified in Fig. 1 C–E. (D) Component map identical to the one presented in Fig. 1B using the component spectra in Fig. 1A. The boxes indicate the regions magnified in Fig. 1 C–E. (E) PIC map of the same region in D, indicating different crystal orientations with different colors. Noncrystalline pixels are black. Additional images for this region are in Fig. S2, and the repeat component map is in Fig. S3.

Fig. S2.

The coral tissue–skeleton interface is analyzed in detail in Fig. 1 and Fig. S1. (A) Average of all PEEM images acquired across the Ca L₂₃ edge. (B) Cross-polarizer micrograph showing the spine, the tissue immediately adjacent to it, ectoderm and endoderm, and the symbiont zooxanthellae.

Fig. S3.

Component maps from repeat movies for the areas in Figs. 1–4. A, C, E, and G are first movies; B, D, F, and H are second movies. Insets show small portions of the same maps at higher magnification, such that individual pixels are visible. Comparing A with B, some ACC-H₂O transformed into ACC, and some transformed into aragonite. The same occurs when comparing E with F or comparing G with H. No appreciable change is observed between C and D, which was acquired in mature coral skeleton.

Fig. S4.

Component spectra normalized by subtracting from each entire spectrum the intensity at the top of the peak at 352.6 eV (labeled “1”); therefore, all spectra coincide at the top of peak 1. This shows that peak 1 is sharpest and most intense for the most crystalline mineral, aragonite, and shortest and broadest for ACC-H₂O. Thus, peak 1 intensity is another reliable indicator of crystallinity or lack thereof. Peak 2 in ACC is similar to peak 2 in calcite, not aragonite (23). The three little peaks around 348 eV (peak 4 region) are characteristic of aragonite, and are clearly present in the pAra spectrum, whereas peak 2 around 351.5 eV in pAra is similar to peak 2 in ACC, and peak 1 is less intense than in aragonite but more than in ACC. These three observations concur to indicate that pAra is a poorly crystalline aragonite phase.

Fig. 5.

(A) Confocal micrograph of a living spat. Calcein fluorescence is displayed in blue; native green fluorescent protein (GFP) and chlorophyll are in green and red, respectively. (B) Map of the same spat, where regions of FM and LT are filled with blue and green, respectively. Notice that they are interspersed, with LT in FM and FM in LT. (C) Differential interference contrast micrograph acquired with crossed polarizers, showing a similar region of the same spat, where a basal plate (BP), a growing septum (S), and a layer of stinging cells (SC) are better recognizable, and therefore, they could be placed in B. (D) Outlines of all 2,726 blue-stained particles analyzed, which vary in size between 0.4 and 9.4 µm.

Fig. 6.

PIC maps showing nanoparticles with different orientations (A) at and around CoCs in an S. pistillata coral skeleton and (B) in synthetic aragonite spherulite grown via particle attachment. Insets show lower magnification PIC maps of the same regions in A and B. Crack bridging in B provides additional evidence that the spherulite is formed by particles. Fig. S5 has details on particle sizes in A and B.

Fig. S5.

PIC maps of the same regions as in Fig. 6. White squares numbered 1–8 indicate similarly sized particles in all panels. Each particle is identified by a square (above) and a number (below) and is precisely between the square and the number, except for particle 7 in D, which is below the square and left of the number 7. The white squares are 400 nm in A and B and 100 nm in C and D. (Zoom in on the online figure if the particles or the squares are not visible to you.) (A) Mature coral skeleton region at the CoCs. Notice the eight labeled and many other 400-nm particles. (B) The same region of coral at lower magnification. Away from the CoCs, domains of identical crystal orientation (color) are larger, but their edges are jagged at the 400-nm scale, as shown by the eight labeled and many other 400-nm edge wiggles. This is consistent with attachment of 400-nm particles. (C and D) The smallest particles identifiable in coral (C) and synthetic aragonite spherulite (D) are labeled 1–8, with 100-nm squares. Notice that this is not the particle size, because PIC mapping is surface-sensitive [∼5 nm at the oxygen K edge where the PIC maps are acquired (96, 97)], and both coral skeleton and synthetic spherulites were polished surfaces; thus, these may have been larger particles with their center above or below the imaged plane. The precise sizes of the particles in C and D were analyzed by measuring the FWHM across the eight labeled particles. These were 143 ± 52-nm particles in coral skeleton and 72 ± 26 nm in synthetic spherulite (mean ± SD). These are in agreement with previous observations of ∼100-nm particles in coral skeletons (20, 44) but not with those observed in LT or with the most frequent 400-nm particles observed in these same PIC maps; thus, we deduce that these particles appear smaller, because their centers were away from the polished and imaged plane.

Fig. S6.

SEM images of cryofractured synthetic aragonite, biogenic aragonite from coral, and geologic aragonite. (A–D) Micrographs at increasing magnifications show a synthetic spherulite grown by particle attachment using the ammonium carbonate diffusion method and an Mg:Ca ratio of 5:1. Particles ∼100 nm in diameter are visible along with larger structures. (E–H) Micrographs of cryofractured fibers in an S. pistillata coral skeleton. Various regions imaged at high magnification exhibit nanoparticles at the tips of fibers (E–G), at fractured fiber cross-sections (E and G), and at the sides of the fibers (F–H). Particle with ∼100-nm size are visible, but so are other structures at the ∼400-nm scale (E) and even greater than a micron (F); thus, as expected, SEM alone cannot provide evidence of particle sizes. Insets in E–H show the same regions at lower magnification. (I–L) Micrographs of cryofractured geologic aragonite at increasing magnifications. These micrographs are representative of 30 other regions also acquired from geologic aragonite samples, cryofractured in liquid nitrogen, and coated with 20 nm Pt. They all show flat surface and straight edges resulting from cryofracturing; they never show nanoparticles either 100 or 400 nm in size, as do synthetic aragonite spherulites (A–D) and coral skeletons (E–H).

Fig. 7.

Model for the formation of coral skeleton or other calcium carbonate marine biomineral. Divalent cations, mostly Ca²⁺ but also, Mg²⁺, and Sr²⁺ ions are represented as blue dots at all stages of biomineral crystal formation, which takes place in five steps (i–v) as described in the text. The carbonate ions –CO₃²⁻, which originate from within the LT, are represented by green dots here. LT separates seawater from the growing biomineral; thus, as long as the animal is alive, the biomineral is not exposed to seawater and will not dissolve, even at low pH. Abiotic overgrowth is also excluded. Seawater is endocytosed (in the upper left) into vesicles and enriched in carbonate ions, and therefore, ACC-H₂O and anhydrous ACC precursors are precipitated and stabilized in these vesicles. The vesicles are transported to the side of the tissue in immediate contact with the growing biomineral surface, and their ACC content is exocytosed there. After a day or so, all of the ACC transforms into CCC. CCC is aragonite in coral or nacre and calcite in foraminifera or sea urchin spines, spicules, or teeth.

Fig. S7.

Energy landscapes in the tissue and the CoCs (A) and in the coral skeleton (B). These phases are progressively more stable and thus, energetically downhill (32, 33). (A) In the tissue, both barriers are high; thus, both ACC-H₂O and ACC are long-lived. (B) In the coral skeleton, the first is high, and the second low; thus, once deposited in the skeleton, ACC-H₂O is long-lived, and ACC is short-lived. This is deduced from the fact that in the skeleton we find many red pixels (R), many blue pixels (B), and mostly magenta (M = R + B) where the two coexist. In the skeleton we infrequently find green pixels, hence ACC is short-lived, and the activation barrier between ACC and aragonite must be lower than that between ACC-H₂O and ACC.

Fig. S8.

Comparison of RGB and RGBY maps from details of the regions in Figs. 1 and 2, the only ones that contain any pAra phase. Both maps were obtained analyzing each pixel with a linear combination of all four components, but in RGB and RGBY maps, only three or all four of them are displayed, respectively. Notice that, in the RGB maps, there are no Y pixels (Upper) or nearly no Y pixels (Lower), which if present, would result from R + G = Y. Thus, the presence of Y pixels in the RGBY map can unambiguously be interpreted as pAra.

Fig. S9.

Column 1 shows the RGBY maps for each of Figs. 1–4, and columns 2–5 show the separate R, G, B, and Y, respectively. Each of the separate maps shows the spatial distribution of R, G, B, and Y pixels on a grayscale from 0 to 255, corresponding to black (0), and pure R, G, B, or Y to white (255), respectively. Table 1 shows the numeric data on these maps.