Gilbert
et al
.,
Sci. Adv.
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, eabl9653 (2022) 9 March 2022
SCIENCE ADVANCES
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BIOPHYSICS
Biomineralization: Integrating mechanism
and evolutionary history
Pupa U.
P. A.
Gilbert
1,2
*†, Kristin D.
Bergmann
3
, Nicholas
Boekelheide
3
, Sylvie
Tambutté
4
,
Tali Mass
5
, Frédéric
Marin
6
, Jess F.
Adkins
7
, Jonathan
Erez
8
, Benjamin
Gilbert
9,10
,
Vanessa Knutson
11
, Marjorie
Cantine
3,12
, Javier Ortega
Hernández
11
, Andrew H.
Knoll
11
*
Calcium carbonate (CaCO
3
) 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 re-
sponses 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 CaCO
3
skeleton formation
mechanisms with constraints from evolutionary history, omics, and a meta-analysis of isotopic data to develop a
plausible model for CaCO
3
biomineralization applicable to all phyla. The model provides a framework for under-
standing 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 CaCO
3
biomineralizing organisms.
INTRODUCTION
Minerals made by organisms are called biominerals (
1
), and their
formation mechanisms are collectively termed biomineralization
(
2
–
4
). A major innovation in the history of life, biomineralization
transformed the functional biology, evolutionary trajectory, and
biogeochemical impact of numerous clades among animals, plants,
and protists. While predation likely played a major role in the evo-
lution of biomineralized structures, a variety of other functions accrued,
including locomotion, buoyancy, grinding, reproduction, and de-
tection of gravity, magnetic fields, or light. Biominerals may even
have multiple functions at once, such as calcium carbonate (CaCO
3
)
armors that also serve as lenses in chitons (
5
), microbial shields in
ants (
6
), or detoxification in most phyla (
7
). The establishment of
genetic recipes for complex functional biominerals from the same
basic ingredients is a remarkable product of evolution.
Ever since CaCO
3
biomineralization became widespread, during
the Cambrian (
8
,
9
) and Ordovician (
10
) radiations of marine ani-
mals and algae, it has played a major role in the carbon cycle (
11
),
affecting and being affected by the ambient environment on geologic
time scales (
12
,
13
). Because of their persistence in the fossil record,
biominerals in general, and CaCO
3
biominerals in particular, provide
a major archive of the evolutionary history of life and environments
on Earth. Looking forward, CaCO
3
biomineralization is challenged,
for some phyla more than others, by 21st century global change.
Phylogenetic evidence (Fig. 1) shows that biominerals appeared
in the fossil record long after the different phyla had diverged from
one another (
14
). Because the biomineralizing organisms in various
phyla do not have a common ancestor that was itself biomineraliz-
ing, they must have evolved strategies to form carbonate biominer-
als independently (
15
). These strategies are remarkably similar in
ingredients and recipes across phyla; therefore, they evolved con-
vergently (
15
).
A mechanistic understanding of how organisms make CaCO
3
biominerals is essential for understanding the evolutionary record
of CaCO
3
biomineralization, elucidating its consequences for Earth
surface environments, and clarifying the sensitivity of CaCO
3
bio-
mineralizers to both past and future environmental changes (
16
–
22
).
Here, we attempt to unify all known CaCO
3
biomineralization
mechanisms into a single model applicable to all phyla, emphasizing
the commonalities across diverse and divergent species. We show
that insights on CaCO
3
skeleton formation pathway (
15
,
23
–
26
),
trends and constraints from evolutionary history (
27
), genetics,
transcriptomics, proteomics, and isotope geochemistry (
9
) all point
toward a simple but powerful plausible conceptual model for CaCO
3
biomineralization.
HOW DO CALCIUM CARBONATE BIOMINERALS FORM?
Carbonate biominerals grow from a chemically complex aqueous
solution termed calcifying fluid (CF; see Table 1 for this and all other
acronyms used throughout the text) in a biologically controlled
privileged space that is bounded by a phospholipid membrane
within cells and by epithelial cells in multicellular organisms. Bio-
logical processes modify the chemistry of the CF, including pH and
the concentrations of calcium and carbonate ions, to increase the
thermodynamic driving force for precipitation of CaCO
3
, quanti-
fied by the saturation state of the solution,
, relative to the final
mineral phase. Since the Ca concentration of seawater is ~10 mM and
CO
3
2−
concentration is ~0.1 to 0.3 mM, calcifiers need to concentrate
1
Departments of Physics, Chemistry, Geoscience, and Materials Science, University
of Wisconsin-Madison, Madison, WI 53706, USA.
2
Chemical Sciences Division, Lawrence
Berkeley National Laboratory, Berkeley, CA 94720, USA.
3
Department of Earth, At-
mospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge,
MA 02139, USA.
4
Centre Scientifique de Monaco, Department of Marine Biology,
98000 Monaco, Principality of Monaco.
5
University of Haifa, Marine Biology Depart-
ment, Mt. Carmel, Haifa 31905, Israel.
6
Université de Bourgogne–Franche-Comté
(UBFC), Laboratoire Biogéosciences, UMR CNRS 6282, Bâtiment des Sciences Gabriel,
21000 Dijon, France.
7
Geological and Planetary Sciences, California Institute of
Technology, MS 100-23, Pasadena, CA 91125, USA.
8
The Hebrew University of Jerusalem,
Institute of Earth Sciences, Jerusalem 91904, Israel.
9
Energy Geoscience Division,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
10
Department of
Earth and Planetary Science, University of California, Berkeley, Berkeley, CA 94720, USA.
11
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge,
MA 02138, USA.
12
Goethe-Universität Frankfurt, 60438 Frankfurt am Main, Germany.
*Corresponding author. Email: pupa@physics.wisc.edu (P.U.P.A.G.); aknoll@oeb.
harvard.edu (A.H.K.)
†Previously publishing as Gelsomina De Stasio.
Copyright © 2022
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).
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Gilbert
et al
.,
Sci. Adv.
8
, eabl9653 (2022) 9 March 2022
MS no: RVabl9653/AD/BIOPHYSICS, PALEONTOLOGY
SCIENCE ADVANCES
|
REVIEW
2 of 16
dissolved inorganic carbon (DIC) and elevate the pH to achieve
higher CO
3
2−
concentration and therefore higher
values. The or-
ganism also regulates the concentration and speciation of inorganic
ions, polysaccharides, and proteins, all of which can select the min-
eral polymorph in the final biomineral (calcite, aragonite, or vater-
ite) and can vary activation barriers (and hence rates) for particle
formation, phase transitions, and growth.
Nanoparticles of amorphous calcium carbonate (ACC) are a
precursor phase for CaCO
3
biominerals formed by diverse organ-
isms (
15
), including species from clades that diverged long before
evolving calcification (Fig. 2). Abiotic studies have shown ACC pre-
cursors to be a common intermediate in the nucleation of CaCO
3
minerals in solution and on surfaces because this phase nucleates
preferentially at a lower
value compared to crystalline poly-
morphs (
26
). Although the molecular pathways for the formation of
a final crystalline carbonate via ACC are not fully established, calo-
rimetry studies have clarified that the pathway is thermodynamical-
ly downhill (
28
). Crystalline vaterite is another transient phase, thus
far observed only during test formation in foraminiferans (
29
). Fol-
lowing the formation of a solid-phase carbonate mineral (whether
amorphous or crystalline), growth by ion attachment (IA) has far
smaller activation barriers than further nucleation. These observations
in natural biominerals and synthetic systems suggest that CaCO
3
biominerals form through a combination of ACC particle attach-
ment (PA) and IA processes at the growth surface.
Extracellular and
intracellular privileged spaces
All marine biominerals are formed within a biologically controlled
compartment first termed “privileged space” in studies of sea urchin
embryos (
30
). In multicellular organisms, the privileged space in
which the final biomineral (e.g., skeleton) grows is bounded by a
monolayer of specialized epithelial cells and contains extracellular
CF (ECF). In corals, the ECF, also known as extracellular calcifying
medium (ECM), lies between the calcifying cells and the growing
Fig. 1. Phylogenetic distribution of CaCO
3
biomineralization in animals.
Besides
animals (shown), there are species (not shown) that make CaCO
3
skeletons in the
foraminiferans, coccolithophorids, green algae, red algae, dinoflagellates, and even
a few amoebozoans and brown algae. CaCO
3
skeleton–forming animals are shown
in turquoise font; dark gray font indicates clades that form nonskeletal CaCO
3
bio-
minerals, and light gray font indicates those that do not form CaCO
3
at all. Skeletons
confirmed to be formed in part by particle attachment (PA) are underlined, and the
age of the oldest unambiguous fossil found to date is in blue font. All fossil age
estimates have an uncertainty of a few million years. For hemichordates, biominer-
alized fossils have not yet been identified. Data are from (
15
,
215
,
242
–
246
). All
clades started biomineralizing after they diverged from one another.
Table 1. Acronyms used throughout the text.
CaCO
3
Calcium carbonate, which comprises any of the
anhydrous polymorphs of CaCO
3
: calcite,
aragonite, or vaterite
PA
Particle attachment
IA
Ion attachment
CF
Calcifying fluid
ICF
Intracellular calcifying fluid. Its existence is deduced
but not yet observed.
ECF
Extracellular calcifying fluid, well documented in a
variety of organisms
ECM
Extracellular calcifying medium. The ECF is termed
ECM by other authors. The two are identical and
interchangeable. We chose ECF because “fluid”
includes liquid, dense liquid, and flowing gel
phases. ECF cannot be confused with extracellular
matrix, often termed ECM.
DIC
Dissolved inorganic carbon
Saturation state of a solution. Minerals precipitate
and grow in supersaturated solutions (
> 1).
ACC
Amorphous calcium carbonate
ACC-H
2
O
Amorphous calcium carbonate, hydrated
CCC
Crystalline calcium carbonate, which could be any
of the anhydrous polymorphs of CaCO
3
, calcite,
aragonite, or vaterite, since all three exist as final
biominerals.
CO
2
Carbon dioxide
HCO
3
−
Bicarbonate ion
CO
2
(aq)
Aqueous, dissolved carbon dioxide
CA
Carbonic anhydrase, an enzyme that rapidly
catalyzes the conversion of CO
2
to bicarbonate ions
(HCO
3
−
). CA is a membrane protein, which can also
occur intra- and extracellularly and thus can be in
the cytosol and presumably in the ECF and ICF.
CA
or CA-like proteins were shown to be an integral
part of the skeletal matrix.
CoCs
Centers of calcification in coral skeletons; these are
the regions of the skeleton from which all acicular
crystalline fibers radiate to form plumose
spherulites and are most obviously nanoparticulate.
GRNs
Gene regulatory networks
[Ca],
pH,
DIC,
and
CaCO3
Difference observed when comparing the CF with
seawater, in calcium concentration, pH, DIC, and
supersaturation with respect to relevant CaCO
3
mineral
EGF
Epidermal growth factor
VEGF
Vascular endothelial growth factor
LCDs
Low-complexity domains (also called
compositionally biased regions) observed in
proteins and characterized by the dominance of
one or two amino acids. In addition, several LCDs
may exhibit tandem repeats.
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, eabl9653 (2022) 9 March 2022
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skeleton (
31
); in mollusks, the ECF is the extrapallial fluid in a com-
partment between the mantle and the growing shell (
32
); in sea
urchin embryos, the ECF is a spicule-shaped compartment termed
syncytial envelope in which a calcite spicule grows (
33
,
34
). In fora-
miniferans, which are single-celled amoeboid protists, the test forms
from the ECF within a specialized membrane-bound compartment,
formed by endocytosis of seawater (
35
,
36
). Coccolithophorids, single-
celled algae that are major contributors of calcium carbonate to the
deep seafloor, also have specialized membrane-bound compartments,
likely derived from preexisting organelles such as the Golgi apparatus
(
18
), that provide the privileged space in which each mineralized
coccolith grows (
37
,
38
).
It is well established that intracellular vesicles play key roles in
concentrating the ions required for biomineralization, in transporting
them to the ECF, and likely in mineral deposition. The existence of
intracellular reservoirs of DIC used for skeleton deposition, termed
“DIC pool,” was first proposed on the basis of the comparison of
14
C and
45
Ca uptake kinetics (
35
,
39
–
42
). Subsequent imaging and
microchemical studies have revealed the intracellular vesicles and
vacuoles providing DIC pools in numerous organisms. For exam-
ple, in foraminifera, vacuoles contain chemically modified seawater
(
35
,
36
), as well as smaller Mg- and Ca-rich vesicles (
43
). Elevated
Ca in intracellular vesicles is observed in coral cells (
44
), sea urchin
embryos (
45
–
47
) and spines (
48
), and coccolithophorids (
49
).
Vesicles can also be locations where initial biomineral-forming
ACC nanoparticles are nucleated, as first hypothesized by Cohen
and McConnaughey (
50
) for the case of coral skeleton formation.
Now, evidence for such intracellular precursor nanoparticles is over-
whelming in the tissue adjacent to the forming surface of coral skel-
etons (
25
,
51
–
53
), in sea urchin embryonic cells forming spicules
(
47
), and in the tissue regenerating adult sea urchin spines (
48
). Cells
extracted from coral polyps cannot form a tissue-bounded privileged
space with an ECF, yet they are able to form carbonate crystals, likely
explained by calcification in intracellular vesicles (
54
). Thus, some,
Fig. 2. The same amorphous precursors across phyla: Cnidarians, mollusks, and echinoderms.
(
A
)
S. pistillata
coral in the Red Sea (photo credit: T.M.). (
B
) California
red abalone
Haliotis rufescens
(photo credit: P.U.P.A.G.). (
C
) California purple sea urchin
Strongylocentrotus purpuratus
(photo credit: P.U.P.A.G.). (
D
,
G
, and
J
) X-ray absorp-
tion spectra from nanoscale regions of fresh forming biominerals:
S. pistillata
skeleton,
H. rufescens
nacre, and
S. purpuratus
embryonic spicules. Three distinct spectral line
shapes at the Ca L-edge, and thus, three distinct mineral phases or “components” occur in each biomineral: hydrated ACC, anhydrous ACC, and crystalline calcite or aragonite.
(
E
,
H
, and
K
) Component maps showing abundant amorphous pixels in the forming parts of each biomineral and submicrometer amorphous particles in nearby cells.
(
F
,
I
, and
L
) Color legend for both component spectra (D, G, and J) and component maps (E, H, and K). (
M
to
P
) Scanning electron micrographs showing that modern and
fossil biominerals show nanoparticulate texture after cryofracturing (M to P), whereas nonbiogenic minerals do not (Q). Insets in (M) to (Q) show photographs of each sample.
(M and N) Modern aragonite biominerals: coral skeleton from
S. pistillata
(M) and nacre from
H. rufescens
(N). (O) Calcite sea urchin spine from
S. purpuratus
. (P) Phosphatized
Ediacaran
Cloudina
(550 Ma before present) from Lijiagou, China. (
Q
) Nonbiogenic aragonite from Sefrou, Morocco. Data are from (
15
,
23
–
25
). arb. u., arbitrary units.
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possibly all, biomineralizing organisms use vesicles containing in-
tracellular CF (ICF) to initiate CaCO
3
precipitation.
Privileged space chemistry is under biological control
Marine biominerals form from seawater, but they do not form in
seawater. The composition of the CF in the privileged space (ICF
and ECF) is seawater modified chemically and isotopically by active
biological control. Microelectrode studies of corals (
31
,
55
,
56
) have
provided the most compelling and direct characterization of the
chemical conditions of the privileged space. Sevilgen
et al.
(
31
) demon
-
strated that pH and CO
3
2−
and Ca concentrations in the ECF of
Stylophora pistillata
corals are all elevated relative to surround-
ing water, enabling the saturation state with respect to aragonite
(
aragonite
~12) to be calculated. Further evidence for biological con-
trol comes from measurements of boron isotopes in CaCO
3
that are
sensitive to the fluid pH during carbonate formation. Tropical, tem-
perate, and cold-water coral species maintain an approximately
constant pH difference between seawater and ECF (
57
,
58
). The
capability of maintaining elevated pH in the CF at the sites of calci-
fication in artificially acidified waters was recently demonstrated
across a range of phyla (
59
). Studies of single-celled organisms are
more challenging, but Taylor
et al.
(
18
) used electrophysiological
and genetic approaches to demonstrate that coccolithophorids ele-
vate coccolith vesicle pH using voltage-gated membrane H
+
chan-
nels. Ter Kuile
et al.
(
39
,
40
) and Bentov
et al.
(
35
) showed that
foraminifera ECF has greater DIC and pH with respect to seawater.
Higher DIC and pH produce higher supersaturation with respect to
calcite
calcite
≈ 30 to 40 in foraminifera ECF.
Kahil
et al.
(
47
) iso-
lated spicule-forming single cells from sea urchin embryos and demon
-
strated that each cell contains hundreds of intracellular vesicles
and that the ICF in these vesicles is much richer in Ca compared
to seawater.
Privileged spaces are never completely isolated from
seawater
Marine biomineralizers may use a combination of active and pas-
sive mechanisms, transcellular and paracellular pathways, to control
the chemistry of the privileged space. Active mechanisms include
solute passage through transmembrane transporters and fluid trans-
port by pinocytosis, as observed in foraminiferans (
35
,
36
) and corals
(
60
). Passive mechanisms include water passage through aquaporins,
CO
2
diffusion through membranes, and paracellular transport between
cells. Regardless of the mechanism, the maintenance of a thermody-
namic gradient between seawater and the privileged spaces requires
the expenditure of energy by cells. A notable example of privileged
space partly open to seawater was found in coral, where junctions be-
tween epithelial cells enable passive diffusion of ions and molecules
selected by charge and size but always smaller than approximately
20 nm in adult corals (
61
) or larger in primary coral polyps (
52
). The
degree to which the privileged space is open to the environment affects
the control of chemistry (
62
) and likely affects the susceptibility of
marine biomineralizers to changes in seawater conditions.
CaCO
3
biomineral growth by amorphous particle and
ion attachment
Many previous models for biomineral formation described two con-
trasting mechanisms: IA from a CF (
63
–
65
) or PA of ACC precursors
from intracellular vesicles (
15
,
25
,
26
,
45
,
47
,
48
,
53
,
66
). IA is unavoid-
able in the presence of a CF that is rich in ions and supersaturated
with respect to the final biomineral (
31
,
64
,
67
,
68
), yet evidence for
PA of ACC precursors is abundant (
15
,
25
,
26
,
45
,
47
,
48
,
53
,
66
,
69
).
Here, we propose a mechanistic model that includes both PA and IA.
ACC precursors to
CaCO
3
biominerals
In 1997, Beniash
et al.
(
70
) demonstrated that embryonic sea urchin
spicules form via an ACC precursor. Later, ACC was identified by
Weiss
et al.
(
71
) in mollusk embryonic shells and by Politi
et al.
(
72
)
in regenerating sea urchin spines. In 2008, Politi
et al.
(
73
) demon-
strated using synchrotron spectromicroscopy that, at the nanoscale,
there are two amorphous precursors to crystalline calcite in sea
urchin spicules, hydrated (ACC-H
2
O) and anhydrous ACC.
The
same two ACC precursor phases were then detected during the for-
mation of sea urchin teeth (
74
); in sea urchin spicules, again, to in-
dicate the sequence of phases and phase transitions (
24
); in mollusk
shell nacre (
23
); in corals skeletons from
S. pistillata
(
25
); and from
five additional reef-forming coral species (
51
).
The unexpected discovery of the same precursor phases to ara-
gonite and calcite biominerals, in phyla drawn from three major
branches of eumetazoan phylogeny (cnidarians, mollusks, echino-
derms) (
8
), which diverged from one another long before they started
making biominerals (Fig. 1) (
15
), suggests that this biomineralization
strategy emerged independently in these distantly related clades.
Because skeletons formed in this way preserve a nanoscale textural
record of the ACC PA by which the skeletons formed, Gilbert
et al.
(
15
) were able to show that this mineralization pathway was already
used by Cambrian mollusks (~500 Ma before present) and even
by the iconic Ediacaran fossil
Cloudina
, one of the first animals to
form a CaCO
3
skeleton, some 550 million years ago. These results
are summarized in Fig. 2.
PA
+
IA growth is rapid, space-filling, and
makes
tougher biominerals
Crystal growth by PA is much faster than by IA.
A notable example
is provided by eggshells, which grow faster than any other known
biomineral: Hen eggshells grow to 300
m in thickness in 24 hours,
with PA of 100- to 300-nm ACC particles that subsequently crystal-
lize to calcite (
75
). Ostrich eggshells grow to 2 mm in thickness in
48 hours (
76
), making them the fastest-growing of all biominerals.
Coral skeletons of
S. pistillata
grow, on average, ~40
m/day (
77
–
79
)
by PA of 100- to 400-nm ACC particles, which then crystallize to
aragonite (
25
). By contrast, the rate of abiotic aragonite growth by
IA from seawater is on the order of 0.01 to 0.1
m/day (
80
–
83
). Five
other reef-forming coral species were recently demonstrated to
form skeletons and fill space by PA + IA (
51
). The impact of PA is
perhaps best illustrated by the Great Barrier Reef, the largest of all
biomineral structures, which is visible from outer space, and stretches
continuously for 2300 km from north to south along the coast of
Queensland, Australia.
Identical particles at any scale, from the nano- to the macroscale,
can never fill three-dimensional space (
84
), not even in the most
space-filling hexagonal close packing. This is the configuration of
identical cannonballs stacked on one another in three-dimensional
arrays: A considerable amount of space, 26%, remains unfilled as
cannonballs fill 74% of space. Particles of different sizes can fill >74%
of space but never 100% (
84
). However, surface area data show that
sea urchin spines and coral skeletons are 100% space-filling as are
abiotic single crystals of calcite or aragonite, respectively (
51
,
85
,
86
).
In the PA + IA model, PA fills part of the space, and ions fill the
remaining interparticle voids as recently concluded by Walker
et al.
(
37
) for coccolithophorids and by Sun
et al.
(
51
) for coral skeletons.
The mechanical properties of biominerals are greatly improved
by PA, even when the final biomineral is single crystalline, and the
originally ACC nanoparticles crystallize to become coherently aligned
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Gilbert
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.,
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, eabl9653 (2022) 9 March 2022
MS no: RVabl9653/AD/BIOPHYSICS, PALEONTOLOGY
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(
87
). Crystalline defects accumulating at nanoparticle interfaces deflect
and dissipate cracks and therefore toughen the biomineral (
87
–
89
).
Morphogenesis by PA
+
IA of
mesostructured biominerals
Despite the apparent universality of the PA + IA pathway, the car-
bonate minerals in skeletons vary widely in form, orientation, size,
and mineralogy, sometimes within a single individual. Termed me-
sostructure, the size, shape, crystal orientation, and spatial arrange-
ment of crystals underpin biomineral function. The controlled
crystal growth manifest in mesostructural diversity is mediated by
an array of structural proteins, including enzymes, glycoproteins,
and polysaccharides, all of which are present in the privileged space,
are incorporated in the biomineral, and are therefore termed “or-
ganic matrix.” De Yoreo and Dove (
90
) showed that growing crys-
tals can be shaped by organic molecules, and other experiments have
demonstrated polymorph selection by the entire organic matrix mix-
ture of organic molecules (
91
,
92
), by single proteins (
93
–
96
), or by
short peptides (
97
). The latter not only served as templates for the
formation of aragonite under calcite growth conditions (no Mg) but
also self-assembled into organic layers that alternated with lamellar
aragonite, similar to nacre (
97
). Recent work by Mummadisetti
et al.
(
98
) documented the function and fine-scale spatial organization of
these matrix molecules in stony corals.
The organic matrix, presumably, must also control the cessation
of crystal growth, that is, inhibit or poison it, as proposed by Addadi
and Weiner (
99
). In this regard, Yang
et al.
(
100
) recently demon-
strated that a previously unidentified matrix protein called PfX binds
to specific crystal faces in developing shells of
Pinctada fucata
, thus
helping to shape carbonate crystal growth. Inhibiting nucleation and
growth of crystals at the wrong place and time is also a key role for
organics (
99
,
101
). How organics control crystal orientation tilting,
however, is not understood (
102
–
104
) nor is the role of organics-
associated cations in crystal orientation control (
105
).
An integrated model for
CaCO
3
biomineralization
A mechanistic model for marine CaCO
3
biomineralization is pre-
sented in Fig. 3. The model builds upon prior proposals for individ-
ual species or phyla [e.g., corals (
50
,
106
–
108
), coccolithophorids
(
18
,
109
), and foraminiferans (
35
,
110
)] but hypothesizes that the
fundamental geochemistry of carbonate species and minerals plus
the functionally equivalent strategies for transport and mineral for-
mation result in an evolutionarily convergent shared framework
despite the diversity of marine biomineralizing organisms. In par-
ticular, the model hypothesizes that amorphous particle formation
in vesicle ICF and biomineral growth by IA in the ECF, along with
PA, is a general strategy. Amorphous precursors or PA have not been
observed in brachiopods or foraminiferans; they are simply hypoth-
esized here. We propose that variation in the contributions and rates
of the mechanisms in Fig. 3 could explain observed differences in
carbonate biomineral isotopic and elemental composition. Although
more work is needed to quantify these contributions, and to incor-
porate photosynthesis, this model provides the framework for under
-
standing the ability of marine calcifiers to act as paleoenvironmental
proxies, their vulnerability or resilience to stressors associated with
past or present climate change, and the commonality of nanostruc-
tures observed in the fossil record across phyla (
15
). For example, in
corals, the pH, calcium, and carbonate ion concentrations in the ECF
are greater than those in seawater (
31
,
67
,
111
,
112
), as indicated by
the magenta deltas in Fig. 3. A similar observation is expected in
other organisms as well (
113
). Whether these are constant offsets,
varying as the seawater values vary (
112
,
114
,
115
), or are maintained
constant at homeostatic values (
116
,
117
) may affect the energy cost
of calcification.
The extent of PA versus IA varies across organisms. Extremes
are coccolithophorids, skewed toward IA (
37
,
38
,
49
), and sea ur-
chin spicules, preferentially growing by PA (
24
,
26
).
To test or falsify the model, two principal directions are possible.
One is to explore whether carbonate biomineralization involves a se-
quence of thermodynamically downhill carbonate phases in many
more organisms. Recent observations of a transient liquid precursor
to sea urchin spine formation, or a transient vaterite phase during test
biomineralization by foraminiferans (
29
), are consistent with the model
but require a richer number of phases and phase transitions currently
not included. Another option is to establish compositional relation-
ships between seawater, CF, precursor particles, and the final skeleton,
consistent across organisms, natural biominerals, and reproducible
in laboratory experiments. For example, if future synthetic experi-
ments find that formation via amorphous (
51
) or crystalline (
29
)
precursors markedly affects isotopes in the laboratory, but bio-
minerals are isotopically more similar to seawater than to synthetic
precursors, then the model with PA + IA must be reconsidered.
A weakness of the model is that ICF is only reasonably deduced
to exist in most organisms; it has only been observed and measured
directly during the formation of sea urchin spicules (
47
) and indi-
rectly during sea urchin spine (
48
) and coral skeleton (
53
) forma-
tion. Thus far, the best characterized CF is the ECF in one coral
species,
S. pistillata
(
31
,
67
,
111
,
112
). Only carbonate chemistry was
measured in
S. pistillata
, whereas the pH was measured in the ECF
of more coral species and found to differ significantly between day
and night (
53
,
112
), highlighting the other weakness of the model:
the lack of photosynthesis and related pathways.
As the model in Fig. 3 shows, biomineral formation takes place
in a privileged space, which may be intra- or extracellular and is
separated from but partly open to seawater, and its chemical com-
position is biologically controlled. All organisms actively concen-
trate Ca and DIC into ICF and ECF and actively remove protons
from both. Passive diffusion of carbon dioxide [CO
2
(aq)] occurs
through all cell and vesicle membranes. At the same time, mito-
chondria respire and produce CO
2
, which in part diffuses toward sea-
water, ECF, and ICF and in part is rapidly converted to bicarbonate
ions (HCO
3
−
), as catalyzed by carbonic anhydrase (CA) in the cyto-
sol and in ICF and ECF.
This additional bicarbonate is also pumped
actively into ICF and ECF, where, at higher pH, its speciation
changes to carbonate ions CO
3
2−
that readily bind Ca
2+
. Besides cal-
cium and carbonate, organic molecules synthesized by the organ-
ism are also injected into the vesicles and the ECF and form the
organic matrix occluded in the biomineral.
ISOTOPE AND
TRACE ELEMENT SUPPORT FOR
AN
INTEGRATED
MODEL OF BIOMINERALIZATION
Marine calcifiers incorporate elements and isotopes into their bio-
minerals at levels that are partly determined by seawater concentrations,
providing the basis for paleoenvironmental proxies, and partly de-
termined by the biological and geochemical processes that modify
the chemistry and isotopic composition of the ICF and ECF.
As a
consequence of such processes, termed “vital effects,” biomineral
compositions may depart from those observed in abiotically precipitated
minerals under the same seawater conditions. These vital effects not
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