-
doi: 10.1098/rspb.2000.1415, 771-778268 2001 Proc. R. Soc. Lond.
B
Antonio G. Checa and Alejandro Rodríguez-Navarro Unionidae
(Bivalvia: Mollusca)the self-organization of shell microstructures
in Geometrical and crystallographic constraints determine
Referenceshttp://rspb.royalsocietypublishing.org/content/268/1468/771#related-urls
Article cited in:
Email alerting service heretop right-hand corner of the article
or click Receive free email alerts when new articles cite this
article - sign up in the box at the
http://rspb.royalsocietypublishing.org/subscriptions go to:
Proc. R. Soc. Lond. BTo subscribe to
This journal is © 2001 The Royal Society
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/content/268/1468/771#related-urlshttp://rspb.royalsocietypublishing.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=royprsb;268/1468/771&return_type=article&return_url=http://rspb.royalsocietypublishing.org/content/268/1468/771.full.pdfhttp://rspb.royalsocietypublishing.org/subscriptionshttp://rspb.royalsocietypublishing.org/
-
Geometrical and crystallographic constraintsdetermine the
self-organization of shellmicrostructures in Unionidae (Bivalvia:
Mollusca)Antonio G. Checa1* and Alejandro Rodr|̈guez-Navarro2
1Departamento de Paleontolog|̈a y Estratigraf|̈a, Universidad de
Granada, Granada 18002, Spain2Savannah River Ecology Laboratory,
University of Georgia, Drawer E, Aiken, SC 29802, USA
Unionid shells are characterized by an outer aragonitic
prismatic layer and an inner nacreous layer. Theprisms of the outer
shell layer are composed of single-crystal ¢bres radiating from
spheruliths. Duringprism development, ¢bres progressively recline
to the growth front. There is competition between prisms,leading to
the selection of bigger, evenly sized prisms. A new model explains
this competition processbetween prisms, using ¢bres as elementary
units of competition. Scanning electron microscopy andX-ray texture
analysis show that, during prism growth, ¢bres become progressively
orientated with theirthree crystallographic axes aligned, which
results from geometric constraints and space
limitations.Interestingly, transition to the nacreous layer does
not occur until a high degree of orientation of ¢bres isattained.
There is no selection of crystal orientation in the nacreous layer
and, as a result, the preferentialorientation of crystals
deteriorates. Deterioration of crystal orientation is most probably
due toaccumulation of errors as the epitaxial growth is suppressed
by thick or continuous organic coats on somenacre crystals. In
conclusion, the microstructural arrangement of the unionid shell
is, to a large extent,self-organized with the main constraints
being crystallographic and geometrical laws.
Keywords: biomineralization; bivalve shell; microstructure;
nacre; composite prisms; self-organization
1. INTRODUCTION
Molluscs fabricate shells that are composites of calcite
oraragonite crystals embedded within an organic matrix.The crystals
display varied morphologies and structuralarrangements and are
described as having di¡erentmicrostructures. In most cases, the
shells are constructedof two or more superimposed layers with
di¡erent micro-structures, which may even be composed of
di¡erentcalcium carbonate polymorphs (i.e. calcite or
aragonite)(Taylor et al. 1969).
Shell formation starts with the secretion of an outerorganic
membrane or periostracum (¢gure 1). Thisorganic membrane acts as
the substrate where shell-forming crystals (calcium carbonate in
either the form ofcalcite or aragonite) nucleate. The
shell-secreting epi-thelium or mantle supplies the Ca2 + and HCO¡3
ionsnecessary for the growth of calcium carbonate crystals.The
mantle is separated from the inner shell surface by aspace. The
organism can maintain a supersaturatedsolution (extrapallial £uid)
within this space, therebyallowing shell-forming crystals to
precipitate (Simkiss &Wilbur 1989). There is evidence from in
vitro experimentsthat speci¢c proteins composing the shell organic
matrixplay an important role in controlling the polymorphicphase,
morphology and orientation of individual crystals(Addadi et al.
1987; Falini et al. 1995; Belcher et al. 1996).However, little is
known about the role these proteinsplay in how crystals assemble
into a given microstructure.Since di¡erent species have speci¢c
shell microstructuresit is generally believed to be genetically
directed (Addadi& Weiner 1992). Even more striking is the
transitionbetween superimposed layers with di¡erent micro-
structures. For instance, in Unionidae the mantle secretestwo
shell layers with di¡erent microstructures. Themantle has an
oblique disposition with respect to theperiostracum, depositing the
two di¡erent layers simulta-neously (¢gure 1). The more marginal
areas of the mantlesecrete the outer shell layers. This is usually
explained byassuming a zonation of the metabolic properties of
theshell-secreting mantle surface (Beedham 1958). In thisway,
di¡erent parts of the mantle could induce the devel-opment of
di¡erent microstructures.
While understanding biomineralization processes hasbeen the main
focus of studying shell growth, the know-ledge may also be applied
to the fabrication of superiormaterials composed of highly
orientated crystals of thesame size and morphologies (Aksay et al.
1996). Forinstance, the structure of mollusc shell, in
particularnacre (which is formed by layers of aragonite tablets
sand-wiched by an organic membrane), has excellent mechan-ical
strength and fracture toughness, exceeding that of thearagonite
single crystals composing it by several orders ofmagnitude (Currey
1977).
Most studies on mollusc shell growth have focused onthe
physiological factors controlling shell growth(Saleuddin &
Kunigelis, 1984; Simkiss & Wilbur 1989;Addadi & Weiner
1992) and the role of speci¢c proteinsin the control of crystal
growth (Lowenstam & Weiner1989). However, much less attention
has been paid tofundamental issues such as the geometrical and
crystallo-graphic factors constraining the growth of crystals
andthe development of aggregate micro-architectures. Forinstance,
simple geometrical laws govern the growth ofcrystals. Crystal habit
depends entirely upon the relativegrowth rates of crystal faces,
which are in turn controlledby both crystal structure and growth
conditions (i.e.supersaturation, temperature and impurities
(proteins))
Proc. R. Soc. Lond. B (2001) 268, 771^778 771 © 2001 The Royal
SocietyReceived 10 November 2000 Accepted 5 December 2000
doi 10.1098/rspb.2000.1415
*Author for correspondence ([email protected]).
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
(Sunagawa 1987). The growth of a polycrystalline aggre-gate
(i.e. shell) is much more complex as crystals growingtogether
impinge on each other and compete for theavailable space, but its
growth is still governed by crystal-lographic and geometrical laws
(Grigor’ev 1965;Rodr|̈guez-Navarro & Garc|̈a-Ruiz 2000). In
this paper,we focus on the evolution of the morphology and
crystal-lographic orientation of crystals during shell growth
inunionids in order to evaluate the importance of geome-trical and
crystallographic factors versus biological factorson the
development of shell micro-architectures.
2. MATERIAL AND METHODS
(a) MaterialShells of the Unionidae were studied including Unio
elongatulus
Pfei¡er, 1825, (Canal Imperial de Aragön, Navarra, Spain),
Uniocrassus Philippson, 1788 (River Meuse, Hastie© res,
Belgium),Ambleminae Potomida littoralis (Lamarck, 1801) (Canal
Imperialde Aragön, Zaragoza, Spain), Lamprotula sp. (locality
unknown,China) and Caelatura bakeri (Adams, 1866) (Nyanza,
Kenya).
(b) Optical and electron microscopyThin sections (30^40mm thick)
of U. elongatulus and Lamprotula
sp. shells cut along a dorsoventral radius were prepared
fortransmitted light microscopy. The mantle shell system wasstudied
in living specimens of U. elongatulus and P. littoralis.Samples
were initially ¢xed in 10% formalin and preserved in70% ethanol.
Square pieces of the shell margin (usually theventral area) were
cut very carefully in order to avoid damagingthe adjacent mantle
margin.The mantle was later cut with preci-sion scissors.The
samples were later completely decalci¢ed, ¢xedin bu¡ered cacodylate
(0.1 M and pH 7.4) 2.5% glutaraldehydeand critical point^CO2 dried.
They were later embedded inepoxy resin, sectioned to 1 mm (Ultracut
S, Leica, Solms,
Germany) and stained with 1% toluidine blue. Samples
weresuper¢cially decalci¢ed with 6% ethylenediaminetetraaceticacid
for 20 min at room temperature for scanning electronmicroscope
(SEM) examination. Outer and inner valve surfaces(sometimes with
the periostracum partly removed with 5%NaOH) as well as transverse
radial fractures of shells (intact oretched in 1% HCl for less than
1min) from all species (exceptP. littoralis) were observed in an
SEM (DSM 950, Zeiss, Jena,Germany) after being sputtered with gold
for 4 min.
(c) X-ray texture analysisThe orientation of the crystals of U.
elongatulus and Lamprotula
shells was determined using an X-ray texture
di¡ractometer(X’pert, Phillips, Amelo, The Netherlands) (Cullity
1977). Poledensities for 002 and 112 re£ections of aragonite were
registeredin order to do this. These pole ¢gures show the
three-dimen-sional distribution of orientation of the [001] and
[112] crystaldirections. The stereographic projections of the pole
¢gures aredisplayed as counter plots. The scattering or degree of
preferen-tial orientation of crystals can also be quanti¢ed from
theseplots as full width at half maximum (FWHM) values of thepeaks
in the pole ¢gures. The lower the FWHM value, thegreater the
alignment of crystals. In order to study the evolutionof the
orientation of crystals during shell growth, samples weremanually
ground to di¡erent levels parallel to the outer shellsurface and
the pole ¢gures registered.
3. RESULTS
(a) The internal structure of prismsUnionid shells consist of an
outer aragonitic prismatic
layer and an inner nacreous layer with the prismatic
layercomprising only ca. 10% of the total shell thickness.
Shellgrowth occurs via spheruliths (spherical aggregates
ofradiating crystal ¢bres) that nucleate within the gelatinous
772 A. G. Checa and A. Rodr|̈guez-Navarro Self-organization of
shell microstructures
Proc. R. Soc. Lond. B (2001)
shell growthdirection
shell margin
outerperiostracum
innerperiostracum
growth lines
mantle
shell margin
(a)
(b)
prismatic layer
nacreous layer
Figure 1. (a) Drawing of a unionid shell with the main reference
directions indicated and showing where shell pieces were cut forthe
texture analysis. (b) Schematic of a cross-section of a shell
showing the disposition of the di¡erent layers constructing
it.Modi¢ed from Checa (2000).
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
inner periostracal surface (¢gure 2a,g). Initially, spheru-liths
grow independently from each other but, as growthcontinues beyond
the boundary of the inner perio-stracum, they start to impinge on
one another anddevelop into polygonal prisms. Prisms grow
vertically(inward) and later they usually become graduallyreclined
towards the shell margin (¢gure 2b). In thisway, the prism main
axis remains perpendicular to thegrowth front (see below). Its
orientation changes gradu-ally from being inclined dorsal-wards to
nearly parallelto the periostracum at the very margin. Prisms
arecomposed of elongate crystals (¢bres) fanning out inthree
dimensions from the main axis of the prismtowards the depositional
surface. Longitudinallyfractured prisms, as observed through the
SEM, showparticularly conspicuous ¢bres towards the edges ofprisms
(¢gure 2c, f ). During prism growth, ¢bres fanout (giving the
prisms a feather-like appearance in cross-section) (¢gure 2b,e) and
become thicker, reaching up to2 mm in thickness at their most
distal ends (¢gure 2c).Each ¢bre is a single, curved crystal with
the c-axisparallel to the long axis as revealed by the
undulatingextinction pattern with polarized light microscopy(¢gure
2b). As prisms grow longer, the mean divergenceof ¢bres with the
main or long axis of the prismsdecreases. Values of 20^258 at the
lower boundary of theprismatic layer are typical. At the same time,
¢bresachieve greater lengths and become straighter(¢gure 2b,c). The
distribution of ¢bres within a prism isasymmetric. Fibres running
forwards (pointing to theshell margin) grow more than those
diverging backwards(¢gure 2c). In this way, while ¢bres remain
strictlyperpendicular to the growth front, the prism long
axisbecomes progressively advanced with respect to the axisof ¢bre
divergence and the shell margin. Prisms showprominent concentric
growth lines (¢gure 2a,e) that aremore or less centred in the
spheruliths. They are perpen-dicular to the ¢bres and remain as
organic sheets afterdecalci¢cation. These growth surfaces most
probablyre£ect successive positions of the mantle.
Interestingly,the deposition of these organic sheets does not seem
tointerrupt or alter ¢bre growth and orientation.
(b) The size and shape of prismsDuring growth of the prismatic
layer, bigger prisms
expand laterally at the expense of smaller ones, therebyblocking
their space for growing. Prismatic units areprogressively lost so
that only a few, big, evenly sizedprisms reach the transition to
the nacreous layer (¢gure2a,e, f ). The thickness of the prismatic
layer as well as thesize of the prisms (both in height and width)
increases ina radial section from older to newer parts of the shell
(i.e.the shell margin). The spacing among neighbouringprisms also
increases in the same direction.
The variation in the aspect ratio (height to width) ofprisms is
shown in ¢gure 3 as a function of their height.It can be observed
that prisms show a trend of increasingelongation (higher aspect
ratios) with increasing height.However, there is a tendency for the
value of the aspectratio to saturate as the prisms’ size (height)
increasesfurther. Furthermore, as the aspect ratio of the
prismsincreases, the limit angle or maximum angle of diver-gence of
¢bres decreases.
(c) Transition to the nacreous layerLarge distal ¢bres of the
surviving prisms become
transversely divided by organic sheets at the transitionzone
from the prismatic to the nacreous layer and gradu-ally transform
into stacked nacreous tablets (¢gure 2c).Fibres are highly aligned
(within a range of 208) at thetransition zone. The ¢rst nacreous
tablets grow epitaxiallyonto the distal ends of ¢bres, from which
they inherittheir crystallographic orientation, having their
c-axisperpendicular to the tablet surface. The transition tonacre
is initiated in the rear part of a radial section of ashell within
individual prisms and proceeds to the frontpart of the prism that
is pointing towards the shellmargin (¢gure 2c). The ¢rst nacre
sheets usually have aslightly wavy disposition that results from
the divergingarrangement of ¢bres. Waviness soon fades out and
thenacre sheets become £at as the nacreous layer growsthicker.
Similar gradual transformations were observedby Dauphin et al.
(1989) in Haliotis and Mutvei (1972) inNautilus.
(d) The inner surface of the shellThe inner surface of the inner
nacreous layer shows a
terraced disposition of the aragonite sheets, which growtowards
the shell margin (¢gure 2d). Sequential stages ofthe nacreous
tablet nucleation and growth can beobserved when moving towards the
shell edge. The shapeof aragonite tablets is rhombic, with the
longest diagonalcoincident with the b-axis and the shortest
coincidentwith the a-axis. The elongate crystal shape re£ects a
fastergrowth rate along the b-axis than the a-axis. Note
thatcrystals forming a nacre lamella or sheet are orientatedwith
their longest diagonal (b-axis) towards the directionof shell
growth and the shorter diagonal (a-axis) trans-versal to it and
parallel to the shell margin. However,exceptions are sometimes
noted in isolated tablets or evenclusters of tablets with
orientation di¡ering signi¢cantlyfrom the common orientation
(b-axis perpendicular to theshell edge), sometimes even being
transverse (¢gure 2d ).
(e) Evolution of the orientation of crystals duringshell
growth
The evolution of the orientation of crystals during shellgrowth
was studied by registering pole ¢gures at di¡erentthickness within
a shell of U. elongatulus. The pole ¢guresfrom the outer surface of
the shell (thickness 0%), whichcorrespond to the initial stages of
shell formation, show auniform distribution of intensity indicating
that crystalsnucleate with a random orientation. At increasing
thick-ness, corresponding to later stages of growth, the pole¢gures
gradually show better-de¢ned peaks, indicatingthat crystals are
progressively more aligned (¢gure 4).The degree of alignment of
crystals is assessed by theFWHM value for these peaks (¢gure 4).
The FWHMw-value in ¢gure 4c (left-hand graph), which wasmeasured
from the 002 pole ¢gures, decreases rapidlyacross the prismatic
layer, reaching a minimum just afterthe transition to the nacreous
layer. It increases againacross the nacreous layer indicating that
the scattering ofthe orientation of the c-axis of crystals follows
the sametrend. Curiously, the scattering of the c-axis is greater
inthe direction parallel to than the direction perpendicularto the
shell margin. The spread of the orientation of the
Self-organization of shell microstructures A. G. Checa and A.
Rodr|̈guez-Navarro 773
Proc. R. Soc. Lond. B (2001)
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
774 A. G. Checa and A. Rodr|̈guez-Navarro Self-organization of
shell microstructures
Proc. R. Soc. Lond. B (2001)
(a) (b)
(c) (d )
(e) ( f )
(g) (h)
Figure 2. (Caption opposite.)
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
a- and b-axes, which was measured as the FWHM ¿-value from peaks
of 112 pole ¢gures, follows the sametrend as that of the c-axis
(¢gure 4c, right-hand graph),though the alignment of crystals along
these axes isalways poorer than along the c-axis. These
observationsimply that crystals that initiate shell growth are
randomlyorientated. Crystals become rapidly aligned during
thegrowth of the prismatic layer. However, their
orientationdeteriorates slightly during the growth of the
nacreouslayer.
Intraprismatic growth lines, which are formed byorganic
material, cross cut the di¡erent prisms indicatingpulsating growth
of the whole layer (¢gure 2a,e). Interest-ingly, the deposition of
organic material does not seem tointerrupt the epitaxial growth of
crystals from one layerto the next. In fact, SEM and X-ray texture
analysisshows that, during shell growth, there is always
conti-nuity in the preferential orientation of crystals and
thatonly the degree of alignment changes.
4. DISCUSSION
(a) Competition between prismsCompetition between prisms was
described and inter-
preted by Ubukata (1994) in several bivalves, includingunionids.
He developed geometrical constructions basedon Grigor’ev’s (1965)
model of the growth of aggregateminerals in order to show the e¡ect
of several factors onprism selection. The main problem when these
models areapplied to the composite prisms of unionids is that
theyimply a free-growing surface (as in the formation of,
forexample, quartz crystals growing on the wall of a rockcavity or
geode). However, in unionids the mantle surfacelimits prism growth,
as evidenced by growth lines. There-fore, unlike Grigor’ev’s (1965)
model, the growth front ofprisms is permanently constrained by the
position of themantle, thereby precluding the possibility of
di¡erencesin the longitudinal growth rate among prisms. Taking
thisinto consideration, we have developed a model in whichprism
selection is not due to competition between prisms,but between the
¢bres of di¡erent prisms meeting at theinterprismatic surfaces
(¢gure 5). When a prism out-
competes another it is apparent that its ¢bres run lessinclined
to the growth front (¢gure 2 f ). A lesser inclina-tion angle
implies a faster growth in parallel to thegrowth surface, which
explains why less-inclined ¢bresoutcompete more-inclined ones.
Therefore, competitionbetween prisms can be reduced to competition
betweentheir constituent ¢bres. A growth rate normal to thegrowth
surface is a negligible factor since ¢bre growth inthis direction
is constrained by the rate of mantle displa-cement, which is
uniform for ¢bres of all prisms growingat the same time.
(b) Genesis and evolution of crystal orientationIt has been
suggested that crystal orientation is
imposed by epitaxial nucleation on the organic matrixsurface
(Weiner & Traub 1980, 1984). However, thedegree of orientation
of the organic constituents and therange over which they are
orientated (a few microns) are
Self- organization of shell microstructures A. G. Checa and A.
Rodr|̈guez-Navarro 775
Proc. R. Soc. Lond. B (2001)
Figure 2. (a) Oblique view of a partly dissolved inner
periostracum and fractured outer prismatic layer of Lamprotula sp .
Growthsurfaces cross cut adjacent prisms. Growth direction, top
left to bottom right. Magni¢cation£ 420. (b) Polarized light
micrographof the outer prismatic layer of U. elongatulus. Prisms
are composed of ¢bres diverging from the prism main axis.
Extinction bandsusually displace rearwards (right) during prism
growth (to the bottom) indicating that ¢bres directed forwards to
the shellmargin become progressively more developed at the expense
of those directed backwards. Prisms curve to remain perpendicularto
the growth front. Magni¢cation£ 190. (c) Transition between the
prismatic and nacreous layers in Lamprotula sp.Prism-composing
¢bres gradually align parallel to the long axis of the prism.
Transition to the nacreous layer occurs when thedegree of
convergence reaches a certain threshold. Fibre distribution is
asymmetric with those running forwards to the shellmargin (right)
being more developed. This causes transition to the nacreous layer
to proceed from the rear of the prism forwards.Magni¢cation£ 1800.
(d ) View of the step-like arrangement of nacreous sheets at the
internal growth surface of the shell ofP. littoralis. Rhombic
nacreous tablets are orientated, with minor exceptions, with their
longest diagonal (b-axis) parallel to theshell growth direction
(upper left). Magni¢cation£ 1800. (e) Thin section through the
outer prismatic layer of P. littoralis.Intraprismatic growth
surfaces are perpendicular to ¢bres and, hence, concave towards the
initial spherulith. They continueacross the di¡erent prisms, which
indicates synchronized growth. Magni¢cation£ 190. ( f ) Prismatic
shell of Lamprotula sp. Twoprisms outcompete a third trapped
in-between. When ¢bres meet, those of the bigger prisms always form
a higher angle with thelong axes of prisms. Magni¢cation£ 1800. (g)
Outer shell surface of Lamprotula sp. with the periostracum
removed. Initialspheruliths are statisticallyplaced towards the
rear of prisms (lower right), which indicates an asymmetric
development ofprisms. Magni¢cation£ 850. (h) Section through the
prismatic and nacreous layers of U. elongatulus. Growth halts are
markedby discontinuities. Growth resumes with prismatic layers that
intrude into the nacreous layer and gradually wedge outinternally.
Prism size decreases in the same direction, so that the
relationship of width to height is kept. Magni¢cation£ 95.Scale
bars: (a) 50 mm, (c,d, f ) 5 mm and (g) 10 mm.
0 20 40 60
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.080 100 120
height (m m)
aspe
ct r
atio
(H
/W)
140 160
H/W = 2.523°
H/W = 2.7521°
H/W = 3
limit angle of 19°
180 200 220 240
Lamprotula sp.Unio elongatulus
Figure 3. Aspect ratio (height to width (H/W)) of prisms as
afunction of their size (height) measured for Unionidae
shells.Values were obtained from SEM views of sections
eitherparallel (U. elongatulus) or transversal (Lamprotula sp.) to
themargin. Prisms become more elongate with size.
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
much less than those of aragonite crystals (in the order
ofmillimetres) (Weiner & Traub 1984). In addition, such
amechanism does not explain the evolution of the orienta-tion of
crystals during shell growth and, contrary toobservation, it
implies that the orientation of crystals isalready selected in
early crystal growth stages. Conversely,we observed that
preferential orientation of crystals in theshells of unionids
develops during growth of the prismaticlayer, starting from
randomly orientated crystals.
This progressive alignment of crystals during prismgrowth is due
to geometric selection of crystals. Theprogressive alignment of
¢bres and the c-axis of crystalswith the main axis of the prisms is
merely a geometricfeature. Initially, ¢bres of the spheruliths
(from whichprisms initiate) radiate in all directions. As
growthprogresses these ¢bres impinge on ¢bres of
neighbouringspheruliths, thereby constraining further growth of
¢bresto the sides. Fibres only have free space for
developingvertically. As the prismatic layer becomes thicker
theorientation of ¢bres is further selected and only
¢bresorientated vertically (growing inward) continue to grow(¢gure
6). Sylin-Robert (1986) described a similar modelin explaining the
development of a preferential orienta-tion of crystals in eggshells
along their c-axis. However,in order to explain the orientation of
crystals with theira^b-axis also aligned another mechanism must
beinvoked. The horizontal displacement of the mantle at theshell
margin frees some space for the expansion of ¢bres
in the a^b-plane towards the shell margin. This
allowsgeometrical selection of ¢bres orientated with their
hori-zontal, fastest, growth direction (b-axis) perpendicular tothe
shell margin (¢gure 6). This process results in thecrystal ¢bres
having their three axes aligned. However,the degree of alignment of
crystals along the a^b-axes isless than that along the c-axis. The
selection mechanism ismore e¤cient along the c-axis since the
orientation ofcrystals is due to di¡erences in growth rates
alongdi¡erent crystal directions and the c-axis is the
overallfaster crystal growth direction.
(c) Transition to the nacreous layerInterestingly, the
transition to nacre does not occur
until the angular divergence of ¢bres (and that of c-axes)within
prisms falls below a certain threshold (withinca. 208). It seems as
if a uniformly orientated substrate (orat least one with a reduced
range of dispersion) is neces-sary for nacre to grow by epitaxy.
This is not surprisingsince a main characteristic of nacre is that
the tabletshave their c-axis aligned and orientated perpendicular
tothe shell surface (e.g. Hedegaard & Wenk 1998). It isprobably
not accidental that molluscan nacre is, in mostcases, preceded by
some kind of ¢brous or prismatic(either aragonitic or calcitic)
layer (Taylor et al. 1969).
Since ¢bres curve outwards, bigger prisms will attainthe c-axis
dispersal threshold relatively late and couldgrow longer (higher
aspect ratios) (¢gures 2 h and 3).
776 A. G. Checa and A. Rodr|̈guez-Navarro Self- organization of
shell microstructures
Proc. R. Soc. Lond. B (2001)
0 20
60
50
40
30
20
10
40outersurface
innersurface
shell thickness (%)
a-axis
b-axis
c-axis
112 face
002
c c cD
f fD
112a
b(a)
(c)
(b)
001 face
FW
HM
c (d
eg)
60
50
40
30
20
10F
WH
Mc
(deg
)
nacreous layer nacreous layer
alignment of c-axisshell margin
alignment of a- and b-axis
shell growth directionpri
smat
icla
yer
pris
mat
ic la
yer
60 80 100 0 20 40outersurface
innersurface
60 80 100
Figure 4. (a) Single crystal of aragonite. (b) Distribution of
crystal orientations measured at the boundary between theprismatic
and nacreous layers. The subcentral position of unique maxima in
the 002 pole ¢gure indicates that the crystals areorientated with
their c-axis perpendicular to the shell surface. The positions of
the four maxima displayed in the 112 pole ¢gureindicate that the
crystals orientate with their b-axes parallel to the shell growth
direction (horizontal line) and a-axes parallel tothe margin. (c)
Evolution of the alignment of crystals across the shell thickness
(left-hand graph) along the c-axis and (right-handgraph) along the
a- and b-axes. Interestingly, the crystals become aligned within
the prismatic layer (FWHM values decrease),whereas their alignment
deteriorates as the nacreous layer becomes thicker (FWHM values
increase).
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
More remarkable is the fact that the transition to nacreoccurs
in the rear part of the prism ¢rst rather than inthe front part of
the prism (¢gure 2c). The asymmetricaldistribution of ¢bres within
prisms causes the c-axisdispersal threshold to be reached ¢rst at
the back, withthe subsequent secretion of the ¢rst nacreous
tablets, andto propagate forward later. The asymmetric
developmentof prisms can be traced back to the initial
spherulithstage. Spheruliths are usually more developed
anteriorlysince growing spheruliths have more free space to
expandforwards than backwards where older spheruliths arealready
expanding (¢gure 2g).
Nacreous tablets inherit the crystallographic orienta-tion of
the most distal ¢bres of prisms, which act asepitaxial mineral
substrates. As the nacreous layer growsthicker the orientation of
crystals deteriorates, as revealedby X-ray texture analysis and
through the SEM (seeabove) (¢gure 4). In our opinion, this is
probably due totwo factors. First, the orientation of crystals is
not furtherselected by competition since crystals nucleate
separatelyand only establish contact when fully grown (¢gure 2d
).Second, there may be an accumulation of errors in whichthe
epitaxial growth of crystals from one nacre sheet tothe next is
suppressed. There are porous organic coatsbetween nacre sheets that
allow physical contact and,hence, epitaxy between crystals of
di¡erent layers(Scha¡er et al. 1997). However, a thick or
continuousorganic coat could prevent epitaxial growth and cause
thenew crysal to have a random orientation (¢gure 7).
Finally, in our opinion, genetic factors must determinethe shape
of the mantle and periostracum, which de¢nethe geometry of the
cavity where shell mineralizationoccurs. In addition, cellular
processes might be responsible
for the secretion of speci¢c organic components (i.e.proteins),
which must certainly modulate the crystalgrowth. However, it should
be noted that, once crystalgrowth is initiated, the subsequent
arrangement of crys-tals is mostly determined by crystallographic
constraintsand space limitations, with the resulting aggregate
micro-structure being self-organized.
5. CONCLUSIONS
The microstructural development of the shell in Union-idae can
be understood when the monocrystalline ¢bres(instead of prisms,
into which they aggregate) of the outershell layer are considered
as elementary units. In parti-cular, we have explained (i)
competition and selectionbetween prisms, (ii) the development of
the orientation of
Self-organization of shell microstructures A. G. Checa and A.
Rodr|̈guez-Navarro 777
Proc. R. Soc. Lond. B (2001)
(a) (b)
r0/r’0 = 2
ttr’n
r’n+1r’n+2
rnrn+1rn+2
r’0r0
r’0r0
r0/r’0 = 1.5
Figure 5. Model for competition between composite prisms
inUnionidae. When two ¢bres (rn and r0n) meet, the subsequentpair
of impinging (rn + 1 and rn + 1’) can be obtained at ¢xeddistances
(t) measured from their ends, perpendicular to theradii. When two
prisms of di¡erent size collide, the ¢bres ofthe bigger prism
always meet those of the smaller prism at alesser angle to the
horizontal (growth front). Displacement isfaster as the di¡erence
in width between prisms (r0/r00) isgreater. Two examples are
provided for comparison.(a) r0/r00 ˆ 1.5 and (b) r0/r
00 ˆ 2.
(a)
(b)
(c)(d)
advance of the shell growth frontt = 6
t = 1
front
b-axis
front
rear
Figure 6. Competitive growth model for the alignment ofa- and
b-axes of ¢bres during prism growth. (a) Sketch of alongitudinal
section of prisms parallel to the shell growthdirection of the
shell margin (right). See also ¢gure 1g.Prisms initiate as
asymmetric spheruliths, with a moredeveloped frontal part, towards
the shell margin. This causesthe rear prism to displace the one in
front of it progressively,while, in turn, it is displaced by the
prism behind (not shown).As a consequence, the ¢bres running in a
frontal directionbecome progressively more developed. (b) View of
prismsparallel to the shell surface. (c,d ) As ¢bres grow longer
andthicker, competition in the a^b plane (cross-section of
¢bres)causes selection of those ¢bres having their fastest
horizontalgrowth direction (b-axis) orientated parallel to the
shellgrowth direction (arrow).
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://rspb.royalsocietypublishing.org/
-
crystal ¢bres within the prismatic layer starting
fromspheruliths, (iii) the transition from the prismatic to
thenacreous layer, and (iv) increasing scattering of
crystalorientation with addition of new nacreous sheets, as
meregeometrical and crystallographic processes. These pro-cesses
are regarded as epiphenomena arising from theanisotropic growth
rates of biogenic aragonite crystals. Insummary, the inner
periostracum serves as the substratefor nucleation of spheruliths,
whereas the mantle suppliescalcium carbonate and organic components
to the shellgrowth front via the extrapallial space. Finally, our
obser-vations suggest that the physical factors determining
theself-organization of shell microstructures deserve as
muchattention as genetic ones in explaining the formation ofthe
bivalve shell.
This study was ¢nanced by research project PB97-0790 of
theDirecciön General de Ensen¬ anza Superior e
InvestigaciönCient|̈¢ca (Ministerio de Educacion y Ciencia, MEC),
researchgroup RNM-0178 (PAI, Junta de Andaluc|̈a) and an
award(DE-FC09-96-SR18546) from the US Department of Energy tothe
University of Georgia Savannah River Ecology Laboratory.We also
thank the postdoctoral program of the MEC (Spain).We are very
grateful to Professor Chris Romanek (SavannahRiver Ecology
Laboratory), Robert C. Thomas (SavannahRiver Ecology Laboratory)
and Professor Russell Messier (PennState University) for their
useful comments and support.
REFERENCES
Addadi, L. & Weiner, S. 1992 Control and design principles
inbiological mineralization. Angew. Chem. Int. Ed. Engl. 31,
153^169.
Addadi, L., Moradian, J., Shay, E., Maroudas, N. G. &
Weiner, S.1987 A chemical model for the cooperation of sulfates
andcarboxylates in calcite crystals formation. Proc. Natl Acad.
Sci.USA 84, 2732^2736.
Aksay, I. A., Trau, M., Manne, S., Honma, I.,Yao, N., Zhou,
L.,Fenter, P., Eisenberger, P. M. & Gruner, S. M. 1996
Biomimetic pathways for assembling inorganic thin ¢lms.Science
273, 892^898.
Beedham, G. E. 1958 Observations of the mantle of
theLamellibranchia. Q. J. Microscopial Sci.
Belcher, A. M., Wu, X. H., Christensen, R. J., Hansma, P.
K.,Stucky, G. D. & Morse, D. E. 1996 Control of crystal
phaseswitching and orientation by soluble mollusc-shell
proteins.Nature 381, 56^58.
Checa, A. 2000 A new model for periostracum and shell forma-tion
in Unionidae (Bivalvia, Mollusca). Tissue Cell. (In thepress.)
Cullity, B. D. 1977 Elements of X-ray di¡raction, 2nd edn,
pp.127^131. Reading, MA: Addison-Wesley.
Currey, J. D. 1977 Mechanical properties of mother of pearl
intension. Proc. R. Soc. Lond. B196, 443^463.
Dauphin, Y., Cuif, J. P., Mutvei, H. & Denis, A.
1989Mineralogy, chemistry and ultrastructure of the
externalshell-layer in ten species of Haliotis with reference to
Haliotistuberculata (Mollusca: Archaeogastropoda). Bull. Geol.
Inst.Univ. Uppsala 15, 7^38.
Falini, G., Albeck, S., Weiner, S. & Addadi, L. 1995 Control
ofaragonite or calcite polymorphism by mollusk shell
macromo-lecules. Science 271, 67^69.
Grigor’ev, D. P. 1965 Ontogeny of minerals. Jerusalem:
IsraelProgram for Scienti¢c Translations.
Hedegaard, C. & Wenk, H.-R. 1998 Microarchitecture
andtexture patterns of mollusc shells. J. Moll. Stud. 64,
133^136.
Lowenstam, H. A. & & Weiner, S. 1989 On
biomineralization.NewYork: Oxford University Press.
Mutvei, H. 1972 Ultrastructural relationships between the
pris-matic and nacreous layers in Nautilus. Biomineral. Res. Rep.
4,81^86.
Rodr|̈guez-Navarro, A. & Garc|̈a-Ruiz, J. M. 2000 Model
oftextural development of layered aggregates. Eur. J. Mineral.12,
609^614.
Saleuddin, A. S. M. & Kunigelis, S. C. 1984
Neuroendocrinecontrol mechanisms in shell formation. Am. Zool. 24,
911^916.
Scha¡er, T. E. (and 11 others) 1997 Does abalone nacre form
byheteroepitaxial nucleation or by growth through mineralbridges?
Chem. Mater. 9, 1731^1740.
Silyn-Roberts, H. & Sharp, R. M. 1986 Crystal growth and
therole of the organic network in eggshell biomineralization.Proc.
R. Soc. Lond. B 227, 303^324.
Simkiss, K. & Wilbur, K. M. 1989 Biomineralization: cell
biologyand mineral deposition. San Diego, CA: Academic Press.
Sunagawa, I. 1987 Morphology of crystals, pp. 511^581.
Tokyo:Terra Scienti¢c Publishing Co.
Taylor, J. D., Kennedy, W. J. & Hall, A. 1969 The shell
structureand mineralogy of the Bivalvia. Introduction.
Nuculacea^Trigonacea. Bull. Br. Mus. Nat. Hist. Zool. 3(Suppl.),
1^125.
Ubukata, T. 1994 Architectural constraints on the morpho-genesis
of prismatic structure in Bivalvia. Palaeontology 37,241^261.
Weiner, S. & Traub, W. 1980 X-ray di¡raction study of the
in-soluble organic matrix of mollusk shells. FEBS Lett.
111,311^316.
Weiner, S. & Traub, W. 1984 Macromolecules in mollusc
shellsand their functions in biomineralization. Phil. Trans. R.
Soc.Lond. B 3042, 425^435.
As this paper exceeds the maximum length normally permitted,the
authors have agreed to contribute to production costs.
778 A. G. Checa and A. Rodr|̈guez-Navarro Self- organization of
shell microstructures
Proc. R. Soc. Lond. B (2001)
orientated crystalinner shell surface
randomly orientated crystalorganic sheet
Figure 7. Model of accumulation of defects within thenacreous
layer. Tablets of a new nacre sheet will growepitaxially on the
previous sheet, provided that pores of theorganic sheet allow them
to establish direct contact. Other-wise, new tablets will nucleate
with random orientation.These defects accumulate since new nacre
tablets can nucleateepitaxially on randomly orientated
crystals.
on 18 September 2009rspb.royalsocietypublishing.orgDownloaded
from
http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0027-8424^28^2984L.2732[aid=981416]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0036-8075^28^29273L.892[aid=691746,nlm=8688064]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0036-8075^28^29271L.67[aid=981419,csa=0036-8075^26vol=271^26iss=5245^26firstpage=67]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0260-1230^28^2964L.133[aid=981420,csa=0260-1230^26vol=64^26iss=1^26firstpage=133]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0935-1221^28^2912L.609[aid=981421]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0003-1569^28^2924L.911[aid=981422]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0014-5793^28^29111L.311[aid=981424]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0027-8424^28^2984L.2732[aid=981416]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0935-1221^28^2912L.609[aid=981421]http://pippo.ingentaselect.com/nw=1/rpsv/cgi-bin/linker?ext=a&reqidx=/0014-5793^28^29111L.311[aid=981424]http://rspb.royalsocietypublishing.org/