A New Model for Periostracum and Shell Formation in Unionidae (Bivalvia, Mollusca)

Home , Mantle (mollusc), Periostracum

Tissue & Cell, 2000 32 (5) 405Ð416 © 2000 Harcourt Publishers Ltd doi: 10.1054/tice.2000.0129, available online at http://www.idealibrary.com Tissue&Cell A new model for periostracum and shell formation in Unionidae (Bivalvia, Mollusca)

A. Checa

Abstract The periostracum in Unionidae consists of two layers. The outer one is secreted within the periostracal groove, while the inner layer is secreted by the epithelium of the outer mantle fold. The periostracum reaches its maximum thickness at the shell edge, where it reflects onto the shell surface. Biomineralization begins within the inner periostracum as fibrous spheruliths, which grow towards the shell interior, coalesce and compete mutually, originating the aragonitic outer prismatic shell layer. Prisms are fibrous polycrystalline aggregates. Internal growth lines indicate that their growth front is limited by the mantle surface. Transition to nacre is gradual. The first nacreous tablets grow by epitaxy onto the distal ends of prism fibres. Later growth proceeds onto previously deposited tablets. Our model involves two alternative stages. During active shell secretion, the mantle edge extends to fill the extrapallial space and the periostracal conveyor belt switches on, with the consequential secretion of periostracum and shell. During periods of inactivity, only the outer periostracum is secreted; this forms folds at the exit of the periostracal groove, leaving high-rank growth lines. Layers of inner periostracum are added occasionally to the shell interior during prolonged periods of inactivity in which the mantle is retracted. © 2000 Harcourt Publishers Ltd

Keywords: biomineralization, periostracum, shell, microstructure, bivalves, Unionidae

Introduction periostracum (e.g., Hillman, 1961; Dunachie, 1963; Bevelander & Nakahara, 1967; Neff, 1972; Bubel, 1973; Saleuddin, 1974; Richardson et al., 1981) or to periostracum A number of studies on periostracum and shell formation and shell formation (Kennedy et al., 1969; Taylor & have been conducted in gastropods (see reviews in Kennedy, 1969; Taylor et al., 1969; Nakahara & Saleuddin, 1979; Wilbur & Saleuddin, 1983; Lowenstam & Bevelander, 1971; Waller, 1980) in several taxa. Detailed Weiner 1989). For bivalves, there is a good documentation studies were conducted and published (mainly in this on shell microstructures, but relatively few studies on the journal) by Petit et al. (1978, 1979, 1980a, b) and Petit biomineralization process, these being restricted either to (1981), on fresh-water Unionidae (mainly the genus Amblema). They developed a mechanism for biomineralization in unionid bivalves, which was later made extensive to those bivalves with prismato-nacreous shells (Saleuddin & Departamento de Estratigrafía y Paleontología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain Petit, 1983). It can be summarized as follows: Received 8 March 2000 (1) The periostracum is initiated as a pellicle by basal cells Accepted 19 July 2000 located deep in the periostracal groove (between the Correspondence to: Antonio Checa, Departamento de Estratigrafia y Paleontología, Facultad de Ciencias, Universidad de Granada, 18071 outer and middle mantle folds). The pellicle is coated Granada, Spain, Tel.: 958 243201; Fax: 958 248528; on both sides and increases in thickness, thus forming E-mail: [email protected] the outer periostracum. Growth lines are visible on its

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Fig. 1 Petit et al.’s model for shell biomineralization in Amblema (Unionidae). The periostracal groove secretes a thin outer periostracum and a thicker middle periostracum (inset 1). When this periostracum is extruded, the inner surface of the outer mantle fold secretes a third layer (the inner periostracum) below the middle periostracum (inset 2). The inner periostracum later separates from the middle periostracum and develops folds, which disappear before the whole periostracum reflects at the shell margin. During reflection, the middle periostracum becomes vacuolized and forms a cavernous structure called the antrum (inset 3). In time, antrum cavities become filled with aragonite and originate the outer prismatic layer. The inner periostracum also foliates and becomes mineralized, giving rise to the inner nacreous shell. Some free nacreous units, which form within the extrapallial fluid, later adhere to the nacreous layer.

external surface. Still within the periostracal groove, a These free units may later add to the nacreous layer and multi-layered middle periostracum is secreted inside contribute to its growth (Fig. 1, inset 4). the outer periostracum by the glycocalyx of the In summary, the periostracum is composed by three columnar epithelial cells lining the periostracal groove layers. The outer and middle layers are formed within the (Fig. 1, inset 1). periostracal groove, while the inner layer is secreted by the (2) Upon extrusion from the periostracal groove, an addi- mantle epithelium. The middle layer is responsible for tional layer (inner periostracum) is secreted below the secretion of the outer prismatic shell layer through middle periostracum, by the glycocalyx of the processes of vacuolization and antrum formation. Similar columnar cells that line the outer mantle epithelium processes create the nacreous layer from the inner perios- (Fig.1, inset 2). At a short distance from the periostracal tracum. groove, the inner periostracum separates from the rest I have carried out a detailed study of the structure and of the periostracum and becomes highly folded. These mutual relationships of the mantle, periostracum and shell, folds or loops disappear before reflection of the perios- in several species of Unionidae. In the present paper, on the tracum at the shell edge. basis of these new data and observations, I propose a new (3) The extruded periostracum (free periostracum stage) is model for periostracum and shell formation, which is rather long and extends from the exit of the periostracal intended to be of general applicability to unionid bivalves groove to the shell edge, forming a wide extrapallial and which is drastically different from that proposed for space. During reflection of the periostracum, vacuoles Amblema. appear in the middle layer, which begin to be filled with spherical subunits of needle-like crystals. Material and techniques Subsequently, these vacuoles enlarge and fuse, giving rise to elongated columns (Fig. 1, inset 3). With time, Material this system of cavities (antrum) becomes filled with Living specimens of Unio elongatulus Pfeiffer, 1825 spherical crystalline units, which, in packing, finally (Unioninae) and Potomida littoralis (Lamarck, 1801) give rise to the characteristic prisms of the outer shell (Ambleminae) were collected in 1997 at Canal Imperial de layer. Aragón (provinces of Navarra and Zaragoza, Spain). They (4) The inner periostracal layer also thickens at the shell were initially fixed in 10% formaline and preserved in 70% edge and suffers foliation processes in which nucle- ethanol. Quadrangular pieces of the shell margin (usually ation of aragonite needles begin; these later pack into the ventral area) were cut with a small rotating disk (24 mm typical nacreous bricks (initial nacre). Organic in diameter). Care was taken not to damage the mantle proteinaceous layers can also be released into the extra- margin, which was later cut with precision scissors. In this pallial fluid from the inner periostracal layer, serving as way, the entire mantle-shell system could be examined at templates for the crystallization of nacreous layers. the same time. PERIOSTRACUM AND SHELL FORMATION IN UNIONIDAE 407

Given the importance of the shell as information source, scattering or degree of preferential orientation of crystals well-preserved valves of other Unionidae were also studied: can be deduced from the width of intensity maxima of pole the Unioninae Unio crassus Philippson, 1788 (river Meuse, figures. The smaller the width, the less scattered are the Hastières, Belgium) and U. pictorum (Linnaeus, 1758) crystals and the higher the degree of preferential orientation. (Nijvel, Belgium), and the Ambleminae Lamprotula sp. The broadness of the central maxima of the {001} pole (locality unknown, China) and Caelatura bakeri (Adams, figure will account for the average tilting of the c-axis of 1866) (Nyanza, Kenya). crystals. The broadness of the maxima of the {112} pole figure indicates how much a- and b- crystal axes are rotated Optical and scanning microscopy around the c-axis. Samples were prepared so that the equa- Samples of U. elongatulus and P. littoralis were surficially torial plane of the Lambert projection would be parallel to decalcified with 6% EDTA for 20 min at room temperature the outer shell surface. To obtain values of the two shell for SEM observation (Zeiss DSM 950). They were later layers, samples were ground manually parallel to the fixed in cacodylate buffered (0.1 M, pH 7.4) 2.5% external shell surface to the desired level. glutaraldehyde for 4 h at 4°C. Once washed in the same All the material is deposited in the Department of buffer, they were dehydrated in increasingly concentrated Stratigraphy and Palaeontology of the University of ethanols and critical point-CO2 dried. Gold coating in a Granada (labelled SPUG). Polaron 5000 sputtering for 4 min followed. For optical microscopy, samples were completely decalcified, fixed and dehydrated according to the above routine. Observations and Discussion They were included in epoxy resin (Aname Epon 812) after previous infiltration and polymerized at 60°C for 24 h. Sections of 1-µm-thickness (made with a Leica Ultracut S) Outer and inner periostracum were mounted and stained with 1% toluidine blue. The initial pellicle of Petit et al. (1978, 1979, 1980a) has Treatments were performed in the Centre for Scientific been easily recognized by SEM and thin sectioning. The Instrumentation of the University of Granada. process of thickening within the periostracal groove is also Samples of Unio elongatulus, U. crassus, U. pictorum, evident (Fig. 2) and, upon extrusion from the periostracal Lamprotula sp. and Caelatura bakeri were studied with groove, the periostracum has an approximate thickness of SEM. Observations were made on outer and inner valve 2Ð3 µm in Unio elongatulus and Potomida littoralis (Figs 3, surfaces (sometimes with the periostracum partly removed 6), for normal-sized adult animals (75Ð85 mm of anteropos- with 5% NaOH), as well as on transversal radial fractures terior diameter), lower values being typical of P. littoralis. (intact or etched in 1% hydrochloric acid for less than 1 These figures are much lower than those reported by min). Saleuddin and Petit (1983) of 25Ð50 µm in Amblema, but In order to study crystallographic properties with polar- coincide roughly with the thickness of their pellicle, ized-light microscope, shell samples of Unio elongatulus inferred from Petit et al. (1979, Fig. 1; 1980a, Fig. 11). As in and Lamprotula sp. were embedded in epoxy, thin- Amblema, tiny growth lines have been recognized on the sectioned to 30Ð40 µm (with a Buehler Petro-Thin) perpen- outer surface of this layer in the species studied (Fig. 3). dicular to the shell surface along a dorsoventral radius, and Upon extrusion from the periostracal groove a new layer polished (Struers Planopol-V). is secreted below the outer periostracum. This layer is composed internally of micron-sized sublayers (Fig. 4) and Texture analysis is clearly non-tanned. It seems to initiate at the very exit of The three-dimensional orientation of crystals in shells of the periostracal groove and thickens progressively in a Lamprotula sp., Unio elongatulus and Potomida littoralis ventral direction (Fig. 5; compare Figs. 3 and 6), to attain was determined by X-ray diffraction, using a texture 30Ð40 µm at the very shell edge in U. elongatulus and diffractometer (Phillips X’pert). The diffractometer brings 20Ð25 µm in P. littoralis. On the basis of thickness data, this the sample into different orientations and the recorded layer can be confidently identified with the middle perios- intensities are proportional to the number of lattice planes tracum of Petit et al. The only plausible interpretation is that {hkl} that are in Bragg orientation. In our case, the Bragg the inner periostracum is secreted by the inner epithelium of diffraction peaks {001} and {112} were recorded over an extended outer mantle fold. Dunachie (1963) showed almost a full hemisphere (Fig. 12A), from the substrate how the inner surface of the outer mantle fold, outside the normal to a maximum angle of 85° from normal. The inten- periostracal fold, is able to secrete periostracal material in sity data were registered by rotating in 2.5° steps, and tilting Mytilus edulis, but he did not recognize any zonation of the sample 2.5° per complete rotation. Data treatment periostracal layers along this surface. In our samples, this included absorption and background corrections, and isoline fold is dramatically retracted even in previously relaxed calculus. The three-dimensional orientation of the corre- animals, and this effect was enhanced during critical drying. sponding poles can be represented in two dimensions by its However, if, as in all processes of periostracum formation (Lambert) projection in the equatorial plane (Fig. 12A). The (see, e.g., Saleuddin, 1979), epithelial contact is needed, it 408 CHECA

Fig. 2 Light micrograph showing increased thickness of the pellicle within the periostracal groove in Potomida littoralis. The pellicle is firmly attached to the outward-facing surface of the periostracal groove. Growth direction to the left. × 192. Fig. 3 SEM view of a fold formed by the pellicle in Unio elongatulus, after extrusion from the periostracal groove. The area shown is very close (some 150 µm) to the exit of the periostracal groove. Correspondingly, the inner periostracum is extremely reduced. Tiny growth lines formed within the periostracal groove are visible on the outer periostracal surface. Growth direction to the top left. × 340. Fig. 4 Light micrograph of the periostracum of a decalcified shell of Lamprotula sp. The outer periostracum is included within the translucent layer and is folded in a complex fashion. The inner periostracum connects directly with the prisms of the outer shell layer (of which only the organic sheaths remain) and is layered internally. Growth direction to the left. × 384. Fig. 5 Light micrograph of a decalcified shell of Unio elongatulus, showing the free and calcified periostracum stages. The boundary is indicated by an arrow. Folds are formed exclusively by the outer periostracum, just after extrusion from the periostracal groove (not shown). Thickening of the periostracum during the free stage is achieved solely at the expense of the inner periostracum. × 48. Fig. 6 Same specimen and sample as in Figure 2. SEM view of the periostracum at a point closer to the shell edge (some 350 µm) and far from the periostracal groove (some 1300 µm). The fold consists exclusively of outer periostracum, and the inner periostracum is thick (compare to Figure 2). Growth direction to the top right. × 725. Fig. 7 SEM view of a partly decalcified shell margin of a fully-grown specimen of Potomida littoralis. A complex bundle of periostracal folds formed at the shell edge due to continued emission of outer periostracum (sometimes overlying a thin inner periostracum), while shell and inner periostracum secretion was not active. Some bundles continue into the shell as intra-shell layers of inner periostracum. Growth direction to the bottom left. × 150. PERIOSTRACUM AND SHELL FORMATION IN UNIONIDAE 409

Fig. 8 SEM oblique view of a partly dissolved inner periostracum and a fractured outer prismatic layer of Lamprotula sp. Prisms initiate as spheruliths within the inner periostracum and expand transversely to coalesce. Competition among prisms causes disappearance of smaller units. Growth surfaces are continuous among adjacent prisms, indicating synchronized growth. Growth direction to the bottom right. × 340. Fig. 9 Detail of Figure 7 to show the spherulithic initial part of prisms embedded within the inner periostracum. Fig. 10 SEM micrograph of the transition between the prismatic and nacreous layers in Lamprotula sp. The internal fibrous structure of prisms is also evident. With growth, fibres align progressively with the long axis of the prism. Some prisms that disappear by competition are still visible in the outer part. Growth direction to the left. × 725. Fig. 11 Polarized-light micrograph of the outer prismatic layer of Unio elongatulus. Prisms are composed of fibres diverging from the main axis. Divergence becomes smaller towards the nacreous layer (right). Under polarized light, only those crystals with a parallel orientation of their c-axes extin- guish synchronously. Undulating extinction then demonstrates that prisms are polycrystalline aggregates, each fibre being a single crystal. Growth direction to the top right. × 192.

can be assumed that the outer mantle could be extended to backwards at the shell edge just prior to the onset of the shell edge to fill most of the extrapallial space and biomineralization. In aquarium-maintained unionids the secrete the inner periostracum (Fig. 17A). This would also mantle margins advance and protrude slightly from the shell solve the mechanical problem of folding the periostracum in a normal-feeding position. 410 CHECA

Fig. 12AÐD A. The pole of a given {hkl} lattice plane ({112} in the example) is the Lambert projection of its Bragg reflection onto the equatorial plane. When the sample is rotated over a full hemisphere, a record of intensities (pole figure) is obtained for the selected {hkl}. B, C, D. Pole figures for the prismatic and nacreous layers of a shell of Unio elongatulus. Darker colours indicate increasing intensities. The broadness of intensity maxima of {001} pole figures gives an indication of c-axis scattering. It is high in the prismatic layer, which fits in with the fan-like arrangement of prism-forming fibres (see Figure 10). Scattering is low in the nacreous layer, which implies that monocrystalline tablets have their c-axes evenly oriented (see Figure 15). Four defined intensity maxima of {112} pole figure for nacre indicate that, in addition to the c-axis, tablets have parallel a- and b-axes (as in A, the b-axis coincides with the horizontal axis and the a-axis with the vertical one).

An important difference with respect to Amblema is that Thickness data and layering indicate that the middle I have found no traces of an innermost third periostracal periostracum of Petit et al. would correspond to our inner layer. periostracum. However, on a positional basis, with PERIOSTRACUM AND SHELL FORMATION IN UNIONIDAE 411

Fig. 13 Light micrograph of the prismatic and part of the outer nacreous layer of Unio elongatulus. Internal growth lines plunge backwards and are continuous across the prismatic layer and into the nacreous layers. An intra-shell periostracal layer (arrow) originates a small prismatic layer. Both the intra-shell layer and the associated prismatic layer wedge out dorsally. Growth direction to the right. × 95. Fig. 14 SEM micrograph of a partly decalcified shell of Potomida littoralis, showing the contact between the prismatic and nacreous layers. Several intra-shell periostracal layers (with their associated prismatic sheets) can be seen intruding the nacreous layer. Growth direction to the bottom. × 145. Fig. 15 Transition between the prismatic and nacreous layer in Lamprotula sp. Nacreous tablets adapt to the diverging arrangement of basal fibres of a prism. This supports the hypothesis that the first nacreous tablets grow by epitaxy directly onto the fibres. Growth direction to the left. × 1450. Fig. 16 SEM view of the internal growth surface of the shell. Nacreous sheets are arranged step-like. Individual plates are rhombic in outline and, with minor exceptions (arrow), are consistently oriented with their b-axes aligned with the longest diagonal of the rhombi and with the growth direction (upper left). The a-axis is transversal, along the shortest diagonal. × 950. formation outside the periostracal groove, Petit et al.’s inner Periostracal folds periostracum and ours would be equivalent. Given the role Petit et al. (1979, 1980a) mentioned loops in the inner played by the different layers during calcification (see periostracal layer in front of the periostracal groove. I find below) and the uncertainty in recognizing secretion sites in folds in the same area in U. elongatulus (Figs. 3, 5, 6), and Petit et al.’s model, we favour the first possibility. Lamprotula sp. (Fig. 4), but formed by the outer periostracum. Sometimes multiple, these folds are sealed at their 412 CHECA

Fig. 17AÐE A, B. Proposed model for the formation of the periostracum and shell in Unionidae (compare to Figure 1). A. During active shell secretion the outer mantle fold extends and fills the whole extrapallial space. The periostracal groove secretes the outer periostracum (inset 1), and the inner periostracum is secreted by the inward-facing epithelium of the outer mantle fold below the outer periostracum (inset 2). The periostracum reflects at the shell edge, and calcification initiates with the prismatic layer at the shell edge (inset 3) and continues with the nacreous layer a certain distance backwards from the margin (inset 4). B. When the mantle enters an inactive period, both the shell and inner periostracum cease to be secreted, while the outer periostracum continues to be extruded from the periostracal groove (inset 1). Since the periostracal conveyor belt remains motionless, the outer periostracum folds upon extrusion from the periostracal groove (insets 2a and 2b). Folds may be later sealed basally by an inner periostracal deposit when shell secretion resumes. They are later incorporated onto the shell surface as high-rank growth lines. The degree of retraction of the outer mantle fold is uncertain. C, D. E. Formation of intra-shell periostracal layers. C. During prolonged periods of inactivity, the mantle fold retracts and the outer periostracum detaches from the periostracal groove and ceases to be secreted. The free periostracum is expelled from the shell interior, forming a marginal bundle. D. When growth resumes, the periostracal groove and the outer fold epithelium begin to secrete their corresponding periostracal layers at the same time. Therefore, a transi- tory inner periostracum lacking its outer periostracal cover is secreted initially by the outer mantle fold, which is replaced with time by a two-layered normal periostracum (inset 1). As the outer mantle fold extends, the isolated inner periostracum is transferred to the outward-facing face of the fold and to the internal surface of the shell (inset 2). E. Once the outer mantle fold is fully extended, calcification proceeds on the inner periostracal layer (inset).

necks by the inner periostracum (Figs. 5, 6). Since they are periostracum folds because it continues to be extruded at a not present within the periostracal groove, they must form at constant rate from the periostracal groove while the perios- the very exit of the periostracal groove, before or at a very tracal ‘conveyor belt’ ceases to move forwards, in coinci- incipient stage of inner periostracum formation. They are dence with halts in inner periostracum and shell secretion later incorporated onto the shell surface (Fig. 5) and disap- (Fig. 17B). They form at the site of lowest periostracal pear with age due to wear of the periostracum. There are thickness and, hence, lowest resistance to folding. (2) For three ways in which these folds can form. (1) The outer the same reasons, folds can form by compression of the free PERIOSTRACUM AND SHELL FORMATION IN UNIONIDAE 413 periostracum when the mantle margin retracts between outer-mantle-fold epithelium begins to secrete an isolated calcification periods. (3) Formation may result from a inner periostracum (since the outer periostracum is still combination of these first two ways. emerging from the periostracal groove). On sliding along Similar folds are found in P. littoralis, a particularly the fold surface the extreme of this periostracal layer closest intricate bundle of loops being visible at the shell edge of a to the mantle edge soon leaves the secretory fold epithelium fully-grown specimen (Fig. 7). Folds consist of outer perios- and remains extremely thin, but the zones of the mono- tracum, whether or not sealed basally by a drastically layered periostracum which subsequently reach the mantle thinned (3Ð4 µm) inner periostracum. This fits in with edge thicken progressively. Maximal thicknesses were hypothesis (1) above since this pattern implies that growth reached when the mono-layer was replaced by the two- stopped for an extended period, while the outer perios- layered normal periostracum progressing forwards along the tracum continued to be expelled from the periostracal mantle-fold surface (Fig. 17D). To explain how the mono- groove. The presence of a thin inner periostracum associ- layered periostracum attaches to the internal shell surface I ated with some bundles could imply restricted growth assume that the outer mantle fold is retracted within the bursts. The pattern is so complex that it is difficult to discern shell and that the mono-layer moves from the inward- to the individual folds and also which periostracal layer is outward-facing surface of the outer mantle fold. In this way, involved in their formation. In their third dimension, folds it could reach a point of the internal shell surface, somewhat are roughly parallel to the margin and, although they some- back from the aperture, and be progressively added to the times merge with adjacent folds, they reach long extensions. internal shell surface when the outer mantle fold progresses Their regularity and marginal extension point to possibility forwards (Fig. 17D, E). Secretion by a retracted mantle fold (1) above. They are the growth lines usually discerned on is reasonable since, as commented on above, intra-shell the shell surface at low magnification, and are unrelated to layers represent prolonged growth halts. Under these condi- microscopic growth lines of the outer periostracal surface tions, detachment of the periostracum from the periostracal (Petit et al., 1980a, Fig. 11; Saleuddin & Petit, 1983, Fig. groove and from the fold epithelium could occur (Fig. 17C), 15; Fig. 3). The location and aspect of folds in Potomida is so that, when growth resumed, the whole periostracum has so similar to those referred in Amblema (Petit et al. 1979, to be fabricated de novo. The detached old periostracum can Fig. 5, 1980a, Fig. 14) that it is tempting to conclude that the also be expelled from the shell interior forming periostracal origin is the same in both cases. The above-mentioned, dras- bundles similar to those observed in P. littoralis (Fig. 7), tically thinned or absent inner periostracum in Potomida, which connect with intra-shell periostracal layers. prevents the periostracal layer from thickening appreciably after extruding from the periostracal groove. If the speci- Prismatic layer mens examined by Petit et al. were also fully grown, they The outer prismatic layer initiates with spheruliths near the could not appreciate coarsening of the periostracum during base of the inner periostracum, but still within it (Figs. 8, 9). the free stage and easily arrive at the conclusion that the During the bivalve’s life this layer must have behaved in a middle periostracum was secreted within the periostracal viscous fashion, since no cracks associated to spherulith groove. growth have been found. Spheruliths grow inwards and extend beyond the inner boundary of the periostracum. On Intra-shell periostracal layers coalescing, they become polygonal and transform into In some shells, the periostracum is interrupted from time to prisms (Fig. 8). A similar process was described by Taylor time, and it is relieved by a new periostracal band, which et al. (1969) and Waller (1998), and is inferred from the initiates inside the outer prismatic layer and, on reaching the descriptions and illustrations of the Unionidae shell surface, bends forwards to continue the former shell Margaritifera (Mutvei, 1991) and Anodonta (Ubukata, profile. That part of the periostracum internal to the shell is 1994). At this point, geometric selection (Taylor et al., here called intra-shell periostracum (Figs. 7, 13, 14). These 1969) causes the disappearance of smaller units, so that only intra-shell layers are more common near the adult shell edge a few evenly-sized prisms become fully grown (Fig. 8, 10). and indicate growth halts related to reduced growth rate This process was interpreted by Ubukata (1994) as being typical of maturity. Intra-shell layers consist exclusively of due to competition among prisms, in a way similar to that inner periostracum and wedge out towards the shell interior observed in inorganic crystal growth. (Fig. 13). Given the mode in which the inner periostracum is Petit et al. (1979, 1980a, b) assumed that crystals were formed, this implies that contact with the secretory surface preceded by the formation of vacuoles in the inner perios- of the outer mantle fold was progressively more prolonged tracum, which enlarged and fused to form a cavernous struc- from the interior to the exterior of the shell. An isolated ture, called the antrum (see above). Once fully formed, the inner periostracal band can conceivably be formed if the antrum consisted of hollow organic columns with hexagonal whole periostracum were, for some reason, lost and secre- outlines, exactly representing the template for the subse- tion of the outer (at the periostracal groove) and inner (at the quent prisms. Spheruliths nucleate and pack to finally fill mantle fold surface) periostracum reinitiated suddenly and the whole volume of preformed prisms. This model is concomitantly. In this way, the inward-facing surface of the untenable for the following reasons: 414 CHECA

(a) Aragonitic unionid prisms were defined as simple studied. These include three Ambleminae and it is hardly prismatic by Kennedy et al. (1969) and Taylor et al. (1969), conceivable that Amblema has such a drastically different and also by later authors (e.g., Watabe, 1988). Ubukata calcification mode. (1994) was the first to recognize their composite (non- The thickness of the prismatic layer is clearly related to denticular) character, since they are typically composed of the thickness of the inner periostracum and to shell size. In a smaller units which radiate from the main axis and are shell of U. elongatulus the values of inner periostracum and inclined inwards (e.g., Taylor et al., 1969; Carter, 1980). prismatic layer thicknesses, measured under SEM, are (in The composite character of prisms is evident under SEM µm): 3.5/47, 9/80, 20/133 and 35/241, for a range of anteri- observation of intact fractured shells (Fig. 10) and in petro- oposterior diameters of 15.5 to 72 mm. Intra-shell perios- graphic thin section. Polarized-light examination of thin tracal layers are comprised exclusively by inner sections reveals that prisms are polycrystalline, each fibre periostracum (see above) and have the ability to extend pris- being a single crystal (Fig. 11). Also, {001} pole figures of matic deposits partly into the nacreous layer (Figs. 13, 14). the prismatic layer reveal that scattering of c-axes of fibres These periostracal strips wedge out towards the shell inte- is great (Fig. 12B), but there is no random orientation, as rior, in coincidence with reduction in prism size. The rela- implied by the spherulith hypothesis of Petit et al. With tionship of inner periostracum and prisms and of the relative growth, fibres align progressively with the long axis of the thicknesses of both structures is, therefore, evident. As in prism, until a few, slightly diverging, large-sized units the laboratory (in which gels enhance or inhibit nucleation), remain (Fig. 10, 11). None of the techniques reveal traces of it is conceivable that the gel-like inner periostracum in some second-order spheruliths associated with prisms. In addi- way controls not only the presence but also the density and tion, it is worth pointing out the possibility that the size of prisms. spherulithic structures illustrated by Petit et al. (1979, Figs. 10, 15; 1980a, Figs. 19, 21, 22) formed during sample treat- Nacreous layer ment, since the fixatives they appear to have used may, in According to Petit et al. this layer is initiated by cleavage some circumstances, induce artifactual crystallization (see and vacuolization of their internal periostracum, in a way Watabe, 1990). In any case, it is clear that their spheruliths similar to their middle periostracum in relation to the pris- do not correspond to prism microstructure. matic layer. As mentioned above, the third periostracal layer (b) Prisms display internal growth lines, which run of these authors is clearly absent in our samples. across adjacent prisms, indicating synchronized growth At the base of prisms, there is a several-micron-thick (Figs. 8, 13). Growth lines (or surfaces) plunge towards the shell layer, transitional to the nacre, which was previously shell interior away from the margin (Fig. 13). These are recognized by Petit et al. (1980b, figs. 1, 2, 14). In this area, organic-matter-rich surfaces, which remain as transversal prism fibres begin to septate transversely to transform into sheets after decalcification. Similar growth lines were illus- stacked nacreous (Figs. 10, 15). Stacking is not so evident trated in other unionids by Tevesz & Carter (1980), Watabe deeper into the nacreous layer. A likely explanation is that (1984), Wilbur & Saleuddin (1983) and Ubukata (1994), as nacreous plates begin to nucleate by epitaxy on the most well as by Petit et al. (1980a, b). The latter authors inter- distal ends of fibres. This is supported by small changes in preted these structures as fused vacuoles. Formation of orientation of the first nacre crystals on adjusting to the prisms in independent cavities, as the antrum hypothesis somewhat diverging orientation of basal fibres (Fig. 15). implies, could not explain synchronized growth. These SEM observation and texture analysis of unionid growth surfaces rather reflect the successive positions of the nacreous tablets reveal that they are evenly oriented expanded mantle (see above) during inward growth of monocrystals (Figs. 10, 15, 16). Their c-axis is perpendic- prisms. ular to the tablet plane (Fig. 12C), the b-axis perpendicular (c) Prisms usually curve downwards to accommodate the to the margin (i.e., in the growth direction) and the a-axis change in curvature of the growth surfaces (Figs. 11, 13). parallel to the margin (Fig. 12D). Similar pole figures were The mode in which antrum cavities would be formed is not found by Hedegaard & Wenk (1998) in the pterioid clear, but, if it were by tension, prisms would always be Pinctada margaritifera. Texture analysis fits in with the straight. observed rhombic morphology of growing crystals, which (d) The hexagonal outline of prisms and their mutual align their longest diagonal (b-axis) with the growth direc- competition is not explained in the antrum hypothesis and tion (Fig. 16). They differ clearly in outline and pole-figure result from mere crystallographic processes (e.g., Ubukata, pattern from pseudo-hexagonal twinned crystals of nacre 1994; Checa & Rodriguez-Navarro, in prep.). found in other molluscs (Wise, 1970; Mutvei, 1977, 1978; Vacuole and antrum formation are interpretations, prob- Hedegaard & Wenk, 1998). Their monocrystalline nature ably inspired on the compartment hypothesis of Towe & and their common crystallographic orientation precludes the Hamilton (1968), Bevelander & Nakahara (1969) and Towe possibility that nacreous bricks formed by spherulith coales- (1972) for the formation of nacreous tablets in some cence (see, e.g., Saleuddin & Petit, 1983, p. 227). bivalves, and later rejected by Erben & Watabe (1974). The nacreous layer extends along the inner shell surface From our observations, it is clear that a similar hypothesis and constitutes most of the shell thickness (e.g., 89% in an cannot be applied to the prismatic layer in the species adult shell of U. elongatulus). The different lamellae of PERIOSTRACUM AND SHELL FORMATION IN UNIONIDAE 415 nacre are arranged into steps and grow simultaneously (Fig. periostracal groove and annual or semi-annual growth halts. 16), as has been reported in Amblema (Petit et al. 1980a) The formation of intra-shell periostracal layers (see and in other bivalves (Bevelander & Nakahara, 1969; above) can be explained with a similar model. They indicate Taylor et al., 1969; Wise, 1970; Erben & Watabe, 1974; the onset of biomineralization after prolonged periods of Lutz & Rhoads, 1980; Nakahara, 1991). Nacreous tablets inactivity and retraction of the outer mantle fold. Upon nucleate on previously formed crystals and epitaxial growth mantle retraction, the outer periostracum detaches from the explains the common orientation of nacreous tablets periostracal groove and ceases to be secreted. The loose (Fig. 16). periostracal band is expelled from the shell interior, forming As with the prismatic layer, the position of the mantle a marginal bundle (Fig. 17C). When shell growth is to reini- sets the boundary of the forming nacreous lamellae, and tiate, the internal surface of the outer-mantle-fold epithe- their growth front advances when new room is provided by lium begins to secrete an isolated inner periostracum. This the dorsally-inclined outer surface of the mantle epithelium layer slides along the fold surface with subsequent growth in its forward movement during growth (Fig. 13). and is transferred to the outward-external surface of the outer mantle fold and to the internal surface of the shell (Fig. 17D, inset 2). At the same time, it is being replaced by Conclusions the two-layered normal periostracum progressing forwards along the internal fold surface (Fig. 17D, inset 1). The The main conclusions on the mode in which the Unionidae mono-layered periostracal sheet adheres progressively to produce their periostracum and shell are the following (Fig. the inner shell surface as the outer mantle fold extends 17A): forwards (Fig. 17D, inset 2). Once the mantle fold is fully extended and reaches the shell edge, prisms grow on the (1) The periostracum of Unionidae consists of two layers, substrate provided by the internal periostracal layer an outer, thin layer, which is secreted within the perios- (Fig. 17E, inset). Alternatively, as proposed by Dunachie tracal groove, and an inner, thicker layer, which is (1963) for Mytilus edulis, the inner surface of the outer added by the inward-facing surface of the outer mantle mantle fold could also secrete inner periostracum when fold (Fig. 17A, insets 1, 2). exposed to seawater, after detachment of the old perios- (2) Calcification initiates within the inner periostracum tracum. Calcification may reinitiate once the periostracum with spheruliths, which, during early growth, protrude is regenerated and the extrapallial space is sealed off from from the periostracum and coalesce, transforming into the environment. composite prisms. Prism growth is synchronized and In our model, the periostracum has a more reduced role the common growth surface is constrained by the in shell configuration, in comparison to that in Petit et al.’s mantle epithelium (Fig. 17A, inset 3). Prisms extend in model. While the outer periostracum serves only a tight- length as the dorsally plunging mantle epithelium sealing function for the extrapallial space, there is a clear moves forward during bivalve growth. connection between the inner periostracum and the initia- (3) Nacreous tablets begin to nucleate directly on the ends tion of the prismatic layer. Gels in laboratory may enhance of prism fibers by epitaxy (Fig. 17A, inset 4). or inhibit nucleation and the gel-like inner periostracum Transition from composite prisms to nacre is probably might be responsible for the unusual size of unionid arago- induced by merely crystallographic processes (Checa & nitic composite prisms. The fact that both the inner perios- Rodríguez-Navarro, in prep.). tracum and shell are secreted concomitantly by the outer The existence of folds formed by the outer periostracum and mantle fold points to this connection. The rest of present throughout the shell surface implies that calcifica- microstructural features of the unionid shell may be attribut- tion would be a periodic activity, interrupted by refractory able to the activity of the mantle and to the crystallographic episodes. The cycle is two-phase: (a) The mantle margin properties of mineral matter. extends to the shell margin and its outer fold fills the extrapallial space and touches the inner surface of both the free ACKNOWLEDGEMENTS periostracum and the shell growth front. The conveyor belt movement of the periostracum and shell system would be Special thanks are given to Juan de Dios Bueno-Pérez switched on, at the same time that the outer mantle fold (Unidad de Servicios Técnicos, Universidad de Granada), would add new sub-layers to the inner periostracum and to for his technical assistance, and to Drs Serge Gofás the forming shell (Fig. 17A). (b) Inner periostracum and (Muséum National d’Histoire Naturelle, Paris, and, shell cease to be secreted and only the outer periostracum Departamento de Zoología, Universidad de Málaga) and continues to be extruded from the periostracal groove (Fig. Rafael Araujo (Museo Nacional de Ciencias Naturales, 17A, inset 1), with the subsequent formation of folds (Fig. Madrid) for providing specimens. Texture analysis was 17B, insets 2a, b). The physiological origin of these cycles kindly carried out by Dr. Alejandro Rodríguez-Navarro is not clear, but has a period intermediate between those (Savannah River Ecology Lab, University of Georgia). The growth lines internal to the prisms of the outer shell layer or critical suggestions of an anonymous reviewer have signifi- those developed on the periostracal surface within the cantly improved the paper. 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