The Biological Crystals in Chamid Bivalve Shells: Diversity in Morphology and Crystal Arrangement Pattern

Abstract

Chamid bivalves are marine organisms that live in high-energy environments and are cemented to hard substrates. To avoid shell damage, the organisms form thick, densely ornamented shells. Shell material consists of aragonite, and the ornamentation may be either aragonitic or calcitic. The latter can be developed as scaly spines, rows of blades, or comarginal, radial arched lamellae. We investigated biological crystal morphology and mode of assembly of Chama arcana and Chama gryphoides shells. Structural characteristics were obtained from electron backscatter diffraction (EBSD) measurements, complemented with laser confocal and BSE imaging. We found a wide range of crystal morphologies and sizes, ranging from irregularly shaped calcite and/or aragonite prisms to tiny and thin aragonite laths. We observed four different modes of crystal assembly patterns: 1. strongly interlocked dendritic calcite units forming the ornamentation blades; 2. aragonite laths arranged to lamellae forming the outer shell layer, the layer adjacent to the calcite; 3. aragonite laths arranged into blocks comprising inner shell layers or aragonitic ornamentations; and 4. shell portions consisting of aragonite prisms, structured in size and crystal orientation, at muscle attachment sites. These four different types of crystal arrangements were observed for the shells of the investigated chamid species; however, they had slightly different strengths of structuring and slight variations in crystal organisation. Additionally, we observed unique microstructural features in Chama shells: We report ornamentation crystals resembling idiomorphic calcite and novel, twinned entities found at the changeover between the aragonitic layers. We highlight and discuss these differences and anomalies in this contribution.

1. Introduction

To protect their soft tissue from environmental hazards [1,2,3], bivalves form, in general, multi-layered shells. These have different and elaborate crystal arrangement patterns (microstructures). Among invertebrates, bivalves developed one of the strongest muscle–shell attachment mechanisms for valve movement [4]. At those valve portions where the muscles attach to the valves, characteristic scars are present at both inner valve surfaces, the muscle scars [4,5,6]. The muscle scars have a specific optical appearance resulting from their specific microstructure, the adductor myostracum.

Myostracal microstructure and texture appear conservative for species of many bivalve genera. Crippa et al., le Moine, and Hoerl et al. [7,8,9] investigated with diffraction measurements myostracal microstructure and texture for 22 bivalve species belonging to 10 orders. Except for the myostracum of three bivalve species (Arctica islandica, Tellina planata, and Mytilus edulis), the myostracum of all the other bivalves had a similar microstructure and texture. Crystal organisation was determined with electron backscatter diffraction (EBSD). The specific mode of myostracal crystal assembly demonstrates that myostracal crystal growth and, hence, myostracal microstructure is formed by a specific growth process, namely the process of competitive growth [4,6,7,8,9,10].

The myostracal valve portion and other mineralised parts of the shell are composite materials consisting of minerals and biopolymers. Bivalve myostraca consists solely of aragonite, even when the adjacent shell layers are formed of calcite [8,9]. The above-mentioned studies indicate that myostraca comprises, relative to the other shell layers, little organic substance. The aragonite of the myostracum resembles, for many structural characteristics, non-biological aragonite [11,12,13].

Shells of the genus Chama are prime examples of the fact that microstructures may vary even among closely related species. Chama forms thick (up to 5 mm) shells that are densely covered with ornamentations. These consist of pointy blades arranged for the different species in various ways on the shell surface [14,15]. Chama species are common in warm and tidal waters. Most of them occupy shallow environments, up to a water depth of about 30 m [16]; however, some species, such as C. gryphoides, live in deeper environments, at about 270 to 280 m water depth [17]. Except for a few species that rest freely on loose sediment, most chamid shells are cemented to substrates by a byssal complex [14,18]. Chama shells are about 2–3 cm in diameter and have rather irregular shapes and a circular morphology. Some rock-bound Chama organisms have enlarged umbonal regions on the left side of the hinge [19]. An outstanding characteristic of Chama organisms is the notably elongated adductor muscles that enable a rapid and prolonged valve closure despite their notoriously weak hinge [19,20,21].

With their thick shells, complex shell organisation, use of the two main carbonate phases for shell generation, and a large variety of microstructures, Chama is a very suitable genus to investigate the interrelation of calcitic and aragonitic shell microstructures, together with their specific characteristics among the different species. Similar to the Ostreidae, the Chamidae show a high morphologic variation due to ecological adaptations of the respective species [22,23]. The focus of this study is to highlight, juxtapose, and discuss structural characteristics, such as crystal morphology and mode of crystal assembly, of the different layers of two Chama species. We selected shells of C. arcana (Bernard, 1987) and of C. gryphoides (Linnaeus, 1758). These bivalves are abundant in many marine environments [24,25]; however, they dwell at slightly different water depths: C. gryphoides in deeper (up to 280 m) and C. arcana in shallow (up to 30 m) environments, respectively. Crystal structural characteristics, their shape, and their assembly were gained from high-resolution electron backscatter diffraction (EBSD) measurements, complemented with laser confocal microscopy and field emission scanning electron microscopy (FE-SEM) imaging.

Checa [26] discusses possible modes of molluscan shell microstructure generation. These comprise physical processes, such as competitive growth, chemical controls, such as stereochemical recognition, chemical–biological mechanisms, such as nucleation on and between self-organised polymer templates and solely biological determinants, such as contact recognition at biopolymer and mineral component deposition. These processes enable the overall understanding of mollusc shell microstructure generation; however, they do not provide local structural information on the growth characteristics of carbonate molluscan shells, e.g., the transmission of crystal orientations between adjacent shell layers or the patterns of crystallographic axes alignments of crystals belonging to adjacent shell layers [27,28,29,30] and many more.

Many studies investigated the overall structure of bivalve shells, including Chama shells [26,31,32]; however, few studies focused on small-scale and local carbonate crystal arrangement patterns. The study of Crippa et al. [7] provides this information for Glycymeris, but, to the knowledge of the authors, up to now, there is no such full characterisation for species of the genus Chama. Accordingly, the study presented here provides the following:

(i)

An in-depth characterisation of the different shell, ornamentation, and myostracal microstructures and textures for two Chama species;

(ii)

Juxtaposes these;

(iii)

Indicates the crystal growth mechanisms for the different shell layers;

(iv)

Our study highlights, in particular, structural characteristics of the myostracal valve sections, including pallial and adductor myostraca. While the microstructure and texture of pedal and adductor myostraca are investigated by now with EBSD (for Glycymeris species [7]), the measured pattern of crystal organisation for the pallial myostracum has not been reported yet. The latter is performed in this study;

(v)

Myostracal pillars are prominent structures for Chamidae [5]. We characterise the microstructure and texture of these and trace them from the pallial myostracum to the inner shell surfaces.

2. Materials and Methods

2.1. Materials

Shell samples investigated in this study were chosen to illustrate differences in shell microstructures within and between different Chama species and scanned with EBSD. Several shells were investigated in this project, and we report the results obtained for two specimens for each species. The shell sizes of investigated C. arcana samples range from 3 to 5 cm. For C. gryphoides, the shells each had a size of about 3 cm. The investigated species are, thus, considered to be fully grown, adult animals.

Specimens of C. gryphoides were collected in infralittoral rocks near Benalmádena (Málaga, Spain). The specimens of C. arcana were collected in Newport Beach, CA, USA, and obtained from collections of the Natural History Museum (London, UK).

2.2. Methods

2.2.1. Sample Preparation for Electron Backscattered Diffraction (EBSD) Measurements, Field Emission Scanning Electron Microscopy (FE-SEM), and Laser Confocal Microscope Imaging

The shells were sectioned such that the cut crossed the adductor myostracum and adjacent shell layers. The shells of C. arcana were cut either perpendicular to the hinge (cut 1, Figure S1) or transversely through those shell portions where both the anterior and posterior adductor muscles were attached to the valves (cut 2, Figure S1). For C. gryphoides, the shells were cut obliquely through the hinge and the adductor muscles (cut 3, Figure S1). Shells were sectioned with a low-speed diamond saw. The cut shells were embedded in EPON resin and conventionally polished in several mechanical grinding and polishing steps, cloths, and suspensions. For EBSD, sample surfaces were coated with 4–6 nm of carbon. For FE-SEM imaging, sample surfaces were coated with 6–8 nm of Pt/Pd. We show in this study images with BSE contrast.

2.2.2. Secondary Electron (SE), Backscatter Electron (BSE) Imaging, and Electron Backscattered Diffraction (EBSD) Measurements

FE-SEM imaging and EBSD measurements were carried out using a Hitachi SU5000 field emission SEM, Japan, equipped with an Oxford Instruments NordlysNano II EBSD system. For all analytical techniques, the SEM was operated at 20 kV. EBSD measurements were performed with a step size ranging from 200 to 450 nm. EBSD data were evaluated with the Oxford Instruments AZTEC 6.0 and CHANNEL 5 HKL software and are presented as colour-coded crystal orientation maps and the corresponding pole figures. For each measurement, the band contrast images and the pole figures depicting the individual data points of the crystal orientations are depicted in the Supplementary Materials of this research article. The specimens of C. arcana were scanned with 15 EBSD maps, two of which consisted of three individual measurements that were combined into a single scan. For C. gryphoides, nine different measurement positions were chosen, four on each shell sample A and five on shell sample B.

2.3. Terminology

Subsequently, we define the structural terms that we use in this study. For further information concerning EBSD, see Schwartz et al. [33]. For information related to twin formation, see Hahn and Klapper and Griesshaber et al. [34,35]. To index aragonite EBSD patterns, we used the unit cell setting: a = 4.9614(3) Å, b = 7.9671(4) Å, c = 5.7404(4) Å [36].

Microstructure refers to the sizes, morphologies, co- and misorientations, and modes of interlinkage of grains in a material. It is shown with coloured EBSD maps, where similar colours reflect similar crystal orientations, and different colours highlight differences in crystal orientation.

Pole figures are stereographic projections of crystallographic axes orientations measured for all pixels of an EBSD map. With pole figures, we either show individual orientation data points or the density distributions of the orientation data. Showing data points on the lower hemisphere of the stereographic projection ensures that the pole figures are displayed in the same spatial orientation as the corresponding EBSD map.

Texture or crystallographic preferred orientation relates to the distribution of all crystal orientations within a material. It is illustrated with pole figures that show either the colour-coded orientation data or the contoured version of the density distribution of the a-, b-, and c-axes poles. A cylindrical or axial texture is given when the c-axes of crystals are co-oriented (clustered in the pole figure around a specific location), while the corresponding a- and b-axes vary in orientation on a great circle perpendicular to the direction of the texture axis.

We observe with our study two texture modes: (i) a 3D “single-crystal-like” texture and (ii) an axial/cylindrical texture.

A 3D “single-crystal-like”, also called sheet, texture is present when clear-cut maxima are observed in the pole figures of all crystallographic axes. Accordingly, for calcite, we need to observe in the pole figure one cluster for the c-axes and three clusters for the a-axes; for aragonite, one maximum in the pole figure for the a-, b-, and c-axes.

An axial/cylindrical, also called fibre, texture is developed in the relevant material when c-axes show a cluster in one particular direction and the a-axes scatter in orientation on a great circle, perpendicular to the c-axis orientation.

Crystal co-orientation statistics are derived from Kikuchi patterns measured at each pixel of an EBSD map. The degree of aragonite/calcite co-orientation within individual crystals is obtained from measurements of the orientational density distribution, the MUD value.

The MUD (multiple of uniform (random) distribution) value is calculated by the CHANNEL 5 EBSD software and is an indication of the strength of crystal co-orientation. A high MUD indicates high crystal co-orientation, and low MUD values indicate low to random crystallite and/or mineral unit co-orientation. The parameters for data contouring in the pole figures were fixed to a half width of 5 and a cluster size of 3 to maintain comparability for all measurements shown in this contribution. For a half width of 5 and a cluster size of 3, an MUD value of 700 indicates single-crystallinity, and an MUD value of 1 indicates poly-crystallinity.

The EBSD band contrast map depicts the signal strength of the Kikuchi pattern at each measurement point in the EBSD scan. It is displayed as a grey-scale component in the map; white to light grey colours indicate a high intensity of the Kikuchi signal, corresponding to strong mineralisation. Dark grey and black colours point to a weak or absent Kikuchi signal, e.g., when organic matter is scanned.

We describe the presence of twins in different aragonitic microstructures. Twinning is a structural phenomenon where crystals of the same mineral grow together in a symmetrical mode. This can occur during the initial growth of the crystal, or it might take place after its formation, resulting from stress and temperature changes. A twinned crystal is, then, formed. The two parts of twinned crystals might have a variety of configurations. A twinned crystal is a composite crystal of a similar substance, where the adjacent parts, the twin domains or twin individuals, have different but crystallographically related orientations. If the two domains/individuals of a twined crystal meet in a plane, that plane is the composition or twin plane, and the twinned crystal is a contact twin. If the twin domains meet along a complex surface, then the twined crystal is an interpenetration twin.

Aragonite is twinned on the {110} plane. For aragonite, cyclic twinning of penetration twins (showing more than two domains and more than two orientations) leads to pseudo-hexagonal symmetry. A pseudo-hexagonal prism is formed, which consists of three intergrowing twin domains [35], referred to the latter also as triplets [37]. For aragonite, the misorientation boundary is between 63.4° to 64°. We prove the presence of twinned aragonite with the relevant pole figures.

The shell surface of many Chama species is covered with calcium carbonate ornamentations. These are developed as ribs and are thin spicule- to blade-shaped hard tissue protrusions that are often arranged on the surface of the shell in an ordered pattern [14,38].

3. Results

We describe in this contribution structural characteristics and crystal organisation for the different microstructures of C. arcana and C. gryphoides shells. For each species, we show and discuss two specimens (specimen A and specimen B). For these, we present the changeover between the shell layers and detail the prevailing microstructure and texture. For a comprehensive visualisation of the observed microstructures, each colour-coded crystal orientation map (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8) is complemented with the corresponding, grey-scaled, band contrast measurement map, which is provided in the Supplementary Materials (Figures S3–S5, S7, S8 and S11–S13). Aragonite and calcite textures are shown with pole figures (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7 and Figure 8 provide the contoured version of orientation data; Figures S3–S5, S7, S8, and S11–S13 provide orientation data points). The directions of the cuts and the position of the scans are visualised in overview laser confocal microscopy images, provided in the Supplementary Materials section (Figures S1, S2, S6 and S10).

Figure 1, Figure 2 and Figure 3 and Supplementary Materials Figures S3–S5 visualise the microstructure and texture of the different layers of a C. arcana shell sectioned along cut 1. The outer shell surface of C. arcana is covered with calcitic ornamentations. The microstructure of an ornamentation blade is shown in Figure 1a. A calcitic ornamentation consists of large (up to 50 μm in diameter) crystal units (Figure 1a); these are internally structured (Figure 1b). The crystallites that form the calcitic units of the ornamentation are well co-oriented (Figure 1a,b). Towards the inner shell surface, adjacent to the calcite, is an aragonitic shell layer with crossed-lamellar microstructure (Figure 1b). The boundary between the calcite and the aragonite is undulating, a remarkable feature that we observed for all investigated C. arcana specimens (Figure 1a,b). This characteristic is different from what is found for the boundaries between shell layers of Mytilidae, Glycymerididae, and Anomiidae [8,39,40]. Crystal orientation changes gradually within a calcitic ornamentation as the first crystals grow with their c-axis orientation perpendicular to the outer shell surface (Figure 1a). The calcite within individual calcitic units is, rather, co-oriented (Figure 1b), even though the calcite units are substructured by small-angle boundaries (Figure 1b). The interface between the calcitic ornamentation and the aragonitic crossed-lamellar layer is serrated. At the changeover between calcite and aragonite, we observed that some calcitic units show regular (104) growth morphologies; these units resemble idiomorphic crystals (indicated by yellow arrows in Figure 2a). Hence, they exhibit crystal morphologies characteristic of non-biological calcite obtained from solution (Figure 2a). Idiomorphic shapes for biologically secreted crystals are outstanding and very rarely observed. Adjacent calcite units within an ornamentation blade are interdigitated in 3D, as indicated by the dendrite-like calcite microstructure (Figure 2a). This is also a very specific structural feature that has not yet been reported for other modern bivalves.

Adjacent to the calcite of the ornamentation, the interface zone to the crossed-lamellar shell consists of a thin layer formed of minute (average size 1–2 μm²) aragonite crystallites (Figure 2a,b). These are randomly oriented, appear to be granular, and do not show the characteristic crossed-lamellar texture comprising the two sets of first-order lamellae. The well-developed crossed-lamellar microstructure is observable a few micrometres away from the calcite–aragonite boundary (Figure 2a,b). From EBSD measurements, we could deduce that the orientations of the first-order lamellae of the crossed-lamellar microstructure are transmitted to the crystals of both the adductor myostracum (Figure 2b) and the pallial myostracum (Figure 3a). They can also be transmitted from the pallial myostracum onto the initial complex crossed-lamellar portion of the shell (Figure 3a). Nonetheless, the crossed-lamellar mode of crystal orientation is eventually lost within the complex crossed-lamellar microstructure (Figure 3a). The microstructure of the myostracum (Figure 2b and Figure 3b) is characteristic of crystal growth resulting from a competitive growth mechanism. The first myostracal crystals adopt the texture of the crossed-lamellar layer; however, with distance away from the changeover, a distinct microstructure characteristic evolves for competitive growth (Figure 2b and Figure 3b). The crystal size and co-orientation (quantified by the MUD values) increase with distance away from the changeover, and the microstructure is characterised by prismatic crystals and low organic content (Figure 2b). Close to the myostracum, within the crossed-lamellar shell portion, we observed for the investigated C. arcana shells the incorporation of large (up to 20 μm), irregularly shaped aragonite units (yellow stars in Figure 3a,b). This is a characteristic of C. arcana and is not observed in the shells of other Chama species. These aragonite units are often twinned and may pierce into the myostracum. Their function and/or advantage for the shell is not yet known.

Figure 4, Figure 5 and Figure 6 and Supplementary Materials Figures S7–S9 highlight the structural characteristics of another C. arcana specimen. The latter is sectioned along cut 2 and shows the presence of myostracal prisms in the inner shell layer, in addition to the complex crossed-lamellar microstructure. The outer shell layer of this C. arcana specimen also comprises ornamentations consisting of calcite crystals with spherulite-resembling crystal arrangements due to the crystals retaining a high angle to the shell surface (Figure 4a). We observed large crystal units that are highly co-oriented with their c-axes pointing perpendicular to the outer shell surface of the ornamentation (Figure 4a). Sectioned along a traverse cut, the calcitic crystal units of C. arcana appear regular and prismatic. Figure 4b shows the crystal orientation within the aragonitic myostracal pillars located at the inner shell surface of C. arcana. Myostracal pillars mostly comprise large aragonitic prisms, the texture and microstructure of which resemble that of myostracal layers (Figure 4b). Within the myostracal pillars, thin layers of myostracal and complex crossed-lamellar microstructures may alternate parallel to the inner shell surface (yellow stars in Figure 4b). In general, the spherulitic pillars and the surrounding complex crossed-lamellar layer coexist and do not disrupt each other up to the inner shell surface (Figure 5a). The consistent crystal orientations (red star in Figure 5a) show that myostracal pillar crystal orientations derive directly from the pallial myostracum. Similar to the uneven crystal units observed in the first specimen (e.g., yellow stars in Figure 4a,b), large prisms are also visible in the myostracal changeover sections of the second specimen (Figure 5a,b). They appear close to pallial (red star in Figure 5a) and adductor myostraca (yellow star in Figure 5b) and are strongly twinned along clear-cut planes. These crystals may exceed 100 μm and often traverse both the crossed-lamellar and the myostracal shell portion (Figure 5b).

The microstructure and texture of myostracal pillars in C. arcana shells are very intricate (Figure 6). This is the first study that shows measured crystal arrangements for these myostracal pillar structures. Adjacent to the crossed-lamellar layer, a row of small crystals and prisms increase in size towards the inner shell surface. In cross-section, we observed an alternation between the microstructure obtained by competitive growth and the complex crossed-lamellar microstructure of the remaining valve (Figure 6a). Single myostracal pillars often do not cover the entire shell portion from the pallial myostracum to the innermost shell surface, as continuous sheets of complex crossed-lamellar microstructure can interrupt the growth of myostracal pillars (yellow star in Figure 6b). Following this sheet, a further myostracal pillar may form, inheriting the texture of the primary pillar. It appears that non-continuous notches of the complex crossed-lamellar layer do not disrupt the microstructure of myostracal pillars (white stars in Figure 6b).

Figure 7, Figure 8 and Figure 9 and Supplementary Materials Figures S11–S14 show the structural characteristics and organisation of different microstructures found in two different C. gryphoides shells, sectioned along an oblique direction (cut 3). In contrast to C. arcana, C. gryphoides shells are purely aragonitic and construct their ornamentations of aragonite (Figure 7a,b). This outermost shell layer comprises first-order lamellae consisting of granular aragonite crystals that have a low degree of crystal co-orientation (Figure 7a,b). As it occurs at the outer shell region, we addressed the latter microstructure as a complex crossed-lamellar-type structure [11]. According to Taylor et al. and Kennedy et al. [12,38], the complex crossed-lamellar microstructure is only present at inner valve sections. Towards the inner shell surface, the changeover from a complex crossed-lamellar type to a crossed-lamellar assembly is smooth (Figure 7b). However, the texture in the crossed-lamellar layer is rather 3D “single-crystal-like” and shows higher crystal co-orientation than the strictly axial complex crossed-lamellar-type microstructure (Figure 7b and Figure S14b).

As opposed to the C. arcana specimens (e.g., Figure 3a,b) or to other bivalves [8,9], the first-order lamellae in C. gryphoides run at a high angle to the growth direction (Figure 8a,b). At the changeover with the myostracum, the adjacent myostracal crystals adopt epitaxially the crossed-lamellar crystal orientations (Figure 8a,b). Towards the inner shell surface, crystals become increasingly large and prism-shaped (Figure 9a,b). The myostracal layers in C. gryphoides shells show irregular crystal shapes and little-regulated myostracal crystal arrangement patterns (Figure 8a,b and Figure 9a,b). This contrasts significantly with the crystal characteristics that we found for Glycymerididae myostraca [7,9].

4. Discussion

Bivalve shells vary strongly in shape, size, and internal structure [11,41]. To adapt to demands imposed on the organisms by their environment, many bivalves have developed specific shell microstructures [3,42,43,44]. For carbonate-shelled molluscs, up to now, about 15 basic microstructures are described: granular, fibrous-prismatic, columnar-prismatic, sheeted-nacreous, columnar-nacreous, spherulitic, foliated, crossed-foliated, complex crossed-foliated, lamellar, crossed-lamellar, complex crossed-lamellar, vesicular, myostracal, helical and chalky (e.g., [5,7,28,45,46,47,48,49]). While many shell microstructures appear to be highly conservative and recur among various unrelated bivalve species, their origin lies in different evolutionary pathways [49,50,51]. The parallel evolution of shell architectures and microstructures for different bivalve classes started in the Cambrian and is most probably related to an increase in selective pressure for the development of stronger and more protective shells [52,53]. Subsequently, we discuss the different microstructures and crystallographic features found in the shells of C. arcana and C. gryphoides.

4.1. The Microstructures, Textures, and Changeover between Different Crystal Assemblies

Figure 10 gives an overview of the diversity of crystal arrangement patterns and illustrates the microstructures that we observed for the investigated Chama species. We found five different microstructures. Prismatic calcite (1) is present in the ornamentations of C. arcana; however, it is not present in the aragonitic ornamentations of C. gryphoides. The ornamentations of C. gryphoides are aragonitic and have a complex crossed-lamellar type microstructure (2). In addition to the microstructures of the ornamentations, both investigated Chama species construct their shell of crossed-lamellar (3), myostracal (4), and complex crossed-lamellar (5) aragonite crystal assemblies.

Secretion of bivalve shell crystals takes place by mantle epithelial cells. These are secreted below the periostracum, an organic layer formed of cross-linked proteins, mucopolysaccharides, and lipids, and serve as a template for crystal nucleation [54]. To achieve the generation of the ornamentations, the nucleation template is folded before mediating the growth of the shell crystals. Crystal growth direction is, in general, normal to the marginal parts of the mantle. The first crystals grow epitaxially from the folded periostracum [38,55,56]. Therefore, irrespective of whether the ornamentation is aragonitic or calcitic, ornamentation crystal a- and b-axes orientations scatter on a great circle in the pole figure (Figure 2a, Figure 4a and Figure 7a,b) instead of having co-oriented a- and b-axes distributions. The ornamentation texture pattern deviates considerably from that of the crossed-lamellar shell layers. These have a 3D “single-crystal-like” texture.

The changeover from the ornamentation to the adjacent crossed-lamellar microstructure and texture highlights how bivalves achieve a drastic change in microstructure and texture without losing the structural integrity of their shell. Figure 11 gives an overview of the shell layers found in C. arcana near the adductor myostracum region. The changeover from prismatic calcite (ornamentation) to crossed-lamellar aragonite (shell) is achieved via an undulating interface consisting of calcite crystals (Figure 1, Figure 2 and Figure 11b). Along the interface, many calcitic units show regular (104) growth morphologies resembling idiomorphic calcite (yellow stars in Figure 3a,b). The regular growth morphologies indicate a lack of biological control during the growth of the ornamentation. This observation is in agreement with the conclusions of Harper and Checa [57]. The authors investigated the biological influence on the shell growth of euheterodont and pteriomorph species. They found that calcitic euheterodont microstructures, such as the ornamentations in C. arcana, feature a low organic content due to the absence of an organic envelope encasing the prisms. Hence, rather than being controlled by biological determinants, the fibrillar prisms of the ornamentations seem to predominantly form under physical controls [57]. At the changeover to the crossed-lamellar layer, the spatial restrictions imposed by the neighbouring crystals appear to be cancelled. This could explain the regular (104) growth morphologies found along the serrated interface.

A further specific feature of the above-mentioned changeover from the calcitic ornamentation to the aragonitic crossed-lamellar shell can be observed for the first few micrometres of the crossed-lamellar layer. The microstructure of crossed-lamellar aragonite is usually formed of laths that are assembled into two sets of first-order lamellae. However, we observed instead, at the direct transition from ornamentation calcite into crossed-lamellar aragonite, a thin shell layer (white stars in Figure 2a) that is low in crystal co-orientation and rich in organic substance. The latter might be needed to guide aragonite arrangement to form the characteristic crossed-lamellar pattern, as, here, an organic membrane template controlling crystal orientation is absent, and the crystal growth process changes from low (ornamentation) to high (crossed-lamellar) biological control. Similar structural characteristics have been found for the calcite–aragonite interface in the shell of Pinctada margaritafera [58].

In C. gryphoides, the entire shell is aragonitic, and there is no changeover from calcite to aragonite. Nonetheless, the microstructures of the layers are quite different. The aragonite crystals may appear as large prisms (myostracum), thin laths arranged in first-order lamellae (crossed-lamellar), or granules (complex crossed-lamellar type). The ornamentation in C. gryphoides comprises a complex crossed-lamellar-type microstructure with an axial texture (e.g., Figure 7a,b and Figure S14a). The adjacent crossed-lamellar layer shows a 3D “single-crystal-like” texture (Figure 7b and Figure S14b). Regarding crystal morphology, the changeover between those two layers is rather sharp; however, it is smooth regarding crystallographic texture. Thus, it appears that the crystal orientation pattern of the ornamentation is initially transmitted to the crossed-lamellar layer and subsequently “filtered” during its growth towards the inner shell surface (Figure 7b). This may ensure a strong connection between the two layers while maintaining the tough crossed-lamellar microstructure that always comprises only the two sets of first-order lamellae. The similar crystal orientation patterns of the four different microstructures in C. gryphoides interconnect the shell layers without having to construct a metabolically expensive sheet of organic material or losing the microstructural properties of the individual layers.

Our study shows that, for the investigated Chama species, the shell layers differ in microstructure and that adjacent microstructures do not merge into one another. There is no intermediate between adjacent microstructures. The contrary is observed for the textures of adjacent shell layers. (i) If adjacent shell layers are formed of different carbonate polymorphs, calcite, and aragonite (Figure 1b and Figure 2b), then the texture pattern is not transferred from one shell layer to the other. In this case, there is always a marked inlay, rich in organic substance, between the calcitic and the adjacent aragonitic shell portions. (ii) If adjacent layers are formed of the same carbonate polymorph, as is the case for C. gryphoides solely of aragonite (Figure 3a, Figure 5a and Figure 7b), then the texture of the one layer is transmitted to the adjacent shell layer at the changeovers. Crystallographic a-, b-, and c-axes of crystals of the adjacent aragonitic layers have very similar orientations at and close to the interface. With distance from the changeover regions, each of the textures becomes more distinct, and differences between the shell layers become visible. All aragonitic microstructures of the observed Chama specimens show strong twinning on the (110) plane. Hoerl et al. [9] demonstrated the twinning modes in crossed-lamellar, myostracal, and complex crossed-lamellar layers.

4.2. Ornamentation Morphology and Crystallography

Even though most species of Chamidae form a purely aragonitic shell [12], a few Chama species, such as C. arcana, form their shell of aragonite and the ornamentation of calcite. Bivalve ornamentations not only serve as protection for the soft tissue but also have a variety of other functions. They help the bivalve maintain a stable position in the sediment [59,60,61,62], play an important role during burrowing [63,64,65,66], and add to the general stability of the shell [60,61,67]. Through adaptation to various environments and lifestyles, such as to the site of cementation, exposure to wave action, and encrusting biota, bivalves formed numerous patterns of ornamentation sculptures [38,68,69]. The ornamentations of the two investigated Chama species vary in carbonate mineralogy, crystal morphology, microstructure, and texture and are, thus, well suited to demonstrate the structural variety of chamid shells.

The aragonitic ornamentations of C. gryphoides comprise a complex crossed-lamellar-type microstructure. Due to its microstructure with first-order-lamellar blocks that comprise small crystals arranged into an intricate pattern (Figure 7a,b and Figure S14a), this layer was historically described to have a crossed-lamellar microstructure [18]. To our knowledge, this is the first study to describe the microstructure of the complex crossed-lamellar-type layer and its changeover to the crossed-lamellar microstructure in great detail, using electron microscopy and EBSD. The changeover is rather sharp, and the pole figures show dissimilar textures for the two layers: While the complex crossed-lamellar-type layer has an axial texture with a- and b-axes orientations varying on a great circle perpendicular to the c-axes, the texture of the crossed-lamellar layer is 3D “single-crystal-like” (Figure 7, Figure 10c,d, and Figure S14). Despite the initial transmission of the crystal orientation pattern from ornamentation to the crossed-lamellar layer in C. gryphoides shells, the layers are, thus, distinct, and their crystal growth mechanisms might vary. The homogeneous grain morphology and crystal orientation pattern indicate some degree of organic substance control during the growth of the complex crossed-lamellar-type layer.

In contrast, the calcitic ornamentation layer in C. arcana comprises irregular prisms that lack distinct sheaths around the large (up to 200 μm in length) substructured grains. The low degree of organic content and the distinct microstructure indicate a lack of biological control in the ornamentation layer. The latter might induce the internal substructuring and the complex 3D interdigitating dendrite-like calcite crystals as the growing crystals fight for space in the spatially restricted ornamentation. Similar crystallographic configurations have so far been observed for brachiopods and rotaliid foraminifera that comprise interdigitating calcite crystals characterised by dendritic boundaries and low organic content [70,71]. To our knowledge, this study is the first to report this kind of microstructure for bivalve shells.

The poorly ordered yet effective calcitic ornamentation serves as a primary layer of protection in a life stage where the soft tissue is most vulnerable to predators and environmental threats [72,73]. Once the ornamentation is constructed, most Chama organisms form the highly controlled crossed-lamellar layer. This could explain the structural variety of ornamentation sculptures that have been observed for the Chamacea [38]. Thus, the ornamentation carbonate and layers may not be mechanically optimised but rather the result of predominantly physical control and spatial and chemical restrictions imposed by their surrounding environment. Gránásy et al. [74] support the idea that the physicochemical environment in which the biomineralisation takes place may be controlled and manipulated by the genetically encoded organic material. Seeing that the metabolic cost of the calcitic foliated, prismatic, and chalk structures is lower than for aragonitic structures [75], it might be favourable for some species to form calcitic assemblies of crystals. Rather than forming a genetically controlled microstructure with high organic content (as seen, e.g., in Pteriomorphs [57]), calcitic ornamentations in C. arcana contain little organic material and can possibly be deposited more quickly [57,75]. Thus, calcitic ornamentations can serve as an efficient alternative to aragonite in turbulent environments.

The mechanical drawbacks of calcitic ornamentations might be counteracted by the particular ornamentation geometry that is predefined by the periostracum: In C. arcana shells, the calcitic ornamentations are long and run at high angles to the inner shell surface (Figures S2 and S6a), whereas the fully aragonitic C. gryphoides shows a rather shallow and thin ornamentation layer. Living in different environments, the two observed species, thus, might have developed varying evolutionary strategies for the protection of the soft tissue and survival of the organism.

4.3. Microstructural Anomalies

It is quite common that bivalve shells vary in structural characteristics across different species within the same order [5,12,76,77]. This is also the case for C. arcana and C. gryphoides. Those differences usually result from evolutionary processes that cause certain characteristics to appear, disappear, increase, or decrease. However, it is highly unusual to see significant structural differences in a single specimen that do not result from malformation or spatial limitations. In our study, we report two specimens of C. arcana that drastically vary in their shell structure and properties. The differences are visible both on a microscopic and on a macroscopic scale, as reported for the two following characteristics:

• Myostracal pillars or the complex crossed-lamellar layer along the inner shell surface

For the investigated specimens of C. arcana, we see striking differences in their respective inner shell layers. While specimen A exhibits a continuous complex crossed-lamellar microstructure along its inner shell surface, specimen B features myostracal pillars that reach lengths of up to 1 mm. The myostracal pillars are embedded in a complex crossed-lamellar microstructure and are also reported for other species of the Chamacea [12,38]. However, to our knowledge, this is the first study to measure and analyse those structures using modern techniques such as scanning electron microscopy and EBSD. The myostracal pillars have an elongated, ovoid morphology and seem to arise directly from the pallial myostracum, assuming its texture and microstructure (Figure 4a, Figure 5a and Figure 6). Therefore, they comprise large, irregular prisms that have their c-axis orientation normal to the inner shell surface. Similar to the myostraca, the myostracal pillars have a thin organic matrix separating the grains but do not show well-developed interprismatic protein walls. Their microstructure strongly contrasts the complex crossed-lamellar layer [9,38]. The reason why one specimen shows the presence of pillars and the other one does not might be related to the muscular activity of the organisms: Following the detachment of the pallial muscles in specimen B, other muscles are locally attaching to the shell, initiating the growth of the pillars. Periodic attachment/detachment of those muscles would, therefore, explain the alternating sheets of myostracal pillars and the complex crossed-lamellar layer, as observed in our study in some measurements (yellow stars in Figure 4b and Figure 6b).

2.

Formation of single crystal-like units in the crossed-lamellar layer

For all C. arcana shells, we found the presence of anomalous single-crystal-like aragonite units in the crossed-lamellar layers. They can vary in size from 5 μm to more than 100 μm and are usually oriented with their c-axis perpendicular to the inner shell surface. Most of these crystals are located at or near the changeover into the myostracum and match the texture of the surrounding crossed-lamellar layer. In specimen A of C. arcana, aragonite crystals appear to be clustered in scattered units of around 20 μm diameter (yellow stars in Figure 3a,b). They comprise differently oriented crystals that are twinned along irregular planes. On the other hand, specimen B of C. arcana also shows large crystal entities in addition to the small clusters. Those entities (stars in Figure 5a,b and Figure S9) may exceed a length of 100 μm and a thickness of 25 μm. Similar to the small clusters, they are strongly twinned; however, the twin plane is usually a clear-cut and straight boundary. The direction of the cut may affect the appearance of the entities; however, the large divergence in length and thickness between the specimens points towards a structural difference. To our knowledge, this study is the first to report single-crystal-like entities within crossed-lamellar layers in Chama shells or even bivalve shells in general. Therefore, their exact origins and growth mechanisms are uncertain. Opposed to the myostracal pillars, they probably do not stem directly from muscle–shell attachment: (i) individual crystal morphologies are irregular and can vary in thickness (e.g., Figure 5a,b and Figure S9), (ii) larger crystal entities can penetrate the adductor myostracum without assuming its texture or microstructure and (iii) in contrast to the myostraca, there is no gradient in crystal size, e.g., as is the case for crystal that grow through growth competition (see Figure AP11 in [9]).

Reviews of Taylor et al., Kennedy et al., and Ponder et al. [5,12,39,78] highlight the rich portfolio of shell microstructures that bivalves developed throughout the Phanerozoic. The microstructure diversity for bivalves that we see today is far above that of any other calcifying phylum [46]. Mineralisation by bivalves started in the lower Cambrian with the genera Fordilla and Pojetaia. The microstructure of these two genera was always foliated aragonite [79]. If we take into account all molluscs, then there already was a wide variety of mollusc microstructures in the Cambrian: fibrous, prismatic, crossed-lamellar (weak evidence), calcitic semi-nacre, foliated calcite, and foliated aragonite [52,79,80,81,82]. The nacre in the Cambrian Mellopegma [59] was reinterpreted as calcitic semi-nacre [81], and the evidence of crossed-lamellar microstructure in Yuwenia [59] is very weak. Hence, molluscan nacre was absent in the Cambrian, and the presence of the crossed-lamellar microstructure is very doubtful. Thus, the two main microstructures that we observe today for mollusc shells, the nacreous and cross-lamellar crystal arrangements, were not present in the Cambrian and appeared in the Ordovician. Even though laminated calcite formation by molluscs is reported for the Cambrian [81], the increased production of calcitic bivalve shells started in the late Permian to Early Triassic [83,84,85,86]. Although the cause for mineralised tissue formation is still disputed, one prominent hypothesis is that it was evoked by the onset of predation. The latter called forth the initiation of diverse defensive solutions. These include the development of differently sized and formed shells with various thick shell walls, the formation of layered shells, and the development of diverse crystal orientation patterns for the different shell layers (e.g., [52,81]).

Mechanical property characterisation of bicarbonate microstructures demonstrated that these are variably suitable for protection against mechanical and chemical external threats. For example, the crossed-lamellar microstructure, the most widely used microstructure by Mollusca today, has a higher fracture toughness and ability to dissipate cracks relative to granular and nacreous crystal arrangements (e.g., [39,87,88,89,90,91,92]). However, the crossed-lamellar structure also has an increased organic contact, as each lath of the first-order lamellae is encased by an organic substance (Figure 20d,e in [7]). The organisation of the organic substance in the crossed-lamellar microstructure is more complex and intricate (Figure S20a–c in [7]) relative to the organisation of the organic substance in shells with granular structures (e.g., Arctica islandica, Figures 2b and 3b in [93]) and nacreous microstructures (e.g., Mytilus galloprovencialis, Haliotis glabra, Figures 3–5 in [39]). Furthermore, the functional significance of calcite, relative to aragonite, is uncertain, as calcitic microstructures are, in general, softer than aragonitic [9,39,75,94,95] and do not seem to provide notable benefits regarding shell dissolution or protection from external chemical attacks [96,97,98,99].

Hence, the “best” microstructure for biological hard tissues depends not only on its mechanical properties but also on the metabolic cost and the constraints imposed by the surrounding environment. Thus, we hypothesise that calcite and aragonite seas may play primary roles in determining the initial carbonate mineralogy; however, their subsequent influence on skeletal mineralogy is limited [100,101,102,103,104].

5. Conclusions

For a trade-off between functionality, mechanical properties, and metabolic cost, bivalves can choose from a wide variety of crystal arrangements when forming their hierarchical shell.

Although the Chamidae are mostly sessile, they live in different ecological environments that pose challenges that benefit some subspecies more than others. This phenomenon might explain the significant structural diversity we observed among chamid shells. We found differences not only in the ornamentation, the first-formed layer that is epitaxially controlled by the periostracum, but also in the inner shell layers and the muscle attachment behaviour.

In this contribution, we highlight and discuss differences in shell hierarchy and microstructure between and within different species of Chama. From our structural results, we conclude the following:

• For C. arcana and C. gryphoides, five distinct microstructures can be observed. From the outer to the inner shell surface, these microstructures are Prismatic calcite (only in the ornamentations of C. arcana), complex crossed-lamellar-type aragonite (only in the ornamentations of C. gryphoides), and crossed-lamellar, myostracal, and complex crossed-lamellar aragonite;
• Depending on the type of muscle attachment, myostracal microstructures may appear as thick patches (adductor), as a thin and hollow hemisphere (pallial), or as pillars arising from the pallial myostracum;
• In some, but not all, shells of C. arcana, myostracal pillars traverse the inner complex crossed-lamellar layer. They have a spherulite-resembling shape comprising large aragonite prisms that grow continuously from the pallial myostracum and, most probably, follow a competitive growth mechanism;
• Myostracal pillars adapt to the texture of the pallial myostracum. Muscular detachment and reattachment can cause myostracal pillars to be interrupted by sheets of complex crossed-lamellar shell material. With further growth of the shell, the competitive growth mechanism restarts and assumes at first the texture of the preceding (in this case, complex crossed-lamellar) layer;
• The pallial and adductor myostraca follow a competitive growth mechanism. The crystal orientation pattern of the crossed-lamellar layer is transmitted onto the myostracum and onto the complex crossed-lamellar layer. The changeover region from crossed-lamellar to myostracal layers in C. arcana shells features large (up to 100 μm), single-crystal-like crystals with irregular morphologies twinned along one or few boundaries. These crystals were not yet observed in crossed-lamellar layers, and their origin and growth mechanism are not known yet;
• Ornamentations in chamid bivalves may be either calcitic or aragonitic. Calcitic ornamentations (e.g., in C. arcana) comprise a prismatic microstructure of large (up to 200 μm in diameter) crystals that have their c-axes oriented perpendicular to the outer shell surface defined by the periostracum. The aragonitic ornamentations in C. gryphoides comprise a complex crossed-lamellar-type microstructure that resembles the inner layer featuring first-order-lamellar blocks that comprise small third-order lamellae;
• The crystal orientation pattern is transmitted at the changeover from the aragonitic ornamentation to the crossed-lamellar shell in C. gryphoides. Since the textures of the complex crossed-lamellar-type ornamentation (axial) and of the crossed-lamellar layer (3D “single-crystal-like”) are different, the crystal orientation pattern is lost a few μm after the interface;
• The changeover from the calcitic ornamentation to the crossed-lamellar layer in C. arcana is sharp and features an organic-rich aragonite sheet that might be needed to mediate the biologically controlled growth of the crossed-lamellar layer and to connect the two layers;
• The serrated interface between calcitic ornamentations and the aragonitic crossed-lamellar layer in C. arcana exposes some regular (104) growth faces of calcite, resembling the morphology of idiomorphic crystals. As indicated by the disordered crystal arrangement and substructured, irregular units, the crystal growth mechanism of the calcitic ornamentation seems to be predominantly physical.