Crystal Organisation of Muscle Attachment Sites of Bivalved Marine Organisms: A Juxtaposition Between Brachiopod and Bivalved Mollusc Shells

Abstract

The movement of valves of bivalved invertebrates is enabled through the action of muscles and the interplay between the muscles and the hinge ligament. The muscles that move the valves attach to their internal surface. To promote the structural integrity at the mechanically mismatched interfaces, a specific crystal microstructure and texture are present at the muscle attachment sites. These are different from the crystal microstructure and texture of the rest of the valves. We present here for modern two- and three-layered brachiopod shells (Magellania venosa, Liothyrella neozelanica and Gryphus vitreus) the mode of crystal organisation at sites of adductor and diductor muscle attachments (i) relative to the microstructure and texture that forms the other sections of the valves and (ii) relative to crystal organisation of muscle attachment sites of bivalved invertebrates of other phyla, namely, species of the class Bivalvia. We discuss similarities/differences in Ca-carbonate phase, microstructure and texture between rhynchonellate brachiopods and bivalves, and discuss whether the Ca-carbonate crystal organisation of muscle attachment sites is convergent for bivalved marine organisms. We show significant differences in muscle attachment site architecture and highlight the different structural solutions developed by nature for shells of marine organisms that serve the same purpose.

1. Introduction

The ability to initiate valve movement and to maintain gape is of fundamental importance for bivalved invertebrates, such as bivalves or brachiopods. The latter organisms are filter feeders, and opening their valves and keeping them open for a prolonged period enables the circulation of nutrient-loaded waters between the valves and guarantees their survival [1,2,3]. It is equally important to close the valves quickly and keep them tightly shut in the face of an external threat to protect the soft tissue and the organs [4,5]. Valve movement requires the action of muscles. Brachiopods use their adductor and diductor muscles to control valve opening and closure, and use their (pedicle) adjustor muscles to orient the shell as a whole, relative to the substrate (e.g., [1,6,7,8,9]). For valve activities, bivalves always utilise their adductor and pedal muscles in combination with their hinge ligament (e.g., [10,11,12,13]).

Invertebrate hard tissue secretion is performed by epithelial mantle cells that line the inner valve surface [14,15]. The invertebrate epithelial cell layer comprises different types of secreting cells (e.g., [10,15,16,17,18]). Specialised epithelial cells differ in ultrastructure from those that secrete the crystals of the rest of the shell [19]. It has been shown for bivalves that the morphology and arrangement of crystals of those valve sections where the muscles attach differ significantly from the morphology and organisation of crystals that form the other sections of the shell [20,21,22].

Crystal shape, microstructure, and texture of bivalve muscle attachment sites, the myostraca, have been extensively investigated [20,21,23], and profound knowledge has been accumulated on myostracal crystal formation, morphology and organisation. It has been shown that bivalve myostraca are always aragonitic, even when the shell consists of calcite [21,23] and have, in general, but not exclusively [24], a very specific microstructure and texture [21,23]. The outstanding microstructure and texture of bivalve muscle scars are largely the result of the crystal growth process that governs the growth of the myostracal crystals, namely, crystal growth through growth competition. When crystals form through growth competition, in biogenic as well as inorganic environments, due to high supersaturation, many crystallites form at nucleation and, at growth, compete for space [25,26]. With ongoing growth, only crystals with their growth vector normal to the orientation of the nucleation template grow to large entities [25,26,27]. The effect of the competitive growth process is that, with progressive growth, there is a strong decrease in the number of crystals, accompanied by a strong increase in crystal size. Furthermore, for bivalves, myostracal aragonite c-axes become aligned in parallel and oriented normal to the inner shell surface and parallel to the orientation of organic filaments that connect the muscle fibres to the crystals of the myostraca [23,26,28,29,30,31].

The assembly of competitive-growth-generated myostracal crystals has been observed for species of many bivalve genera [20,32,33] and, accordingly, it has been suggested that bivalve myostracal microstructure is conservative [20]. This is not entirely the case, as: 1. Hoerl [24] surveyed myostracal crystal organisation of more than 30 bivalve species from 11 bivalve orders and, even though many species secreted their myostraca with a competitive growth induced crystal organisation, for some species, the myostracal crystal microstructure did not imply that the crystals grew through growth competition. 2. Recent work demonstrated that the competitive growth-generated bivalve myostracal microstructure is inconsistent [21,23]. Influencing factors for the modulation of the bivalve myostracal microstructure and texture were (i) the microstructure and texture of the template, of the shell layer where the myostracal crystals nucleate, and (ii) adaptation of shell microstructure and texture for survival in different environments [21,23,24]. Hence, when investigated in great detail, even though comparable, myostracal microstructure and texture cannot be considered conservative across the Bivalvia.

Organisms of the class Rhynchonellata (phylum Brachiopoda) are sessile, filter-feeding and form calcitic shells [34,35,36]. They are widespread in many marine environments [35,37,38,39,40,41,42,43,44]. Like bivalves, brachiopods need to open and close their valves quickly and maintain them in an open or closed position for extended periods [45,46]. Furthermore, cell ultrastructural studies have shown that the connection of muscle fibres to the muscle attachment sites and the ultrastructure of the muscle attachment site secreting cells are similar for rhynchonellate brachiopods and bivalves [6,10,19,47,48]. Accordingly, the following questions led to the present study: 1. As brachiopods are also bivalved marine organisms that depend upon valve opening and closure, but are not closely related [49], do we see similarity in carbonate phase, crystal morphology and crystal organisation between brachiopod and bivalve muscle attachment sites? 2. Does the brachiopod muscle attachment site also have a competitive growth-related microstructure and texture? 3. Brachiopod, bivalve and gastropod muscle attachment site crystals are secreted by specialised cells. The attachment of the muscle fibres to the crystals of the muscle scar is comparable for brachiopods, bivalves and gastropods [29,47,50]. Thus, is the structure and crystallography of the muscle attachment section of the valves convergent across the phyla of Brachiopoda and Mollusca?

In this contribution, we highlight and discuss the crystal organisation of muscle attachment sites for two and three-layered modern rhynchonellate brachiopod shells, namely, for Magellania venosa (Dixon, 1789; two-layered shell), Gryphus vitreus (Born, 1778; three-layered shell) and Liothyrella neozelanica Thomson, 1918 (three-layered shell). Rhynchonellate brachiopod shells consist of a maximum of three layers (e.g., [51] and references therein). All rhynchonellate brachiopod shells have a primary and a fibrous shell layer. The primary layer forms the outer shell layer, and the fibrous layer forms the inner shell layer. G. vitreus and L. neozelanica form their shell of three layers: a primary, a fibrous, and an innermost columnar layer [51,52]. For the G. vitreus shell, we found that the three shell layers are positioned next to each other; for the L. neozelanica shell, we observed a sequential alternation between the fibrous and the columnar microstructure at the inner part of the shell [51,53]. In addition to the questions above, this study examines a possible difference or similarity of the crystal organisation at muscle attachment sites between two-layered and three-layered shells, i.e., when the muscles attach to fibres or columns.

Although there are very many similarities between lifestyle, living environment, the biomineralisation system, valve action and utilisation of muscle types for valve movement between Bivalvia and Rhynchonellata, we found that the muscle attachment sites as a whole, their structural characteristics and imprint appearance differ significantly. However, we found that the microstructure of the muscle attachment site is changed for species of both invertebrate classes (Rhynchonellata and Bivalvia), relative to the microstructure of those shell sections where muscles do not attach. A possible reason for the latter could be that specialised, cuboidal cells secrete the muscle attachment site crystals for species of both invertebrate classes. In contrast, the remainder of the shell is secreted mainly by columnar epithelial cells [6,54].

We found similar structural characteristics of muscle attachment sites between Rhynchonellata and Bivalvia in crystal texture and carbonate crystal c-axis orientation, irrespective of the developed Ca-carbonate phase (calcite or aragonite) of the muscle attachment site crystals. The latter appears to be an important requirement for a tight and strong connection between the muscle strands and the crystals [19]. For species of both invertebrate classes, the strength of the muscle strand-crystal attachment is achieved with the insertion of collagen fibrils, derived from the apical microvilli of the tendon cells, into the crystals. Interestingly, this fundamental process, namely the insertion of polymer fibrils into the muscle attachment site crystals and their morphological orientation, is present for both rhynchonellate and bivalve species. However, it is unclear whether the two classes developed this trait independently [55,56,57,58].

2. Materials and Methods

2.1. Materials

We investigated the occurrence and the distribution of muscles and the structure, microstructure and texture of their attachment sites to the valves for the modern brachiopods Magellania venosa (Dixon, 1789), Liothyrella neozelanica Thomson, 1918, and Gryphus vitreus (Born, 1778). M. venosa was sampled at Comau Fjord, southern Chile, L. neozelanica at Doubtful Sound, New Zealand, and G. vitreus near Montecristo, Italy. Samples were collected alive from different depths, and care was taken not to damage the soft tissue upon collection. We investigated ten specimens of M. venosa, six specimens of G. vitreus and three specimens of L. neozelanica. Only those shells were examined where the valves were fully and tightly shut. This ensured that, at very careful opening of the valves, the muscles were still attached in their original position to the calcite of the valves.

2.2. Sample Preparation

The shells were air-dried and, subsequently, the valves were opened. First, the lophophore and the lophophore support were removed. Then, the coelomic epithelium, the epithelial cover that shields the brachiopod musculature, was removed. For removal of the latter, only tweezers were used; we did not use any chemicals or water to expose the muscles.

The shells were cut dry for electron backscatter diffraction (EBSD) preparation, and the saw blade was not cooled with water. This ensured that, upon embedding into EPON epoxy resin, the muscle fibres remained at/adjacent to the calcite of the valves. The latter also enabled us to discriminate between valve sections where the striated and/or smooth adductor muscle fibres attach. The valves, hinge and muscles were cut perpendicular to the mirror plane of the shell (Figure S1A), and shell/muscle slices were embedded into epoxy resin. We also investigated cross-sections through the anterior part of the valves; here, muscles are not present. All shell muscles of Rhynchonelliformea are concentrated within the posterior part of the shell (Figure S1A). The embedded shell and muscle samples were subjected to several mechanical grinding and polishing steps. The grinding and polishing agents were not water-based, but oil-based. The final preparation consisted of etch-polishing in a vibratory polisher, performed for 15 min. For measurements, the samples were coated with 4–6 nm of carbon.

2.3. Methods

First, we identified the shell regions where the different muscles attached to the valves. This occurred via micro-computed tomography (Phoenix V|tome|x S 240; GE HealthCare, Chicago, IL, USA), as visible in Supplementary Video S1. Subsequently, the valves were imaged with the different types of muscles still in place with a Digital Microscope (VHX-7000, Keyence, Osaka, Japan), a Confocal Laser Scanning Microscope (VK-X1000, Keyence, Osaka, Japan), a Field Emission Scanning Electron Microscope (FE-SEM; SU5000, Hitachi, Tokyo, Japan) and an Environmental Scanning Microscope (Quattro S, Thermofisher Scientific, Waltham, MA, USA). EBSD measurements were carried out with a Hitachi SU5000 FE-SEM, equipped with an Oxford Instruments Nordlys Nano EBSD detector. EBSD scans were taken at 20 kV and were performed with a step size of 300 to 800 nm. EBSD data were evaluated with the Oxford Instruments AZtec Crystal 3.0 and HKL CHANNEL5.0 software. For indexing the calcitic EBSD patterns, we used the unit cell setting: a = b = 4.99 Å and c = 17.07 Å. Rather than providing the crystallographic texture for the bulk sample, data obtained via EBSD measurements does not exceed 50 µm × 50 µm × 10 nm. However, in contrast to X-ray diffraction or neutron diffraction, the texture is not only shown by pole figures for the bulk sample, but can be determined for small regions or layers. For shells of each species, the crystallographic texture is generally consistent, as indicated by additional unpublished measurements and Supplementary Figures S5–S7. The EBSD pole figure coordinate system is indicated in the first shown pole figures and applies to all measurements shown in this study.

2.4. Terminology

Subsequently, we define terms related to microstructure and texture determination that we use in our contribution. For the description of brachiopod soft tissue morphology and histology, we use the terminology given in James et al. [6] and Bubel [54]. In almost all figures, we show a sketch of the topological relationship between the muscles and their attachment to the dorsal and ventral valves. The sketch was inspired by Figure 22 of James et al. [6], but redrawn and modified to highlight the specific muscles shown in the figure. Our study refers to modern species of rhynchonellate brachiopods. The terminology used for structural aspects of brachiopod and molluscan is given by MacKinnon [47] and Chantler [59], respectively. For further information concerning EBSD analysis, see [60].

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

Pole figures are stereographic projections of crystallographic plane-normal or axes orientations measured for all pixels of an EBSD map or selected areas (subsets). The viewing direction of the pole figures is the same as the viewing direction of the corresponding EBSD maps. All pole figures shown here display the lower hemisphere. 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. With pole figures, we show either individual orientation data points or the density distributions of the orientation data.

Texture or crystallographic preferred orientation relates to the distribution of all crystal orientations within a material. It is illustrated with pole figures, which show either the colour-coded orientation data or the contoured version of the density distribution of crystal orientation poles. In our study, we observed mainly axial/cylindrical textures, which are characterised by the calcitic c-axes clustering in one particular direction and the a*-axes scattering 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 multiple of uniform (random) distribution (MUD) value.

The MUD value is calculated by the EBSD evaluation softwares 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. For the parameters fixed for our study (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 given MUD values indicate the crystal co-orientation strength for the described EBSD scan (or a subset of it) and do not apply to the entire volume of a microstructure.

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.

Biological convergence is the process by which unrelated or distantly related organisms develop similar traits or characteristics independently. This happens because these organisms face similar requirements due to their lifestyle, environmental conditions, or ecological roles that require similar solutions, leading to the natural selection of comparable adaptations.

In recent rhynchonellate brachiopods and bivalves, we can differentiate two main types of muscle fibres [47,59]. The twitch-type striated muscles, causing quick, incomplete closure, feature small muscle cells that interconnect in a banded pattern at a small angle to the fibre axis. In contrast, smooth muscles comprise distinct units of unstriated, separated fibrils that contract slowly and shut the valves. Compared to bivalves, this delayed relaxation or catch mechanism is much weaker in the rhynchonellates [61,62].

3. Results

In this study, we investigated the structural organisation and crystallography of the crystals at the attachments of valve-moving muscles to the valves. First, we describe the different muscles that move brachiopod valves. Subsequently, we describe the crystals to which the muscles attach. M. venosa is the largest modern rhynchonellate brachiopod; it secretes the thickest shell and is the largest of the three investigated species. Accordingly, it forms large-sized muscles, relative to the muscles of L. neozelanica or G. vitreus (Figure 1) or other modern rhynchonellate species. As the large-sized muscles were easier to localise, we concentrated our ultrastructural description of the muscles on the muscles of M. venosa, complemented, however, by findings on L. neozelanica and G. vitreus. When describing the different muscles in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figures S2–S4, a modified sketch of James et al. [6] was added to illustrate the arrangement of the different muscles relative to each other, highlighting their attachment positions onto the valves. We investigated the attachments of all three brachiopod muscles to the valves and examined the latter for both valves.

Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figures S2–S4 highlight ultrastructural characteristics of the muscles used by modern rhynchonellate brachiopods for valve and shell movement. The adductor muscles close the valves, the diductor muscles open them, the (pedicle) adjustor muscles move the shells as a whole for orienting them appropriately to, e.g., currents and/or light [6,62,63,64]. Figure 7, Figure 8, Figure 9, Figure 10, Figure 11 and Figure 12 visualise the crystal organisation in those valve sections where the muscles attach. This study focuses on the morphological, arrangement, and crystallographic characteristics of valve crystals at attachments of adductor muscles, whose function—the closure of the valves—induces higher stresses at these attachment sites. This work analysed the crystallographic microstructure, carbonate phase, crystal texture, and co-orientation strength. The investigated shells were sectioned in one direction, perpendicular to the mirror plane through the shell (Figure S1A). Cross-sections were obtained through the valves and the muscles. When investigating the crystals at muscle attachments, we scanned with EBSD those valve portions where the attaching muscles could still be seen in SE micrographs (e.g., Figure 9E and Figure 10B, SE micrographs of Figure 12B,C). This ensured that the EBSD measurements were performed at valve sections where the muscle bundle was attached. Nonetheless, for each investigated species, we scanned with EBSD also valve portions that were distant from the muscle attachment sites (e.g., Figure 7A, Figure 10A and Figure 12A). These scans were used as a reference for shell microstructure and texture, which is intrinsic to the hard tissue of a particular brachiopod species.

Figure 1. The shell, valves and different muscles of the brachiopod species investigated in this study: Magellania venosa (B), Liothyrella neozelanica (C) and Gryphus vitreus (D). (A): Sketch, modified after [6], depicting the two brachiopod valves and indicating the structure and attachment positions of adductor, diductor and adjustor muscles to the valves as well as the attachment location of the tendon of merged the dorsal valve adductor striated and smooth muscles. The valve-moving muscles of rhynchonellate brachiopods develop in pairs. Thus, within the shell cavity, there is always a pair of adductors (red arrows in (B–D)), a diductor and an adjustor muscle bundle. In addition, each adductor muscle of the pair of adductors consists of two branches (white and blue arrows in (B–D)). For all three investigated species, the pair of adductor muscles branches is well visible, positioned left and right of the hinge (blue and white arrows in (B–D)) and consisting of the smooth and the striated adductor muscle fibres (shown in Figure 6 and Figure S2).

Figure 1. The shell, valves and different muscles of the brachiopod species investigated in this study: Magellania venosa (B), Liothyrella neozelanica (C) and Gryphus vitreus (D). (A): Sketch, modified after [6], depicting the two brachiopod valves and indicating the structure and attachment positions of adductor, diductor and adjustor muscles to the valves as well as the attachment location of the tendon of merged the dorsal valve adductor striated and smooth muscles. The valve-moving muscles of rhynchonellate brachiopods develop in pairs. Thus, within the shell cavity, there is always a pair of adductors (red arrows in (B–D)), a diductor and an adjustor muscle bundle. In addition, each adductor muscle of the pair of adductors consists of two branches (white and blue arrows in (B–D)). For all three investigated species, the pair of adductor muscles branches is well visible, positioned left and right of the hinge (blue and white arrows in (B–D)) and consisting of the smooth and the striated adductor muscle fibres (shown in Figure 6 and Figure S2).

Figure 2. Bundles of adductor muscle fibres that attach to the dorsal valve of Magellania venosa. The thickening of the dorsal valve at the attachment sites of the muscles and the hinge is well observable from the micrograph (yellow arrows in (A)) and backscattered electron images (B). However, for the investigated species, we observed specifically thickened valve sections not just at the muscle attachment sites, as is the case for the muscle scars of bivalves (Figure 13A and [23]), but also near the hinge (red box in (B)). White arrows in the micrograph (C) point to bundles of striated adductor fibres.

Figure 2. Bundles of adductor muscle fibres that attach to the dorsal valve of Magellania venosa. The thickening of the dorsal valve at the attachment sites of the muscles and the hinge is well observable from the micrograph (yellow arrows in (A)) and backscattered electron images (B). However, for the investigated species, we observed specifically thickened valve sections not just at the muscle attachment sites, as is the case for the muscle scars of bivalves (Figure 13A and [23]), but also near the hinge (red box in (B)). White arrows in the micrograph (C) point to bundles of striated adductor fibres.

Figure 3. (A): 3D image visualising the adductor and adjustor muscle pairs of a Magellania venosa shell, their arrangements within the shell cavity and their attachment positions to the valves. Note that the muscles are positioned in the posterior part of the shell cavity, generated by the hinged dorsal and ventral valves. See the two branches for one of the adductor pairs (blue and white circles in (A)), attached to the dorsal valve and well separated. The branches and two pairs of adductor muscles converge into a tendon that attaches to the ventral valve (yellow circle in (A)). (B,C): Sketch indicating the different muscles and visualising their position on the inner surface of the ventral and dorsal valve. (D): The course of the pairs of the diductor and adjustor muscles, attaching to the ventral valve of a M. venosa shell. The difference in muscle size and attachment position to the valve is well observable. Note the position of the tendon (yellow circles in (B,D)) that originates from the confluence of the ventral valve adductors and their branches.

Figure 3. (A): 3D image visualising the adductor and adjustor muscle pairs of a Magellania venosa shell, their arrangements within the shell cavity and their attachment positions to the valves. Note that the muscles are positioned in the posterior part of the shell cavity, generated by the hinged dorsal and ventral valves. See the two branches for one of the adductor pairs (blue and white circles in (A)), attached to the dorsal valve and well separated. The branches and two pairs of adductor muscles converge into a tendon that attaches to the ventral valve (yellow circle in (A)). (B,C): Sketch indicating the different muscles and visualising their position on the inner surface of the ventral and dorsal valve. (D): The course of the pairs of the diductor and adjustor muscles, attaching to the ventral valve of a M. venosa shell. The difference in muscle size and attachment position to the valve is well observable. Note the position of the tendon (yellow circles in (B,D)) that originates from the confluence of the ventral valve adductors and their branches.

Figure 4. (A): sketch visualising the different types of muscles that initiate movement of the Magellania venosa ventral and dorsal valves. (B): view onto the pairs of adductor, diductor and adjustor muscle bundles. (C–F): view onto the diductor and adjustor muscle bundles and fibres.

Figure 4. (A): sketch visualising the different types of muscles that initiate movement of the Magellania venosa ventral and dorsal valves. (B): view onto the pairs of adductor, diductor and adjustor muscle bundles. (C–F): view onto the diductor and adjustor muscle bundles and fibres.

Figure 5. (A–E): Top and side views of the different muscles of Magellania venosa. (B): Sketch, visualising the positions and attachments of the muscles to the internal surface of the ventral and dorsal valves. (D): Side and top view of the confluence of the dorsal valve adductors into a single tendon (black arrows in (D)). (E): Top view of the adjustor muscle bundles (highlighted by green arrows). (F): Top view of the diductor muscle bundles. Note that these slightly overlap. (C,F): See the surface of the tendon, formed of the dorsal valve adductors (yellow circle in (F)). See the tendon being positioned between the diductors and that the diductors are slightly offset to the tendon. Note that the adjustors are behind the tendons and the diductors and are closest to the valve (insert in (E,F)). Hence, the tendon, diductor and adjustor muscles are positioned close to each other in a row, even slightly overlapping.

Figure 5. (A–E): Top and side views of the different muscles of Magellania venosa. (B): Sketch, visualising the positions and attachments of the muscles to the internal surface of the ventral and dorsal valves. (D): Side and top view of the confluence of the dorsal valve adductors into a single tendon (black arrows in (D)). (E): Top view of the adjustor muscle bundles (highlighted by green arrows). (F): Top view of the diductor muscle bundles. Note that these slightly overlap. (C,F): See the surface of the tendon, formed of the dorsal valve adductors (yellow circle in (F)). See the tendon being positioned between the diductors and that the diductors are slightly offset to the tendon. Note that the adjustors are behind the tendons and the diductors and are closest to the valve (insert in (E,F)). Hence, the tendon, diductor and adjustor muscles are positioned close to each other in a row, even slightly overlapping.

Figure 6. (A,B,D,E): The branches of one Magellania venosa dorsal valve adductors. See the difference in structure between the smooth and striated adductor branch; see also Figure S2. (C): Sketch visualising the muscles that move the valves of modern rhynchonellate brachiopods.

Figure 6. (A,B,D,E): The branches of one Magellania venosa dorsal valve adductors. See the difference in structure between the smooth and striated adductor branch; see also Figure S2. (C): Sketch visualising the muscles that move the valves of modern rhynchonellate brachiopods.

Figure 7. (A–D): Structure, microstructure and texture of calcite fibre arrays of the two-layered shell of Magellania venosa shells cut transversely and longitudinally. (B–D): The structure and microstructure of the fibrous shell layer adjacent to the attachment of the muscle bundles and distant to the attachment of the muscles to the valves. The orange arrows in (A–D) point to the inner valve surfaces. A thin, 120 to 150 μm thick layer of calcite, significantly different in structure to the fibrous microstructure of the rest of the shell, is visible where the muscle bundles attach to the valves (red star in (B), red dashed lines in (C,D)). Within the valve section next to the attachment of the muscles, the typical morphology of brachiopod fibres is not observable (D); the crystals (fibres) are strongly distorted in morphology. (A): EBSD scan, given as band contrast measurement image (left in (A)) and colour-coded for crystal orientation (right in (A), modified after [51]). The fibres are well aligned in the stack and show an axial crystal texture that is more or less parallel to the growth direction (see red arrow in pole figure in (A)). (A,C,D): EBSD band contrast or crystal orientation images. (B): FE-SEM micrograph, SE contrast.

Figure 7. (A–D): Structure, microstructure and texture of calcite fibre arrays of the two-layered shell of Magellania venosa shells cut transversely and longitudinally. (B–D): The structure and microstructure of the fibrous shell layer adjacent to the attachment of the muscle bundles and distant to the attachment of the muscles to the valves. The orange arrows in (A–D) point to the inner valve surfaces. A thin, 120 to 150 μm thick layer of calcite, significantly different in structure to the fibrous microstructure of the rest of the shell, is visible where the muscle bundles attach to the valves (red star in (B), red dashed lines in (C,D)). Within the valve section next to the attachment of the muscles, the typical morphology of brachiopod fibres is not observable (D); the crystals (fibres) are strongly distorted in morphology. (A): EBSD scan, given as band contrast measurement image (left in (A)) and colour-coded for crystal orientation (right in (A), modified after [51]). The fibres are well aligned in the stack and show an axial crystal texture that is more or less parallel to the growth direction (see red arrow in pole figure in (A)). (A,C,D): EBSD band contrast or crystal orientation images. (B): FE-SEM micrograph, SE contrast.

Figure 8. (A,B): Shell fibre structure at muscle attachment sites directly adjacent to muscle fibre bundles that attach to the inner surface of Magellania venosa shells. Left in (A,B): FE-SEM micrographs, SE contrast. Right in (A): EBSD band contrast measurement image. Well visible is the generally distorted morphology of the fibres that form the shell adjacent to the muscle attachment sites. Nonetheless, outlines of undistorted brachiopod fibres are occasionally visible (e.g., red square in (B)). For these, we can deduce the morphological axis of the fibre and, accordingly, even in SE micrographs, the orientation of calcite crystals that form the fibres. The the calcite c-axis orientation directions are perpendicular to the morphological long axis of brachiopod fibres. The latter implies that the fibre calcite c-axis orientation direction of valve sections at muscle attachment sites is parallel to the morphological orientation of the muscle fibre and bundle.

Figure 8. (A,B): Shell fibre structure at muscle attachment sites directly adjacent to muscle fibre bundles that attach to the inner surface of Magellania venosa shells. Left in (A,B): FE-SEM micrographs, SE contrast. Right in (A): EBSD band contrast measurement image. Well visible is the generally distorted morphology of the fibres that form the shell adjacent to the muscle attachment sites. Nonetheless, outlines of undistorted brachiopod fibres are occasionally visible (e.g., red square in (B)). For these, we can deduce the morphological axis of the fibre and, accordingly, even in SE micrographs, the orientation of calcite crystals that form the fibres. The the calcite c-axis orientation directions are perpendicular to the morphological long axis of brachiopod fibres. The latter implies that the fibre calcite c-axis orientation direction of valve sections at muscle attachment sites is parallel to the morphological orientation of the muscle fibre and bundle.

Figure 9. EBSD scans showing the microstructure, texture, and carbonate phase of the fibrous Magellania venosa valve section where muscles attach to the valves. (A,F,H) are EBSD band contrast measurement images. (C,D) show EBSD band contrast (in grey) overlain with crystal orientation (in colour). (E,G): FE-SEM images, SE contrast. The muscle attachment sites of modern rhynchonellate shells consist of calcite (B). Irrespective of fibre morphological axis orientation, for both transversely (A,C) and longitudinally (F) cut fibres, fibre morphology at the valve section where the muscles attach is strongly distorted. The layer with distorted fibre morphology forms a lining at the inner shell surface (Figure 7B,C) that thins out with distance from the shell portion where the muscle bundles attach (H). White arrows in (H) point to the very thin (few µm) seam of fibres with distorted morphology that line the inner surface of the valve. With distance from the muscle attachment sites, the inner valve surface microstructure consists of undistorted rhynchonellate brachiopod fibres. Pole figures in (C,D,F,H) demonstrate the axial nature of the calcite texture, irrespective of whether the EBSD scan was taken at a muscle attachment site or away from the latter. (C,D): there is no difference in crystal co-orientation strength (MUD value) between valve sections at muscle attachments and those away from muscle attachments.

Figure 9. EBSD scans showing the microstructure, texture, and carbonate phase of the fibrous Magellania venosa valve section where muscles attach to the valves. (A,F,H) are EBSD band contrast measurement images. (C,D) show EBSD band contrast (in grey) overlain with crystal orientation (in colour). (E,G): FE-SEM images, SE contrast. The muscle attachment sites of modern rhynchonellate shells consist of calcite (B). Irrespective of fibre morphological axis orientation, for both transversely (A,C) and longitudinally (F) cut fibres, fibre morphology at the valve section where the muscles attach is strongly distorted. The layer with distorted fibre morphology forms a lining at the inner shell surface (Figure 7B,C) that thins out with distance from the shell portion where the muscle bundles attach (H). White arrows in (H) point to the very thin (few µm) seam of fibres with distorted morphology that line the inner surface of the valve. With distance from the muscle attachment sites, the inner valve surface microstructure consists of undistorted rhynchonellate brachiopod fibres. Pole figures in (C,D,F,H) demonstrate the axial nature of the calcite texture, irrespective of whether the EBSD scan was taken at a muscle attachment site or away from the latter. (C,D): there is no difference in crystal co-orientation strength (MUD value) between valve sections at muscle attachments and those away from muscle attachments.

Figure 10. Microstructure, texture and carbonate phase of the three-layered shell of Liothyrella neozelanica at muscle attachment sites (B–G) and away from these shell sections (A). Left in (A–F): band contrast image of EBSD measurement. Right in (A,D): Colour-coded EBSD map showing calcite crystal orientations. EBSD measurement images in (A) are modified after [51]. (C): Colour-coded maps of individual attachment site crystals highlight their complex morphologies. (B): FE-SEM micrograph, SE contrast. We show three EBSD scans for L. neozelanica, their position indicated in (B) with blue, red, and yellow rectangles. One EBSD scan is given in (D–F); the others are given in Figure 11A,B. All three EBSD scans scan the valve adjacent to muscle bundles and cover the crystals adjacent and distant to the muscle attachments. Muscle attachment valve sections of L. neozelanica consist of calcite (E). For L. neozelanica, the calcite of the valves at muscle attachments is formed of elongated crystals that faintly resemble columns (D,F). These interdigitate (C) and have strongly irregular outer morphologies (D,F). For both the fibres and the elongated crystals at muscle attachment sites, we found an axial texture (G) and high crystal co-orientation strengths (MUD values in (G)).

Figure 10. Microstructure, texture and carbonate phase of the three-layered shell of Liothyrella neozelanica at muscle attachment sites (B–G) and away from these shell sections (A). Left in (A–F): band contrast image of EBSD measurement. Right in (A,D): Colour-coded EBSD map showing calcite crystal orientations. EBSD measurement images in (A) are modified after [51]. (C): Colour-coded maps of individual attachment site crystals highlight their complex morphologies. (B): FE-SEM micrograph, SE contrast. We show three EBSD scans for L. neozelanica, their position indicated in (B) with blue, red, and yellow rectangles. One EBSD scan is given in (D–F); the others are given in Figure 11A,B. All three EBSD scans scan the valve adjacent to muscle bundles and cover the crystals adjacent and distant to the muscle attachments. Muscle attachment valve sections of L. neozelanica consist of calcite (E). For L. neozelanica, the calcite of the valves at muscle attachments is formed of elongated crystals that faintly resemble columns (D,F). These interdigitate (C) and have strongly irregular outer morphologies (D,F). For both the fibres and the elongated crystals at muscle attachment sites, we found an axial texture (G) and high crystal co-orientation strengths (MUD values in (G)).

The shell of modern rhynchonellate brachiopods consists of a maximum of three shell layers. Most species only form an outer primary and inner fibrous shell layer; however, some species secrete a third shell layer of prism-to-column-shaped crystals [51]. We investigated the crystals at adductor muscle attachment sites of Magellania venosa, Liothyrella neozelanica and Gryphus vitreus. The shell of M. venosa consists of two layers and two microstructures, namely, an outer primary and an inner fibrous shell layer (this study and [51]). The shells of L. neozelanica and G. vitreus consist of three shell layers and three different microstructures. Both have an outer primary layer followed inward by a fibrous and a columnar shell layer (this study and [51]). However, we found an alternation between the fibrous and the columnar microstructures in L. neozelanica shell cross-sections (Figures 5D and 6 in [53]). This feature is not observed for the shell of G. vitreus. For G. vitreus, the primary layer, fibrous and columnar microstructures are next to each other and do not form sequential alternations, as is the case for L. neozelanica (this study and [51]).

The muscles of M. venosa, L. neozelanica and G. vitreus assemble within the posterior part of the shell cavity, generated by the hinged ventral and dorsal valves (Figure 3A, Figure 5A and Figure 6A). As a mirror plane runs through rhynchonellate brachiopod valves (Figure S1A), the adductor, diductor and adjustor muscles are developed in pairs (Figure 1 and Figure S1B). As illustrated in Supplementary Figure S1B, a pair of adductor muscles attaching to the dorsal valve can be divided into two branches, the striated and smooth adductor muscles (Figure S1C). Here, the combination of one striated and one smooth adductor muscle branch is termed an adductor muscle segment (Figure S1B,C).

A pair of diductor and a pair of adjustor muscles attach their base to the ventral and the dorsal valves (Figure 3, Figure 4 and Figure 5). At the dorsal valve, a pair of specific adductor muscles is attached (Figure 1 and Figure 3). These merge and form one tendon, which attaches to the ventral valve (Figure 3 and Figure 5). For all three investigated species, the pair of adductor muscle that attaches to the ventral valve has a segment attaching to the left and another to the right of the median septum of the hinge (Figure 1 and Figure 2). Each adductor muscle segment consists of two branches. This is well-observable for all three investigated species (Figure 1). The two adductor branches are positioned next to each other, nonetheless, are delimited spatially from each other, as one adductor branch attaches to the valve behind the other adductor branch (Figure 1 and Figure S2). Accordingly, for all three investigated species, we observed different attachment positions for the two adductor branches. Based on muscle ultrastructure, our study reveals that the two branches of the dorsal valve adductor muscles comprise distinct types of muscle fibres. One branch is formed of smooth, the other of striated muscles (Figure 6 and Figure S2).

Figure 11. EBSD scans taken on the valves of Liothyrella neozelanica at muscle attachments (A,B) demonstrate the changed microstructure of shell calcite adjacent to muscle attachments relative to the columns and/or fibres in other valve sections (e.g., Figure 10A and Figure 11D). In these scans, we also observed a seam of elongated crystal units at the inner shell surface, which have a strongly irregular morphology and interdigitate with neighbouring elongated units. As for the two-layered M. venosa, the specific layer developed at muscle attachments to the shell becomes thinner with distance from the muscle attachment sites (C) and, at some point, disappears completely (D). Crystal orientation is indicated with sketched crystals in (A,B,D), and the growth direction up to the inner shell surface is indicated by orange arrows.

Figure 11. EBSD scans taken on the valves of Liothyrella neozelanica at muscle attachments (A,B) demonstrate the changed microstructure of shell calcite adjacent to muscle attachments relative to the columns and/or fibres in other valve sections (e.g., Figure 10A and Figure 11D). In these scans, we also observed a seam of elongated crystal units at the inner shell surface, which have a strongly irregular morphology and interdigitate with neighbouring elongated units. As for the two-layered M. venosa, the specific layer developed at muscle attachments to the shell becomes thinner with distance from the muscle attachment sites (C) and, at some point, disappears completely (D). Crystal orientation is indicated with sketched crystals in (A,B,D), and the growth direction up to the inner shell surface is indicated by orange arrows.

Figure 12. Microstructure, texture and carbonate phase of valve sections at muscle attachment sites for the three-layered shell of Gryphus vitreus. G. vitreus shells comprise a primary, a fibrous and a columnar shell layer (A). However, unlike Liothyrella neozelanica, there is no sequential alternation of the fibrous and the columnar microstructure in G. vitreus shells. Thus, at the inner surface, the shell consists always of an array of columns (A). EBSD measurement images in (A) are modified after [51]. In general, in G. vitreus shells, the columns are large entities and have regular morphologies. Shell crystals are assembled with an axial microstructure with c-axes parallel to the growth direction (red arrows in pole figure (A)). At muscle attachments (B,C), we also observed arrays of columns. Nonetheless, the morphology of the columns becomes slightly irregular (B,C), and the columns interdigitate in 3D. The columns at muscle attachment sites also comprise calcite and have an axial texture (see pole figures in (B,C)). The top micrograph in (B) and the insert in (C) are FE-SEM images, SE contrast. The map coloured in red in (B) shows that the carbonate phase of the crystals at muscle attachment sites is calcite. Grey-scaled in (A–C) are EBSD band contrast maps, complemented with coloured EBSD maps visualising crystal orientation.

Figure 12. Microstructure, texture and carbonate phase of valve sections at muscle attachment sites for the three-layered shell of Gryphus vitreus. G. vitreus shells comprise a primary, a fibrous and a columnar shell layer (A). However, unlike Liothyrella neozelanica, there is no sequential alternation of the fibrous and the columnar microstructure in G. vitreus shells. Thus, at the inner surface, the shell consists always of an array of columns (A). EBSD measurement images in (A) are modified after [51]. In general, in G. vitreus shells, the columns are large entities and have regular morphologies. Shell crystals are assembled with an axial microstructure with c-axes parallel to the growth direction (red arrows in pole figure (A)). At muscle attachments (B,C), we also observed arrays of columns. Nonetheless, the morphology of the columns becomes slightly irregular (B,C), and the columns interdigitate in 3D. The columns at muscle attachment sites also comprise calcite and have an axial texture (see pole figures in (B,C)). The top micrograph in (B) and the insert in (C) are FE-SEM images, SE contrast. The map coloured in red in (B) shows that the carbonate phase of the crystals at muscle attachment sites is calcite. Grey-scaled in (A–C) are EBSD band contrast maps, complemented with coloured EBSD maps visualising crystal orientation.

Figure 13. Comparison of the striated and smooth adductor muscle bundles and attachment site microstructures in Magellania venosa. Smooth (blue arrows in (A,B)) and striated muscles (white arrows in (A,B)) are spatially separated in a linear arrangement next to the hinge (green arrows in (A,B)). Colour-coded EBSD maps of smooth (C) and striated (D) valve sections at muscle attachment sites of M. venosa show comparable microstructures and textures. While the fibrous calcitic shell (indicated by yellow stars in (C,D)) is organised, the muscle attachment site crystals appear irregular and interdigitate in 3D (indicated by white stars in (C,D)). The texture and crystal co-orientation strength of the two sections are similar, with rather low MUD values and c-axes (red arrows in (C,D) oriented parallel to the growth direction (orange arrows in (C,D)).

Figure 13. Comparison of the striated and smooth adductor muscle bundles and attachment site microstructures in Magellania venosa. Smooth (blue arrows in (A,B)) and striated muscles (white arrows in (A,B)) are spatially separated in a linear arrangement next to the hinge (green arrows in (A,B)). Colour-coded EBSD maps of smooth (C) and striated (D) valve sections at muscle attachment sites of M. venosa show comparable microstructures and textures. While the fibrous calcitic shell (indicated by yellow stars in (C,D)) is organised, the muscle attachment site crystals appear irregular and interdigitate in 3D (indicated by white stars in (C,D)). The texture and crystal co-orientation strength of the two sections are similar, with rather low MUD values and c-axes (red arrows in (C,D) oriented parallel to the growth direction (orange arrows in (C,D)).

Following the adductor muscles, the diductors are the next largest, valve-moving, rhynchonellate muscles (Figure 3B, Figure 4 and Figure 5). Their attachment site to the dorsal valve is minute, unlike their attachment site to the ventral valve. We found large diductor muscle attachment sites onto the ventral valves, positioned left and right to the adductor tendon (Figure 3D and Figure 5C,F). When the diductor muscle bases are viewed from above, a pair of slightly overlapping diductors are visible, attaching to the ventral valve (Figure 5C,F). Of the three valve/shell-moving muscles, the smallest muscle is the adjustor muscle (Figure 5E). As is the case for the diductor muscles, the attachment sites of the adjustor muscles onto the dorsal valve are also minute. However, when attaching to the ventral valve, the adjustor attachments increase considerably in size and curve around the attachment of the diductor muscle (Figure 3D). The adjustor muscles run partly behind the diductor muscles (Figure 3D and Figure 5E). Thus, when viewed from above, we found in a row, however, in a stepped arrangement, the attachment of the adductor tendon, that of the diductors and, close to the valve margin, that of the adjustors.

It is interesting to find that, for the investigated species, all three muscles have, on one end, a small, on the other a large thickness (e.g., sketched in Figure 1A). Furthermore, it should be noted that the large base of the adductors and the small bases of the diductors and adjustors attach to the dorsal valve. In contrast, the large bases of the diductors and adjustors attach to the ventral valve. At the same time, the extent of the attachment site of the adductor tendon is rather small when compared to the attachment size of the adductors onto the dorsal valve. Hence, we found a structured distribution of muscle attachments on the two brachiopod valves for the investigated species and the investigated muscles.

Where the adductor muscles attach to the valves, a valve section is thickened (Figure 2A and Figure S1D). However, valve thickening is not limited to the sole attachment of individual muscle bundles; the thickened valve region extends over a larger portion of the valve (Figure 2A and Figure S1D). As for the rest of the shell, the thickened valve region consists of calcite (Figure 9B, Figure 10E and Figure 12B). This thickened valve section might be addressed as an additional shell layer (Figure 7B and Figure 8), as it is distinct in microstructure, relative to the rest of the shell (Figure 7, Figure 8, Figure 9, Figure 10, Figure 11 and Figure 12). We found that for all three investigated species, irrespective of whether the inner layers of the shell are formed of fibres or columns, the innermost layer comprises a particular microstructure at the thickened valve sections. This is a specific microstructure with a strongly irregular appearance and features crystals with fractal-like morphologies (Figure 7D, Figure 8A, Figure 9F, Figure 10F, Figure 11A,B and Figure 12B,C). The latter crystal morphology and microstructure are distinct from those of the crystals that form the other parts (the absolute majority) of the shell (Figure 7A, Figure 10A and Figure 12A). Subsequently, this microstructure formed of the fractal-like crystals is termed ‘distorted’. The valve layer with the distorted microstructure is intimately connected to valve sections that are formed of the conventional and, for brachiopod shells, typical fibrous and columnar microstructures (Figure 7D, Figure 9C,F, Figure 10F and Figure 11A,B). The distorted microstructure, which forms the innermost layer of the thickened shell sections, appears to evolve from the fibrous and/or columnar microstructures (e.g., Figure 9A,F and Figure 11B). Despite the difference in microstructure (described above), it is very interesting to observe that crystal texture does not change. It is axial for shell layers formed of crystals with undisturbed but also with disturbed crystal morphologies (see pole figures in Figure 9C,D, Figure 10F and Figure 12B,C).

The crystals with fractal-like morphologies can neither be addressed as fibres nor columns. Nonetheless, their c-axis orientations are perpendicular to the inner shell surface. In one rare case, we observed for the two-layered shell of M. venosa, fibre-shaped crystals within the “distorted-microstructure-layer”, adjacent to the attachment of muscle fibres (Figure 8). We found that the calcitic c-axis orientations are parallel to the morphological orientation of the muscle fibre bundle. When crystal morphologies, irrespective of whether for fibres or columns, between undistorted and distorted shell sections are compared, we observed for all investigated species that crystal morphologies at innermost thickened valve sections are serrated and fractal-like and that these crystals interdigitate strongly (Figure 8A, Figure 10C and Figure 12B,C). This is not the case for those fibres and columns that form the other parts of the shell, where the “conventional” brachiopod microstructure prevails (Figure 7A, Figure 10A and Figure 12A).

As mentioned above, the thickened shell layer, comprising the layer with the distorted microstructure at its innermost surface, is not just strictly connected to the sites where the muscle bundles attach. This specific layer is thickest at the muscle bundle attachments (e.g., Figure 10 and Figure 11). However, it also covers valve inner surfaces that surround the regions of the muscle attachments. Nonetheless, with distance away from the muscle attachment sites, the layer with the distorted microstructure thins out (Figure 9H and Figure 11C).

4. Discussion

Rhynchonellata (Brachiopoda) and Bivalvia (Mollusca) belong to different phyla. Rhynchonellate brachiopods and most bivalve molluscs are filter-feeding, marine organisms with species of many taxa being sessile and/or cementing to the substrate. Both have a global distribution and are adapted to many, partly comparable, habitats ([10,18] and references therein).

The species of both invertebrate classes secrete mineralised shells, consisting of two valves and a hinge. For species of both classes, shell secretion is performed by the cells of a mantle epithelium, which lines the inner surface of the shell and encloses the soft body and the pallial cavity. The mantle epithelium consists of an outer and inner layer, separated from each other by connective tissue. For species of both classes, the outer epithelium, which performs shell secretion, is single-layered and comprises cuboidal and specialised cells, while the inner epithelium mainly comprises columnar cells [54,65,66]. Although representing different phyla, the biomineralisation system of the Brachiopoda and Mollusca is comparable [18], such that before secretion of the mineralised shell layers, an organic periostracum is deposited ([18,51,67] and references therein). Shell growth occurs for both the Rhynchonellata and the Bivalvia across a thin extrapallial space [16,28,29]. The latter is a few tens of nm thick and is located between the proximal surface of the mineralised shell inner surface and the secreting outer epithelial mantle cells [68,69].

4.1. The Muscles and Their Attachment to the Attachment Site Crystals

Brachiopods and bivalves protect their soft tissue and organs with the structural hard tissue of the two valves, hinged together at the posterior end of the shell. The motion of the valves is accomplished via the muscles. Species of both invertebrate classes are capable of: (i) fast and slow valve opening and closure, as well as of (ii) valve opening and closure for short and prolonged periods [6,10,59,63,70,71,72]. Valve movement is achieved via adductor and diductor muscles in the case of brachiopods. Bivalves utilise their adductor and pedal muscles as well as their hinge ligament for valve action. Rhynchonellata and Bivalvia can move their valves at different speeds. This requires using various types of muscles, either one particular muscle or a combination of different types of muscles [59]. We observed both circumstances for species of Rhynchonellata and Bivalvia. This involves the use of both smooth and striated muscles [47,59]. One main difference between smooth and striated muscles is that, in the case of smooth muscles, no banding pattern is observable when the muscle is imaged with SEM. In contrast, a banding pattern is well visible for striated muscles, where striation arises from the filament structure of the muscle [48,59]. Brachiopods and bivalves utilise striated muscle fibres for phasic catch contractions and achieve quick valve opening and closure. Smooth muscle fibres exert tonic forces and are used for slow but prolonged valve opening and closure [9,10,59,70,73].

The connection of muscle fibres to the muscle attachment site crystals is similar for shelled invertebrates [29,47,50]. The muscles of brachiopods, bivalves and gastropods do not connect directly to the crystals that form the muscle attachment sites. A series of structures is linearly arranged between the apical base of the muscle fibres and the proximal surface of the attachment site crystals [10,19,29,47,50,54]. At muscle attachment sites of shelled invertebrates, the secreting mantle epithelium consists of specialised epithelial cells, the tendon cells [10,47,54]. These are located between the muscle bases and the proximal surface of the muscle site crystals [29,47,50] and are distinct in ultrastructure from the columnar epithelial cells, which secrete the crystals of the rest of the valves. Tendon cells contain bundles of tonofilaments, which extend throughout their length. The apical and basal surface of tendon cells is dotted with hemidesmosomes [29,47,50]. In addition to the hemidesmosomes, the apical surface of the tendon cells is densely seamed with knob-shaped microvilli. However, the tendon cells do not attach their microvilli directly to the muscle attachment site crystals. A layer of organic fibrils lies between the apical surface of the tendon cells and the proximal surface of the attachment site crystals. These fibrils fill the extrapallial space and become incorporated into the attachment site crystals at shell crystal secretion by the tendon cells [19,28,47,50,54]. The basal membrane of the tendon cells is strongly infolded and seamed with hemidesmosomes. The basal ends of the muscle fibres terminate close to the basal membrane of the tendon cells and anchor, also with hemidesmosomes, to the hemidesmosomes of the basal membrane of the tendon cells [28,50,54]. The above detailed mode of attachment of the muscles to the muscle attachment site crystals is similar for shelled invertebrates; thus, it is similar for brachiopods and molluscs.

The attachment of the muscles of bivalved organisms to the inner shell surface belongs to one of the strongest connections in the biological realm [59]. This is due to: (i) the high muscular strength of invertebrate muscles; the latter is the force that muscles can exert against resistance in a single effort [59], and (ii) the mode of connection between the muscles and the crystals, to which the muscles attach [19]. At the muscle attachment site, the tendon cell fibrils associated with their apical microvilli anchor within the crystals. It is well-known that the connection of very different materials, such as inorganic-organic composites, can be prone to interface failure [74,75,76,77]. However, even though very dissimilar materials join at the muscle-crystal interface, due to the anchoring of the fibrils within the attachment site crystals, very little stress accumulates at the biopolymer fibril-crystal junction, and the connection between the muscle strands and the attachment site crystals becomes strongly increased in strength [19].

As visualised in this study, each trunk of the pair of brachiopod adductor muscle (Figure S1B) consists of two branches (Figure 3A, Figure 6 and Figure S1B,E). These branches attach to the inner surface of the dorsal valve, have different appearances, and their attachment site is spatially well-differentiated from each other (Figure S1C,E). The two branches of the brachiopod adductor muscle consist of different types of muscles. The adductor branch closer to the median septum of the hinge is formed of smooth muscles; the branch further away from the median septum consists of striated muscles. The different types of muscles and their arrangement in the shell cavity are also well investigated for bivalves [10,78,79,80,81,82,83,84,85]. However, such a clear-cut spatial distinction (e.g., Figure 13B) between a smooth and a striated muscle component of a specific type of muscle has not yet been observed. Figure 13C,D and Figures S5–S7 show the microstructure and texture of crystals where the smooth and the striated adductor muscle branches of Magellania venosa attach to the inner surface of the dorsal valve. The morphology of attachment site crystals is highly fractal, and the microstructure of the inner valve section is distorted (white stars in Figure 13C,D) and is distinct from those shell regions that are distant from the muscle attachment sites (yellow stars in Figure 13C,D). We found no structural differences in the crystal morphology and microstructure of muscle attachment sites for smooth and/or striated muscle bundles (Figure 13A–D). This is not surprising, as, for both smooth and striated muscle bundles, the muscle attachment site crystals are secreted similarly and by similar types of cells, namely the tendon cells.

4.2. Are the Muscle Attachment Sites of Rhynchonellata and Bivalvia Convergent in Structure?

In the previous section, we described general attributes shared by rhynchonellates and bivalve species. Thus, is the structure, microstructure, and texture of rhynchonellate brachiopod and bivalve muscle attachment site crystals also comparable? Rhynchonellata and Bivalvia overlap in living environment, lifestyle, comparability of the hard tissue mineralisation system, the generation of a hinged valved shell, the movement of the valves via muscles, and the use of similar types of muscles. However, our study shows that the muscle attachment site crystals differ significantly for the investigated species of rhynchonellates and bivalves (this study and [24,86]). Nonetheless, it should be noted that the crystallographic texture of the muscle attachment site portions of the investigated species is similar. The assembly of the muscle attachment site crystals has an axial texture, with calcite and aragonite c-axis oriented perpendicular to the inner shell surface (Figure 9D,E, Figure 11G and Figure 12B,C and Hoerl [24]), for both the investigated species of Rhynchonellata and Bivalvia.

When comparing structural-crystallographic attributes of rhynchonellate and bivalve muscle attachment sites, we found that:

• The muscle scar of bivalves is a prominent, protruding structure (Figure 14A), limited in extent to only those valve sections where the muscle bundles directly attach to the valves. For rhynchonellate brachiopods, we observed extensive thickening of the valve portions at and near muscle attachment sites (see Figure 2 for dorsal valves). However, the latter is not only present at the sites of muscle attachments but also extends to regions that surround those valve portions where the brachiopod muscles attach to (Figure 2A, red rectangle in Figure 2B). The innermost layer of the thickened shell portion shows a disturbed microstructure of fractal-like crystals. With distance from the muscle attachment, the layer with the disturbed microstructure thins out and disappears (white arrows in Figure 9H and Figure 11C).
• One of the most distinctive characteristics is that, in contrast to rhynchonellate brachiopods, the muscle scars of bivalves have a competitive growth-derived microstructure (Figure 14B and [21]). These myostraca consist of prisms with irregular morphologies, assembled in a very specific way that depends upon the microstructure of the adjacent shell layer. This was not observed for the investigated rhynchonellate brachiopod species. As detailed in this contribution, the microstructures of muscle attachment crystals of brachiopod shells are also distinct from the rest of the shell. However, muscle scar crystal formation of brachiopod shells is not generated by competitive growth; the muscle scar crystals have highly irregular morphologies and a very disturbed microstructure (Figure 7, Figure 8 and Figure 14). Irrespective of whether muscle attachment crystals originate from fibres or columns (Figure 11A,B, Figure 12B,C and Figure 13C,D), their morphologies are fractal-like (Figure 10C, Figure 12C and Figure 14C,D) and the crystals interdigitate in 3D,
• The carbonate phase of brachiopod muscle attachment sites is always calcite, while bivalve muscle scars always consist of aragonite, even when the adjacent shell layer is formed of calcite [21]. While the reasons for this are not yet fully understood, it might be due to historical restrictions. The earliest bivalve shells were presumably purely aragonitic, and the ability to secrete calcite microstructures may have developed later in bivalves [87].
• The texture pattern of the brachiopod muscle attachment site crystals is similar to that of the adjacent shell layer, and it is not changed with progressive attachment site crystal growth. At first, bivalves also adopt the texture pattern of the adjacent section of the valves if it contains the same calcium carbonate phase [21]. However, with progressive attachment site crystal growth, the texture of bivalve muscle scar crystals changes slightly [21,23,26].

Nonetheless, one characteristic is similar between rhynchonellate brachiopods and bivalves for the muscle attachment site crystals. For species of both invertebrate classes, the carbonate crystal c-axis orientation is perpendicular to the inner shell surface and parallel to the morphological axis of the muscle fibre bundle (Figure 8B, Figure 9D, Figure 10D, Figure 11A,B, Figure 12B,C and Figure 14B–D and [24]). The analogy of muscle attachment site crystal c-axis orientation for brachiopods and bivalves, relative to the inner shell surface, is rooted in the mode of attachment of the muscle bundles to the attachment site crystals via the tendon cells. It might be required to generate a strong muscle bundle-attachment site crystal connection. The secretion of muscle attachment site crystals by the tendon cells and the orientation of their crystallographic c-axes, relative to the inner shell surface, is a biological convergence trait (for definition see Section 2.4) and was most likely developed independently by bivalved species of two invertebrate phyla. Nonetheless, it should be kept in mind that brachiopods and bivalves have a partly comparable habitat preference and lifestyle [10,47].

Combining the microstructure and texture results of this study on brachiopod shells with the results gained by Hoerl [24] for bivalve shells, we can state that the microstructure and calcium carbonate phase of bivalve and rhynchonellate brachiopod muscle attachment sites are not convergent. Two main reasons account for the latter:

1. Brachiopods and bivalves are species of different phyla, and varying strategies of Ca-carbonate structural biomaterial formation may have been developed for muscle scar generation.

2. However, the main reason is probably that brachiopods and bivalves apply different solutions for valve opening and closure [11,83,88,89,90,91,92,93].

(i) The bivalve shell comprises two valves, connected along one edge by a flexible hinge ligament [12,13]. The bivalve ligament has two parts, an inner and an outer layer [88,90]. The bivalve adductor muscle pulls the valves together and closes them, stretching the outer layer of the ligament and compressing its inner portion [90,91]. At the release of the adductor muscle contraction, the elastic recoil of the inner ligament layer opens the two valves [91,92].

(ii) The brachiopod shell does not have a hinge ligament. Brachiopods have a toothed hinge with a pair of teeth and a pair of sockets [6]. Brachiopods use only their muscle system for closing and opening their valves, the adductor muscles for closing, and the diductor muscles for opening them [6]. In contrast to bivalves, brachiopod valve closure is a two-stage process, consisting of a rapid and a slow but prolonged valve movement. As described in this study, each trunk of the pair of adductors has two branches, a striated and a smooth adductor branch (Figure 6 and Figure S2), that each attach to different sites to the dorsal valve (e.g., Figure 1B). The striated adductor branch is the quick, the smooth adductor branch is the catch muscle. The quick muscle initiates a rapid valve closure, while the catch muscle induces the complete closure of the valves and holds these shut for a long period [63,70,71]. The latter phenomenon is not observed for bivalves.

Hence, even though secreted by similar mantle epithelial cells and utilising similar types of muscles, the muscle imprints generated at attachment to the inner valve surface are not and cannot be identical for rhynchonellate brachiopods and bivalves, as different valve opening and closing mechanisms are developed. Accordingly, with the study of rhynchonellate and bivalve muscle attachment sites (this study and [24]), we showcase that the mechanism of generating specific sections for attaching valve-operating muscles is similar in both classes. However, the distantly related classes Bivalvia and Rhynchonellata approach the structural formation of shell microstructures at valve-muscle attachment sites differently.

5. Conclusions

Morphological analogies, overlapping habitats and similarity in lifestyle between rhynchonellate brachiopods and bivalved molluscs invite comparisons of the physiology of organisms that form these two invertebrate classes [9,70]. Like bivalves, brachiopods open and close their shells with muscles that exhibit phasic and tonic contractions. These enable fast and sustained valve opening and closing. However, unlike bivalves, brachiopods have dorsal and ventral valves that are opened with a pair of diductor muscles and lack the involvement of an elastic hinge ligament, as developed by bivalves ([9] and references therein).

Distinct imprints mark the sites of attachment of brachiopod and bivalve muscle bases on the inner surfaces of their shells. The imprints result from modifications of the ultrastructure and secretory behaviour of epithelial cells that perform the secretion of the shell. Our study aimed to visualise the structure of rhynchonellate brachiopod adductor, diductor and adjustor muscles, to visualise the adductor muscle imprints on the valves and, in particular, to investigate structural characteristics of brachiopod muscle attachment site crystals and of their assemblies. In a wider perspective, an important aim of our study was to discuss the notion of convergence for the microstructure and texture of muscle attachment sites of the Rhynchonellata and the Bivalvia.

We deduce the following conclusions from the results of this study:

• In rhynchonellate brachiopods, the adductor and diductor muscle bases attach to calcite fibres and to calcite columns.
• The attachment site crystals have very irregular, fractal morphologies. Adjacent attachment site crystals interdigitate markedly in 3D. The attachment site portion of the valves is intimately connected to the non-attachment site sections of the shell.
• There is a marked difference in microstructure between the inner shell surface of the attachment site and the other valve portions. The texture of the attachment site and non-attachment site calcite is similar. We found an axial texture for both.
• Attachment site calcite c-axis orientations are perpendicular to the inner shell surface and parallel to the morphological axis of the muscle bundles. This is a finding we observed for species of Rhynchonellata and Bivalvia and is, most probably, necessary for a strong attachment of muscle base-tendon cell polymer fibril to the crystals.
• The difference in microstructure between attachment site and the other valve portions results from the difference in the ultrastructure and secretory behaviour of the secreting cells. A layer of cuboidal, tendon cells secretes the muscle attachment site crystals, while the crystals of the rest of the shell are mainly secreted by columnar cells.
• Regarding the structural convergence for muscle attachment sites of rhynchonellate brachiopods and bivalves, we could find some structural characteristics of muscle attachment sites that are similar for species of the investigated invertebrate classes. For both invertebrate classes, the texture of the adjacent shell layer continues in the microstructure of the muscle attachment layer. This may derive from the determinants of the similar secreting epithelial cells underlying the muscle attachment sites. However, it should be kept in mind that valve actions are realised differently by rhynchonellate brachiopods and bivalves. Bivalve valve movement is not only the result of muscle action, but also the involvement of the hinge ligament. In contrast, rhynchonellate brachiopods do not involve a ligament in valve motion, but solely utilise their muscles. Thus, different constraints operate on muscle involvement when opening and closing the valves for rhynchonellate brachiopods and bivalves and are a determining factor in the generation of the structural differences that were observed between rhynchonellate and bivalve muscle attachment sites.
• The action of opening and closing the valves is realised differently by rhynchonellate brachiopods and bivalves. Bivalve valve movement not only results from muscle action, but also involves hinge ligament action. In contrast, rhynchonellate brachiopods do not involve a ligament in valve motion; they solely employ muscles. Thus, different constraints operate on muscle involvement at opening and closing the valves for rhynchonellate brachiopods and bivalves. These might be determining factors in the formation of the differences that were observed between rhynchonellate and bivalve muscle attachment sites.