Precipitation of Aragonite by Calcitic Bivalves in Mg-Enriched Marine Waters

Mar Biol DOI 10.1007/s00227-006-0411-4

RESEARCH ARTICLE

Precipitation of aragonite by calcitic bivalves in Mg-enriched marine waters

Antonio G. Checa · Concepción Jiménez-López · Alejandro Rodríguez-Navarro · Jorge P. Machado

Received: 30 March 2006 / Accepted: 22 June 2006 © Springer-Verlag 2006

Abstract To understand the relative importance of (the scallop Chlamys varia, the oyster Ostrea edulis, the biological versus physicochemical control over biomin- saddle oyster Anomia ephippium and the mussel eralization, we have tested if the chemical composition Mytilus edulis) survived long enough to secrete signiW- of the medium (i.e., the Mg/Ca ratio) can change the cant amounts of calcium carbonate. The deposits mineralogy of mollusk shells. The shells of mollusks (sometimes extensive) formed on the interior shell sur- are made of calcite and/or aragonite, which are by far faces were predominantly aragonitic. Three individuals the most common CaCO3 polymorphs. Several species of C. varia also increased their length by adding new of bivalves with predominantly calcitic shells have been shell at the margin. Contrary to the internal shell cultivated in artiWcial seawater with a Mg/Ca molar deposits, these margins were high-Mg calcite. This ratio within the range of 8.3–9.2, well above the present implies that the marginal mantle is able to exert a more value for seawater (5.2). Four out of six species used strict control on the secreted mineral phase than the mantle facing the internal shell surface. This is the Wrst report on an in vivo experimentally forced switch in Communicated by O. Kinne, Oldendorf/Luhe bivalve shell mineralogy, from calcite to aragonite due to a change in water chemistry. A. G. Checa (&) Departamento de Estratigrafía y Paleontología, Facultad de Ciencias, Universidad de Granada, Avenida Fuentenueva s/n, 18002 Granada, Spain Introduction e-mail: [email protected] Although the mineralogy and microstructure of mol- C. Jiménez-López Departamento de Microbiología, Facultad de Ciencias, lusk shells is mainly biologically controlled (Watabe Universidad de Granada, Avenida Fuentenueva s/n, and Wilbur 1960; Addadi and Weiner 1992; Belcher 18002 Granada, Spain et al. 1996) the chemistry of the environment can also e-mail: [email protected] aVect to some degree the chemical and mineral compo- A. Rodríguez-Navarro sition of the shell. Nevertheless, little is known on the Departamento de Mineralogía y Petrología, eVect of the chemistry of the medium on shell composi- Facultad de Ciencias, Universidad de Granada, tion and mineralogy in mollusks (and other organ- Avenida Fuentenueva s/n, 18002 Granada, Spain isms). In the case of bivalves, there are only a few e-mail: [email protected] studies that report the inXuence of environmental J. P. Machado parameters on the shell mineralogy. In Mytilus, Laboratorio de Fisiologia Aplicada, increasing water temperature increases the aragonite/ Instituto de Ciências Biomédicas, calcite proportion in the shells (Lowenstam 1954a, b), Universidade de Porto, Largo Profesor Abel Salazar 2, 4099-003 Porto, Portugal while salinity reduces such proportion (Dodd 1964, e-mail: [email protected] 1966; Eisma 1966).

123 Mar Biol

There are also a few experimental studies on the and Piggot (1981) that changes were the result of tec- V e ect of Mg on chemical composition of the carbonate tonically induced changes in atmospheric partial CO2 skeletons. In particular, Lorens and Bender (1980) pressure (pCO2), rather than oceanic Mg/Ca, following demonstrate that the magnesium content in the calcite mid oceanic ridge activity cycles (Mackenzie and secreted by Mytilus edulis in artiWcial seawaters Morse 1992; Hallock 1997). Stanley and Hardie (1998) increases exponentially with the Mg/Ca ratio in the cul- reviewed the inadequacies of the CO2 model and, in ture medium. Moreover, Stanley et al. (2002) found a accordance with Morse et al. (1997), proposed instead strong correlation between the aqueous magnesium that the Mg/Ca ratio controls carbonate mineralogy. +2 (Mg (aq)) contents in a relatively magnesium depleted Stanley and Hardie (1998) followed Hardie (1996) and culture medium and in the carbonate precipitated by a linked variations of Mg/Ca ratios in marine waters dur- coralline alga. Following a similar experimental line, ing the Phanerozoic to Mg removal following conver- Ries (2005) determined that the aragonitic codiacean sion of basalt into greenstone due to mid oceanic ridge alga Penicillus capitatus is able to precipitate about hydrothermalism. Consequently, increasing rates of 22 § 3% of low-Mg calcite in waters with Mg/Ca < 2. ocean crust production lowers the Mg/Ca ratio of sea- To our knowledge, this is the only previous experimen- water. Stanley and Hardie (1998) presented an tal study that focus on the change of the carbonate updated curve of variation of the mineralogy of non- polymorph (aragonite or calcite) induced by the mag- skeletal carbonates throughout the Phanerozoic and nesium content in the culture medium. These results showed that there is a good match between the skeletal are supported by numerous inorganic studies showing mineralogy of major sediment producing algae and that the presence of dissolved magnesium in the dominant reef builders during the Phanerozoic and the medium favours the precipitation of CaCO3 as arago- aragonitic and calcitic periods. They hypothesized that nite from supersaturated seawater and other Mg-rich skeletal mineralogies of anatomically simple organisms aqueous solutions instead of calcite (Kitano and Kana- were directly inXuenced by seawater chemistry. mori 1966; Lippman 1973; Berner 1975; Deleuze and To determine whether the chemistry of the media is Brantley 1997; Morse et al. 1997) and also decreases able to inXuence the mineralogy of mollusk shells and calcite growth rate (Reddy and Wang 1980; Mucci and if so to what extend, we have cultivated bivalves with Morse 1983; Jiménez-Lopez et al. 2004). The inhibitory predominantly calcitic shells in two mediums with ini- eVect of magnesium in calcite nucleation and growth is tial Mg/Ca molar ratios of (a) 8.4, well above the pres- probably related to the smaller size, the higher charge ent ratio in normal (euhaline) seawaters (t 5.2 molar density and the greater hydration energy of Mg2+, ratio), here referred to as high-Mg experiment, and (b) which impedes calcite nucleation and growth until the 5.0, referred to as control experiment. The main idea of dehydration of Mg2+ occurs (Lippmann 1973). Arago- these experiments was to force bivalves to switch from nite probably forms because Mg adsorption and dehy- precipitating calcite to aragonite by introducing mag- dration slows the growth kinetics of calcite compared nesium in the medium in higher proportions. These to that of aragonite (BischoV and Fyfe 1968). experiments will also help us to understand the relative The study of the eVect of the chemistry of the inXuence of biological versus physicochemical pro- medium on skeletal mineralogy has macroevolutionary cesses on shell formation. implications. Water chemistry has changed over geo- logical time and in particular with respect to the Mg to Ca ratio (Hardie 1996; Stanley and Hardie 1998). In Material and methods this respect, the main discussion is on whether secular changes in certain chemical components of marine Species and shell composition waters may have induced changes in the polymorph from which the skeleton is made. Sandberg (1975) orig- The following species were used in the experiments: inally proposed that ancient oolites shifted from being the Pectinidae Aequipecten opercularis (Linnaeus), calcitic to aragonitic in the Mesozoic due to an increase Chlamys varia (Linnaeus) and Pecten maximus (Linna- in the Mg/Ca ratio. Wilkinson (1979) suggested that eus), the Anomiidae Anomia ephippium Linnaeus, the the mineralogical evolution in marine invertebrates Ostreidae Ostrea edulis Linnaeus and the Mytilidae follows that relationship, with increasing oceanic Mg/ Mytilus galloprovincialis Lamarck. Except for M. gallo- Ca inhibiting calcite deposition. Sandberg (1983) provincialis, that has an outer prismatic calcitic layer divided the Phanerozoic Eon into two calcitic and and an inner nacreous layer, all the other species have three aragonitic sea periods and, together with Wilkin- a predominantly calcitic (mostly foliated) shell. In son et al. (1985), he adhered to the idea of MacKenzie these bivalves aragonite is restricted to muscle inser- 123 Mar Biol

Experimental setting

Specimens were collected from the Mediterranean Sea at Centro OceanográWco de Fuengirola (Ministerio de Ciencia y Tecnología, Málaga) and placed in aquaria in aerated conditions at 18 § 1°C. We selected young individuals, with growth potential. The range of relative sizes (compared to adult sizes) varied from P. maximus (mean § SD = 21 § 6.37, mean adult size in the sampling area = 65 mm) to A. ephippium (mean § SD =35§ 7.5, adult mean size = 43 mm). Both the high- Mg and control experiments were run for 56 days, though living specimens began to be collected for analyses beginning on the 48th day. The high-Mg medium was prepared by mixing 10 l of sea water (Mediterranean Sea, pH = 8.42) and 10 l of a master solution with the following composition:

MgCl2 (Panreac, Barcelona, Spain) 127 mM, CaCl2 (Aldrich, St. Louis, MO, USA) 10 mM and NaCl (Sigma–Aldrich) 460 mM. The Wnal solution (20 l) had 2+ the following chemical composition: Mg (aq) 83.6 mM, 2+ + ¡ Ca (aq) 10.0 mM, Na (aq)460 mM and Cl (aq) 599 mM, pH = 7.90. For the control experiment we mixed 8 l of sea water (Mediterranean Sea) and 8 l of a master solu-

tion with the following composition: MgCl2 50.0 mM, W CaCl2 10.0 mM and NaCl 460.0 mM. The nal solution 2+ (16 l) had the following chemical composition: Mg (aq) 2+ + ¡ 83.6 mM, Ca (aq) 10.0 mM, Na (aq) 460 mM and Cl (aq) Fig. 1 Spatial distribution of aragonite produced by calcitic biv- 565 mM, pH = 8.37. The solutions were renewed alves in high-Mg (left) and control experiments (right), as re- vealed by staining with Feigl’s solution; aragonitic areas in control 22 days following the beginning of the experiment. specimens also stain lighter, despite the fact that they remained Animals were fed on a daily basis with the dry oscill- immersed in Feigl’s solution much longer, indicating less inten- atoriacean blue–green alga Espirulina (ALGAMAR®, sive aragonite production also inside the pallial line; a, b Chlamys varia, right valves, specimens killed at 56 days; the aragonitic Redondela, Pontevedra) with the following composi- 2+ deposits in a extended ventrally outside the pallial line and dor- tion of magnesium and calcium: Mg (aq) 4 mg/g, sally, towards both the anterior and posterior auricles and the Ca2+ 10 mg/g. The initial daily dosage (200 mg) was byssal notch, as well as around the resilium; in control specimens, (aq) there was limited aragonite production only behind the pallial adjusted with time to the number of surviving speci- line and in the adductor muscle; the shell margins in both specimens. mens, grown during the experiment, are marked by arrows; c, d Dead specimens were recovered immediately from Ostrea edulis, right valves, killed at 48 and 56 days, respectively; W in high-Mg experiment specimens, the mantle retreated to the po- the medium until all specimens were nally recovered sition of the foliated layer and produced extensive marginal ara- at the end of the experiment. All shells were rinsed gonitic deposits (black marginal areas); there were also aragonitic with distilled water and dried at 40°C for 3 days. An deposits scattered at the shell’s interior (not visible at this scale); W in the control specimens, aragonitic deposits were only formed in aliquot of 100 ml of the medium was pooled, ltered the adductor muscle scar; e, f Anomia ephippium, right valves, (0.1 m) and set aside for chemical analyses. These killed at 36 and 56 days, respectively; like c, the mantle also re- solution samples were taken at the beginning of the tracted in e to a position marked by a band of aragonite, which is not present in the control specimen f; in general, aragonite ex- experiments, at the time the solution was renewed tended inside the pallial line in control specimens, but in high-Mg (22 days) and at the end of the experiments (56 days). experiment specimens there was important aragonite secretion also outside the pallial line; the venter is towards the bottom in all Analyses cases. aa anterior auricle, pa posterior auricle, ams adductor muscle scar, bn byssal notch, pl pallial line, rs resilium The mineralogy of the newly accreted shell material tion areas (Fig. 1b, d, f). In the Pectinidae and Anomii- was determined by X-ray diVraction (XRD; D5000, dae, there are also very thin (crossed lamellar) Siemens, Munich, Germany), and by electron back- aragonite layers inside the pallial line (Fig. 1b, d). scattered diVraction EBSD (detector TSL OIM 3.5, 123 Mar Biol

2+ EDAX Inc., Tilburg, The Netherlands), coupled with a 56 days. Ca (aq) was 10.5 mM initially, 9.4 mM after SEM (XL30; Philips, The Netherlands). EBSD analy- 22 days and 10.1 mM after 56 days. The Mg/Ca molar ses provide information about the mineral phase of ratio in the medium was 5.1 initially, 5.2 after 22 days individual crystals (a few m in size) while X-ray and 5.2 after 56 days. These data indicate that in the diVraction informs about the mineralogy of a wider control experiment, individuals grew by incorporating area of the shell surface. Distribution of calcite and slightly more calcium than magnesium in the shell, with aragonite on the shell inner surface was assessed by respect to the normal proportion. The pH of the Feigl’s staining (Feigl and Anger 1972). Feigl’s solution medium in the control experiment also remained con- speciWcally stains aragonite in black (in 2–4 min) while stant throughout the experiment, changing from 8.37 at calcite remains unstained. In bivalve shells Feigl’s the beginning of the experiment to a Wnal value of 8.28. staining may not manifest the entire spatial distribution This variation is also within the experimental error for of a certain polymorph as staining might be prevented the pH. in crystals that are very small or/and by the presence of organic coatings. The morphology of the newly depos- Shell mineralogy in specimens cultured in Mg-enriched ited crystals was studied by Field Emission scanning waters electron microscopy (FESEM, Leo Gemini 1530). Untreated samples were observed after coating with In general, specimens were able to form new calcium carbon (UHS evaporator, Hitachi, Tokyo, Japan). carbonate, although the amount of precipitate varied Magnesium contents of newly grown deposit were esti- with specimen survival and among species. All animals mated from energy dispersive X-rays microanalyses were aVected by the change in the chemistry of the (EDX; INCA-200, Oxford Instruments, Oxon, UK). medium and reacted by secreting extensive organic The Mg content in calcite was also estimated from deposits prior to any new carbonate deposition. Rates peak shifts in the XRD pattern (Lippmann 1973). of survival (specimens survived to the end of the exper- 2+ 2+ V The concentrations of Mg (aq) and Ca (aq) in the iment) varied among di erent species. Best rates of solutions were measured by atomic absorption spec- survival were obtained with C. varia (Wve of a total trometry, using an air-acetylene Xame atomizer (AAS; of eight), O. edulis (7/14), A. ephippium (13/23) and 5100B, Perkin-Elmer, USA) after acidiWcation with HCl M. galloprovincialis (17/25). Specimens of both to prevent the precipitation of carbonate. Based on the A. opercularis (13) and P. maximus (8) did not survive repeated analyses of standards and samples, the analyti- longer than 22 days. cal uncertainty was § 0.08 mM for Ca(aq) and Mg(aq). Chlamys varia

Results The most noticeable results were obtained with C. varia. Besides the formation of extensive carbonate deposits Chemistry of the culture medium on the shell’s inner surface that occurred in most of specimens, in three of them, which survived to the end The chemistry of the medium kept almost constant of the experiment, also a signiWcant growth of shell throughout the two periods (22 and 34 days) in which beyond the margin was detected (Fig. 1a). Carbonate the solution remained unchanged. In the high-Mg deposits on the shell’s inner surface consisted on rather 2+ experiment, the total amount of Mg (aq) in solution big prismatic aragonite crystals (up to 30 m in size; was 83.5 mM at the beginning of the study period, Fig. 2a–c). Besides having characteristic morpholog- 83.0 mM after 22 days and 82.7 mM after 56 days. The ies of aragonite, their mineralogy was conWrmed by 2+ total initial amount of Ca (aq) was 10.0 mM, 10.1 mM EBSD and Feigl’s staining. Aragonitic deposits were after 22 days and 9.0 mM after 56 days. The Mg/Ca widespread in the dorsal area and also extended ven- molar ratio in the medium was 8.4 initially, 8.3 at trally well beyond the pallial line (which is the bound- 22 days and 9.2 at 56 days, thus indicating that calcium ary for aragonitic deposits in normal conditions), was preferentially removed relative to magnesium. The even reaching the shell margin (Fig. 1a, b). Aragonite pH of the medium kept almost constant throughout the prisms settled directly onto the naturally grown cal- experiment, ranging from an initial value of 7.90 to a citic foliated shell (Fig. 2b, c), usually separated by a value of 7.83 at the end of the experiment. Such varia- thin organic layer (Fig. 2b). It was observed in some tions are within the experimental error (§ 0.05). instances that the growth of lamellae of the foliated 2+ For the control experiment, Mg (aq) was 53.3 mM layer continued with small (< 1 m) aragonite crystals initially, 48.4 mM afer 22 days and 51.9 mM after (Fig. 2d). 123 Mar Biol

Interestingly, the marginal shell secreted in three (Wbrous to foliated calcite) and did not stain with specimens was composed of two layers. The outer layer Feigl’s solution. XRD indicated that in one of the spec- had a microstructure similar to that of the normal shell imens this layer was high-Mg calcite (5% Mg content). 123 Mar Biol

Fig. 2 Aragonitic (a–f and h and i) and calcitic (e, g and j–l) 100 m backwards from the margin; inset, detail of the aragonitic deposits formed by calcitic bivalves both in high-Mg (a–i) and area, which is formed by microcrystals embedded in organic ma- control experiments (j–l); sketches above indicate the approxi- trix; f prismatic aragonite secreted by Ostrea edulis (right valve) mate position and orientation of shell areas photographed below; close to the venter (20 days of the experiment); inset, detail of a ligaments are drawn in black, pallial lines in light grey and muscu- cyclic twin; g calcite secreted by Ostrea edulis as a continuation of lar impressions in dark grey; arrows indicate position of the vent- the former calcite lath after 48 days; inset, detail of rhombohedral ers; a irregular aragonite crystals growing on the ventral area in a calcites; h rice grain-shaped aragonite crystals grown at the centre left valve of Chlamys varia (48 days of the experiment); note the of the left valve of Anomia ephippium (48 days); aragonite crys- diVerent texture compared to the underlying foliated calcite lay- tals seem to emerge from the foliated calcite layer; inset, detail of er; inset, detail of aragonites; b Xat aragonite prisms growing on aragonite crystals; i aragonite crystals grown below the outer the anterior auricle of a right valve of Chlamys varia (48 days of Wbrous calcite layer in Mytilus galloprovincialis (after 20 days); the experiment); inset, detail of a crystal; note organic Wlm sepa- inset, detail of aragonite plates; j irregular calcite deposits grow- rating the previous foliated layer from the aragonite crystal; c ara- ing on the dorsal area of a control specimen (left valve) of Chla- gonite prisms growing besides the resilifer of a left valve of mys varia (56 days); inset, detail of rhombohedral calcite crystals; Chlamys varia (48 days); inset, detail of prisms; d aragonite mi- k internal surface of the edge of a control specimen of Chlamys crocrystals growing between two big prismatic aragonite crystals varia (left valve), grown after 56 days of experiment; it is entirely as a continuation of the original calcitic lath; ventral edge of a left formed by foliated calcite; inset, detail of laths; l calcite granules valve of Chlamys varia (48 days); inset, detail of microcrystals; e grown on the internal surface of a right valve of a control speci- internal surface of the edge grown in a left valve of Chlamys varia men of Ostrea edulis (56 days); inset, detail of a granule which after 48 days of experiment; the very edge of the shell is formed demonstrates that it is formed by foliated calcite. LV left valve, by high-Mg calcite, whereas aragonite begins to be secreted about RV right valve

A thin internal layer of aragonite crystals embedded in Lorens and Bender (1980). Only one specimen (out of organic matter initiated at a distance of 100–200 m the three examined with FESEM), which died 22 days from the margin (Fig. 2e). after the beginning of the experiment, secreted scat- Figure 3 synthesizes the distribution of aragonitic tered nacre-like aragonite platelets below the outer and high-Mg calcitic deposits in shells of C. varia. Wbrous prismatic calcite layer, close to the boundary with the inner nacreous layer (Fig. 2i). Ostrea edulis Control experiment Many specimens of Ostrea edulis survived up to or close to the end of the experiment. The exposure to the Rates of survival for the diVerent species were: C. varia new medium induced them to retreat their mantle and (four out of nine specimens), P. maximus (5/8), O. edu- to form new secretions backwards from the former lis (14/14), A. ephippium (22/22), M. galloprovincialis shell margin (Fig. 1c). These secretions were either (12/12). Only the species A. opercularis (seven speci- thick organic or extensive aragonitic deposits (Figs. 1c, mens) showed zero survival. 2f). Specimens also formed aragonitic crystals scattered We appreciated deposits on the internal shell sur- in the valve interiors. On the other hand, the original face in C. varia, P. maximus, O. edulis and A. ephip- calcitic foliated layer continued to grow locally in inter- pium and sparse crystals in M. galloprovincialis. Only nal shell areas (Fig. 2g). C. varia and P. maximus extended the margin appreciably. Crystal morphology, EDX and Feigl’s staining Anomia ephippium demonstrate that, in all cases, the shell secreted in the control experiment was low-Mg calcite and also, that Individuals of this species also contracted their mantles the distribution of aragonite does not diVer from that permanently after being placed in the experimental of wild animals (attachment scars and interior of pallial tank. They secreted few carbonate deposits (Fig. 1e). line, when present; Fig. 1b, d, f). Scattered aragonite crystals were recognized onto the calcitic prismatic layer of the lower (right) valve as well Chlamys varia and Pecten maximus as growing onto and from within the foliated layer of the left valve (Fig. 2h). Marginal aragonite deposits Specimens of C. varia showed extensive prismatic were not recognized. deposits onto the native foliated layer in the dorsal area, particularly in the hinge area and around the resi- Mytilus galloprovincialis lium (Fig. 2j). These deposits are similar in extension and thickness to the above described for high-Mg exper- This was the species probably most negatively aVected iment specimens, with the diVerence that the deposits by the high-magnesium concentrations as also seen by are aragonitic in high-Mg experiment specimens and 123 Mar Biol just calcitic in the control specimens. P. maximus Present day marine waters have an Mg/Ca molar showed similar prismatic deposits in the dorsal area, ratio of 5.2, close to the point in which inorganic car- although thinner than those of C. varia. Foliated calcite bonates are composed only of aragonite, any propor- continued to grow in the rest of the shell interior in tion of calcite (even high-Mg) being excluded (Stanley both species. All surviving specimens of both species and Hardie 1998). However, marine bivalves easily extended the shell margin appreciably, which showed secrete low-Mg calcite in their outer shell layers (this no change in microstructure with respect to individuals applies particularly to epibenthic bivalves), which grown in natural conditions (Fig. 2k). recalls for a biological control of the polymorphic phase. This is particularly true since there is little or no Ostrea edulis and Anomia ephippium diVerence in the Mg/Ca ratio between marine waters and the extrapallial Xuid of marine species (Lorens and Specimens of both species retracted their mantles and Bender 1980; Wilbur and Saleuddin 1983). continued to secrete foliated calcite either as a continu- While the control on precipitation of calcium car- ation of the former shell or, in O. edulis, as aggregates bonate on these organisms is mainly biological, our onto it (Fig. 2l). results clearly show that the chemistry of the medium aVects not only the chemical composition (Mg-content) Mytilus galloprovincialis of the calcite secreted (as already demonstrated in M. edulis; Lorens and Bender 1980) but also determines Although no marginal shell growth was detected, the the polymorph of calcium carbonate formed by the morphology of the Wbres composing the calcitic pris- organism. The pH is not likely to have inXuenced the matic outer layer indicates that this layer grew slightly. deposition of aragonite since its values remained constant throughout the experiments. The fact that pH values of the high-Mg waters were somewhat lower Discussion and conclusions than those of the control waters cannot be responsible for the mineralogical changes observed since decreas- Mollusks control shell mineralization in an exhaustive ing pH decreases the saturation and aragonite precipi- way. They deposit shells made by two or more superim- tation requires higher saturation values than those posed layers with diVerent microstructures, which may required for calcite (Lippmann 1973). The precipita- even be composed of diVerent calcium carbonate poly- tion of aragonite in bivalves living in high-Mg seawater morphs (i.e., calcite or aragonite) (Taylor et al. 1969). would, thus, be thermodynamically disfavoured com- Even more striking is that superimposed layers with pared to control bivalves. Accordingly, the switch from diVerent polymorphic compositions are secreted by the calcite to aragonite secretion in the inner shell surface mantle simultaneously. Since diVerent species present in all bivalve species above mentioned living in high- speciWc shell mineralogies and microstructures, all these Mg water can only be attributed to the high Mg/Ca processes are generally believed to be genetically ratio of the medium (= 8.4). This high Mg/Ca ratio directed (Addadi and Weiner 1992). However, the main inhibited the precipitation of calcite, the precipitation question is how the mollusk speciWcally controls shell of aragonite becoming kinetically more favourable. mineralogy. There is evidence from many studies that Since a change in the type or concentration of organic they employ specialized organic macromolecules to con- molecules is not expected in our specimens, we control calcium carbonate precipitation. In vitro experi- clude that the change in chemical conditions has over- ments have showed that speciWc soluble organic come the capacity of the organism to control the macromolecules, composing the shell organic matrix conditions for precipitation of calcium carbonate forc- from shell layers with diVerent mineralogies, speciWcally ing a switch in the polymorph secreted. To our knowl- select the same polymorphic phase of calcium carbonate edge, this is the Wrst experimental study to evidence (Belcher et al. 1996; Falini et al. 1996; Thompson et al. how the chemistry of the medium aVects the mineral- 2000). These macromolecules inhibit the formation of ogy of the carbonate secreted by organisms in general one polymorph (such as calcite) and select another poly- and for bivalves in particular. However, this has not morph (such as aragonite) and vice versa. Additionally, been the case in the newly secreted marginal shell of there is a cooperative mechanism between these soluble C. varia, which was still calcitic and even retained the macromolecules and the insoluble organic matrix, which original microstructure, although with relatively high- conforms a favourable substrate or microenvironment Mg content (Fig. 3). This calls for diVerences between for the precipitation of one polymorph or another (Add- the marginal and internal mantle in either the type and/ adi et al. 1987; Manoli et al. 1997; Falini 2000). or the concentration of organic molecules secreted 123 Mar Biol

and A.R.-N. also acknowledge Wnancial support through the Programa Ramón y Cajal (MCyT, Spain).

References

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