Non-equilibrium fractionation of stable carbon isotopes in chemosynthetic mussels

Chemical Geology 387 (2014) 35–46

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Non-equilibrium fractionation of stable carbon isotopes in chemosynthetic mussels K. Nedoncelle a,b, N. Le Bris a,b, M. de Rafélis c, N. Labourdette c, F. Lartaud a,b,⁎ a b c

CNRS, UMR8222, LECOB, Observatoire Océanologique, F-66650 Banyuls/Mer, France Sorbonne Universités, UPMC Univ Paris 06, UMR 8222, LECOB (Laboratoire d’Ecogéochimie des Environnements Benthiques), Observatoire Océanologique, F-66650 Banyuls/Mer, France Sorbonne Universités, UPMC Univ Paris 06, UMR 7193, ISTEP, Institut des Sciences de la Terre de Paris, 4 Place Jussieu, 75005 Paris, France

a r t i c l e

i n f o

Article history: Received 29 January 2014 Received in revised form 22 July 2014 Accepted 1 August 2014 Available online 10 August 2014 Editor: D.R. Hilton Keywords: Hydrothermal vent mussels Stable carbon isotopes Shell growth rate 13 C fractionation Chemosynthetic pathways

a b s t r a c t Chemosynthetic bivalves from deep-sea hydrothermal vents exploit the energy derived from chemical compounds, such as methane, sulfide or hydrogen, using symbiotic bacteria that are able to fix inorganic carbon. Available chemical resources in their habitat vary widely at various scales, from the vent field scale to the micro-habitat scale. Parallel to this environmental heterogeneity, Bathymodiolus species are considered to be flexible in their energy acquisition pathways. The goal of this study was to determine whether the isotopic compositions archived in the shells of hydrothermal vent mussels could trace chemical energy sources and their variability over spatial and temporal scales. Two different species (Bathymodiolus azoricus and Bathymodiolus thermophilus) inhabiting three vent fields with contrasted geochemical features on the Mid Atlantic Ridge (MAR; Rainbow and Menez Gwen) and the East Pacific Rise (EPR; 9°47′N), were considered for carbon isotopes and growth rate variation along the shell length. The study revealed that 13C fractionation between shells and seawater is higher than expected from calcite– bicarbonate equilibrium fractionation, suggesting a significant influence of the chemosynthetic pathway on the shell composition. Furthermore, significant differences in δ13Cshell fractionation with respect to seawater are observed between sites and habitats of the two MAR vent fields, suggesting that different chemosynthetic pathway (e.g. methanotrophic and thiotrophic) could lead to variable enrichments of the shell in 13C. Mussels supposed to rely more largely on methanotrophy (at Rainbow where free sulfide is unavailable) display a lower δ13Cshell values than mussels relying also on sulfide-oxidizing symbiosis (at Menez Gwen). Variability in δ13Cshell between habitats, or between individuals within the same assemblage, could thus reflect differences in the symbiosis activity at a micro-habitat scale. These isotopic signatures could provide useful information on the relationships between micro-habitat properties, symbiont activity and shell mineralization. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The remarkable productivity of deep-sea hydrothermal vent ecosystems relies on the association between chemoautotrophic bacteria, which are able to fix carbon using the oxidation of reduced compounds supplied by the fluid, and invertebrate species taking advantage of this symbiosis (Fisher et al. (2007) for review). Mytilids are among the most successful colonizer of vent environments where they often dominate the macrofaunal biomass (Fiala-Médioni et al., 1986; Van Dover, 2000; Cravo 2007). Chemosynthetic representatives from the Bathymodiolus genus are present in many hydrothermal habitats (Van Dover, 2000). They are characterized by the diversity of their chemosynthetic symbionts (Fisher et al., 1993; Dubilier et al., 2008; Duperron et al., 2008). Some Bathymodiolus species host a dual symbiosis, with a sulfide-oxidizing bacteria (SOx) ⁎ Corresponding author at: Sorbonne Universités, Univ Paris 06, UMR CNRS-UPMC 8222, LECOB, Observatoire Océanologique, F-66650 Banyuls/Mer, France. E-mail address: [email protected] (F. Lartaud).

http://dx.doi.org/10.1016/j.chemgeo.2014.08.002 0009-2541/© 2014 Elsevier B.V. All rights reserved.

and methane-oxidizing bacteria (MOx) coexisting in their gills (Dubilier et al., 2008). The relative abundance of these bacterial phylotypes is suggested to reflect the bioavailability of methane or sulfide, with more SOx bacteria than MOx bacteria when sulfide is abundant (Halary et al., 2008; Riou et al., 2008). Petersen et al. (2011) additionally demonstrated that SOx bacteria are also able to use hydrogen as an energy source when this substrate is available. The diversity of the bacteria population is expected to confer these mussels a high flexibility in the exploitation of available energetic resources and to optimize host adaptation to a range of geochemical environments (Le Bris and Duperron, 2010). This flexibility is further enhanced by the mixotrophic capacity of Bathymodiolus mussels that are also able to filter suspended particulate and dissolved organic matter (Fisher, 1990; Cavanaugh et al., 1992; Fiala-Médioni et al., 2002; Riou et al., 2010). However, it is generally accepted that Bathymodiolus species rely mainly on chemosynthesis-derived material with a negligible contribution of photosynthetic-derived compounds in their diet (Colaço et al., 2009).

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The isotopic composition (carbon and nitrogen) of Bathymodiolus soft tissues was previously considered as proxy of chemosynthetic pathways which could therefore reflect the different energy resources (Trask and Van Dover, 1999; Colaço et al., 2002; Fiala-Médioni et al., 2002). The carbon isotope values of tissues of chemosynthetic bivalves are strongly depleted in 13C compared to photosynthetic bivalves (Dubilier et al., 1998; Colaço et al., 2002; Fiala-Médioni et al., 2002; De Busserolles et al., 2009). Furthermore, tissues were shown to be more depleted in 13C relatively to 12 C in cold seep environments (e.g. − 42 to − 77‰ for Bathymodiolus heckerae) dominated by methanotrophy due to the extremely light composition of methane in these environments whereas the reliance on sulfide-oxidation dominating in hydrothermal vent system is reflected in higher δ13C of tissues (e.g., − 23 to − 34‰ for Bathymodiolus thermophilus) as the carbon source (CO2) is less 13C depleted in this case (Rau and Hedges, 1979; Paull et al., 1985; Colaço et al., 2002). The signature of the two pathways was also suspected for species that rely on dual symbiosis, methanotrophy and sulfide-oxidizing, even at hydrothermal vents where methane is much less depleted in 13C than at cold seeps (Riou et al., 2008). But the organism tissues only reflect an isotopic signal from the last days of life. Other proxies have to be used to identify chemosynthetic pathways from dead shells or investigate potential metabolic changes over the lifetime of an individual. Accretionary growth of mollusk shells archives geochemical proxies in the carbonate increments, and the δ13C of shallow waters bivalve shells is derived from both dissolved inorganic carbon (DIC) in the surrounding water and organic carbon sources (food), the latter being incorporated into the shell via the metabolism (Lorrain et al., 2004; Lartaud et al., 2010b; Sadler et al., 2012). So far, few studies focused on deep-sea shells (Schöne and Giere, 2005; Lietard and Pierre, 2009). The goal of this study is to better describe the processes controlling the carbon isotopic composition in shells from chemosynthetic habitats, in order to determine the variability of this proxy with habitat conditions, particularly for dead or fossil shell assemblages. Our approach focused on two different Bathymodiolus species hosted on three contrasted hydrothermal vent fields: Bathymodiolus azoricus from Rainbow and Menez Gwen on the Mid Atlantic Ridge (MAR) and, for comparison, B. thermophilus from the V-vent site at 9°47′N on the East Pacific Rise (EPR). Menez Gwen and 9°47′N hydrothermal circulation are hosted in basaltic settings while Rainbow is associated with an ultramafic geological context, leading to substantial differences in the endmember fluid composition (Charlou et al., 2000; Tivey, 2007). Although Menez Gwen and Rainbow are located on the same slow-spreading ridge (MAR) (distant of about 270 km), they largely differ in the availability of sulfide, methane and hydrogen (Le Bris and Duperron (2010) for review). Mussels from the MAR sites host both MOx bacteria and SOx bacteria and different energy pathways for B. azoricus symbiosis have been related to these distinct physico-chemical habitat properties (Duperron et al., 2006; Le Bris and Duperron, 2010; Petersen et al., 2011). To the difference, B. thermophilus from the EPR fast spreading ridge harbor a single endosymbiosis relying on thiotrophy alone. The multiscale approach proposed in this study involves for the first time comparison of shell isotopic compositions between vent fields at ridge scale, between distinct sites of a vent field, and between micro-habitats at meter scale. Furthermore, since shell increments give access to the growth pattern of an individual over time (Schöne and Giere, 2005; Nedoncelle et al., 2013), it was investigated whether the variability in the δ13C recorded along shell length can reflect to some extend the dynamics of environment or the chemical energy pathways used by vent mussels. 2. Methods 2.1. Sampling area Three hydrothermal vent field areas were selected for shells sampling, two from the MAR (Rainbow and Menez Gwen) and one from the EPR

(EPR 9°47′N) (Fig. 1). Rainbow fluids hosted in ultramafic basement are characterized by high methane and hydrogen contents but also high metal concentrations, especially iron. The consequence of this high iron content is the formation of sulfide complexes and subsequent unavailability of free sulfide, the only sulfide form that can pass the branchial membrane and be used by the SOx symbionts (Le Bris and Duperron, 2010). Mussels from this site were shown to host both MOx bacteria and SOx bacteria, which suggest that H2 could serve as an alternative energy source for the thiotrophic symbionts, as shown for the Logatchev vent field (Petersen et al., 2011). Live B. azoricus from Rainbow (36°13.8′N–33°54.1′W, 2275 m) were collected during the MoMARDREAM cruise (CNRS, R/V L'Atalante, August–September 2008) using the Victor 6000 ROV and then dissected onboard. Two locations A and B distant of about 30 m were sampled (Fig. 2a and b). They were both located at the periphery of a large mineral sulfide chimney complex, with several meters high black smokers in the center surrounded by numerous smaller edifices, extinct and weakly diffusing. Location A was divided into A1 zone, at the basis of a sulfide structure about 1 meter-high in the immediate vicinity of a small spire-type active chimney, and A3 zone further to the chimney, on the top of the rocky structure 1 m above A1 and characterized by higher mussel density (Fig. 2a). Large mussels from location B were sampled from the wall of a small sulfide chimney (Fig. 2b). On Menez Gwen vent field, both methane and sulfide are bioavailable for symbiotic organisms, end-member fluids being relatively depleted in metals (Charlou et al., 2002; Douville et al., 2002). B. azoricus shells from Menez Gwen were collected during the MenezMAR cruise (MARUM, RV Meteor, September–October 2011) using the Quest 4000 ROV. Two vent sites distant of 80 m were sampled: Cage Site (37°50.6′ N–31°31.2′W, 815 m), characterized by small discretely distributed mussel patches at the top of a hydrothermal area with some active diffuse vents (CS-shells) (Fig. 2c), and White Flame (37°50.7′N–31°31.1′ W, 832 m) hosting an important mussel bed along the wall of an edifice dominated by a large active white smoker (WF-shells) (Fig. 2d). These vent sites have been mapped in detail by photomosaicking (Marcon et al., 2013). To the difference of the MAR, the diffuse flow vents colonized by mussels on the EPR fast spreading ridge are characterized by important free sulfide concentration but low concentration of methane and hydrogen (Von Damm and Lilley, 2004), conferring to the mussels a single endosymbiosis that supposed to rely only on thiotrophy. B. thermophilus were collected from V-vent (09°47.3′N–104°17.0′W, 2513 m) during the MESCAL1 cruise (UPMC, R/V L'Atalante, April–May 2010) using the Nautile submersible. A single large mussel bed (about 50 m2), located on a diffuse vent area on the basaltic seafloor was sampled both at the periphery (P-shells) and close to the center (C-shells) of the patch covering the top of a small outcrop (Fig. 2e). 2.2. Shell preparation for isotopic and growth rate analysis Isotopic and growth rate analyses were performed on the calcite layer of shells, since calcite is less sensitive to alteration and dissolution than aragonite in those specific environments and preserves the pristine signal from diagenetic alterations (Lutz et al., 1994). For the two species (B. azoricus and B. thermophilus), the mean shell length is representative of the most abundant size class. At Rainbow and Menez Gwen, B. azoricus shell samples with similar size were selected in order to obtain homogeneous groups between vent fields and habitats (KW test between A1, A3, B, CS and WF, p-values N 0.05), with a mean length of 53 ± 21 mm (Table 1). At V-vent site, the studied B. thermophilus also have similar shell length (142 ± 9 mm) between the Center and the Periphery of the mussel bed (MW test, p-values = 1). Two carbonate sampling strategies were used in this study. Lowresolution samplings were done on shells from the three sites in order to compare the isotopic composition between organisms located at distinct vent fields and contrasted habitats. Additionally, high-resolution

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Fig. 1. Localization of sampling sites on the East Pacific Rise (EPR) and Mid-Atlantic Ridge (MAR). The bathymetry was prepared using the GeoMapApp free software (www.geomapapp.org).

samplings were performed on shells from Rainbow to investigate the isotopic evolution with shell growth. To study shell isotopic composition, the valve portions were placed in a peroxide solution (H2O2 3.4%) between 6 and 10 h at 60 °C in order to remove the periostracum layer, and then rinsed with demineralized

water. Calcite powder from the prismatic upper layer was sampled from the ventral margin to the hinge along the maximum growth axis with a Dremel device. Special attention was done to avoid the sampling of the nacreous aragonite lower layer situated just below the prismatic calcite upper layer, to preclude any mineralogical effect

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Fig. 2. Sampling sites (MAR and EPR). a and b: rainbow hydrothermal vent habitats (IFREMER-MoMARDREAM08). Site A is divided into two zones (A1 and A3) according to the proximity of the mussels from the small active. c and d: Menez Gwen habitats (MARUM-MenezMAR). c: Cage Site. d: White Flame. e: 9°47′N V-vent habitats (pictures: IFREMER-MESCAL). Arrows represent the location of collected mussels. White scale bars on images b, c, d and e measure 10 cm.

Table 1 Shell references used for isotopic and sclerochronological analysis. Shell length corresponds to the maximum shell size projected on a 2D plane. The number of samples is given according to the sampling resolution of the carbonate. Vent

Rainbow

Site

A1

A3

B

Menez Gwen

Cage Site

White Flame

9°47′N V-vent

Center Periphery

Shell

Shell length

Number of samples for isotopy

Reference

(mm)

Low-resolution

High-resolution

A1-1 A1-2 A1-3 A3-1 A3-2 A3-3 B-1 B-2 B-3 CS-1 CS-2 CS-3 CS-4 WF-1 WF-2 WF-3 C-1 C-2 P-1 P-2

44 77 24 32 41 45 40 35 87 91 80 59 43 47 43 59 142 148 148 128

10 17 5 6 8 10 7 5 19 17 16 12 8 9 10 12 5 7 6 6

47 84 24 27 38 47 36 25 89 – – – – – – – – – – –

on the isotopic values. Indeed, comparatively to calcite, aragonite is enriched by 1.7 ± 0.4‰ in 13C (Romanek et al., 1992). Since B. thermophilus shells are larger than B. azoricus ones, a different sampling interval was chosen for the two species. Lowresolution samples, performed over a 5 mm wide band, were obtained each 5 mm from the umbo to the ventral margin of shells from Menez Gwen and Rainbow, while bands were collected each 2.5 cm on shells from V-vent. Carbonate powders were thus obtained from 7 shells from Menez Gwen (4 at CS and 3 at WF, n = 8–17 per shell), 9 shells from Rainbow (3 at A1, 3 at A3 and 3 at B, n = 5–19 per shell), and 4 shells from V-vent (2 at C and 2 at P, n = 5–7 per shell) (Table 1). An additional higher-resolution carbonate sampling (every 1 mm) was performed on the 9 shells from Rainbow in order to examine the correspondence between the isotopic data and the growth rate patterns. The left valve of each individual was cut along the maximum growth axis using a Buehler Isomet low-speed saw. In order to precisely match growth rate patterns and isotopic data, one part of the left valve was used for sclerochronological analysis and the other part served for isotopic analysis. According to the shell size, between 24 and 89 isotopic data were obtained per individuals (Table 1). Calcium carbonate powder of low- and high-resolution samples were acidified in 100% H3PO4 at 90 °C under vacuum. Formed CO2 was collected and analyzed using a mass spectrometer (VG Instruments

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Isoprime). Isotopic data are reported in the conventional delta (δ) notation relative to Vienna Pee Dee Belemnite V-PDB (Craig, 1957): δ ‰ VPDB ¼

h
 i Rsample –Rstandard =Rstandard  1000

where R is the isotopic ratio of heavy to light carbon isotopes: 13C/12C. The reference used for the analyses was an internal standard calibrated using NBS‐19. The standard deviation for δ13C was ±0.1‰. To characterize the shell growth rate of mussels from Rainbow, the second section of the left valve was embedded in epoxy resin and then cut parallel to the maximum growth axis in order to obtain a 5 mm thick slide. This slide was ground flat (180, 400 and 800 grit powder with distilled water), and then polished (3, 1 and 0.3 μm Al2O3 powder). To highlight the shells' minor- and major growth lines, all the thick polished cross-sections were etched in Mutvei's solution (Mutvei et al., 1994) at 38–40 °C for 4–6 h, as described by Nedoncelle et al. (2013) (Fig. 3). Sclerochronological patterns were analyzed on 9 shells from Rainbow also studied for their isotopic patterns. The growth rate profiles, reflecting as the variation of the width between two successive striae (i.e., increment width) across the shell section, was performed according to Nedoncelle et al. (2013). However, this analysis was not always possible over the entire shell length as the shell curvature frequently impede revealing growth increment close to the hinge portion of the shells. Major growth lines were identified on each shell as major growth line present both in the upper calcitic and lower aragonitic layers, and were often accompanied with a shell deformation on the external surface (Schöne and Giere, 2005). To compare growth rate variability with δ13C fluctuations, increment width was averaged every 1 mm from the ventral margin to the hinge.

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These two sites are distant of 5.7 km. The carbon isotopes values of the total DIC was obtained by acidification of water, according to Pierre et al. (1994). Measurements were made using a VG mass spectrometer. δ13CDIC values were reported in the conventional delta (δ) notation relative to VPDB (Craig, 1957). The standard deviation for δ13C measurements is ±0.1‰. 2.4. Statistical analysis Comparisons of δ13C values between vents, sites or shells were performed by applying the Kruskal and Wallis test (KW) followed by a post hoc test to discriminate the outlying groups (Wilcoxon rank sum test (WRS) coupled to a Bonferroni correction). The KW test is used for comparison of more than two samples to infer if at least one sample is different from the others, whereas the WRS test determines which samples are different from each others. The Mann–Whitney test (MW) was used for comparison between two samples. Before using this non-parametric test, the non-respect of the normality and homoscedasticity conditions were checked using the Shapiro and Bartlett tests respectively. Those tests were also applied for sclerochronological studies on the increment width between the three studied locations at Rainbow (A1, A3 and B). For each shell from Rainbow, comparison between mean δ13C measured every 1 or 5 mm are performed using the MW test. At individual scale, Spearman test was applied on δ13C data to identify the presence/absence of an ontogenic trend in the isotopic profiles of shells from Rainbow. Mean shell growth rate was averaged every 1 mm and compared to isotopic data using the correlation test of Spearman. 3. Results

2.3. Seawater isotopic analysis

3.1. Shell isotope composition between and within vent fields

On the EPR, seawater samples were collected in 100 mL-glass bottle and were immediately fixed by addition of 1 mL saturated solution of HgCl2 for carbon isotopes analysis of the DIC. At V-vent, during the MESCAL 1 cruise, only one seawater sample was collected. This was completed with additional collection at Bio 9 (9°50.3′N–104°17.5′W, 2508 m) during the MESCAL 2 cruise (R/V L'Atalante, UPMC, March 2012), with two samples of seawater and one in the mussel habitat.

The mean δ13C values along the shell of the studied Bathymodiolus individuals are 3.6 ± 0.5‰ for Menez Gwen, 3.1 ± 0.4‰ for Rainbow and 2.4 ± 0.3‰ for 9°47′N (Table 2). These δ13C compositions are distinct between the 3 vent fields (KW test on δ13C values between Rainbow, Menez Gwen and 9°47′N, chi-squared = 89, p-value b 0.05). In order to quantify the importance of 13C fractionation between the shell carbonate and background deep-sea water and compare shells

Fig. 3. Bathymodiolus shell in cross section along the maximum growth axis (black line on the picture) with Mutvei image highlighting details of the microstructures. The upper fibrous prismatic layer of calcite (p) and lower nacreous layer of aragonite (n) are shown. Increments are identified in the prismatic outer layer (p) where growth lines are organized perpendicular to the external surface. Major growth line extends in the nacreous layer. Isotopic measurements have been carried out on the prismatic calcite layer. U. umbo; V.m. ventral margin; d.o.g. direction of growth.

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Table 2 Mean, minimum and maximum δ13C values measured in the calcite layer of B. azoricus and B. thermophilus shells from the three studied sites. Mean, minimum and maximum ΔC = δ13Cshell − δ13CDIC are reported for comparison between vent fields. Vent field

N

δ13Cshell (‰VPDB)

ΔC = δ13Cshell − δ13CDIC (‰VPDB)

Mean ± SD

Min

Max

Mean ± SD

Min

Max

Rainbow Menez Gwen 9°47′N

87 84 24

3.1 ± 0.4 3.6 ± 0.5 2.4 ± 0.3

2.3 2.6 1.7

4.1 4.5 3.3

2.4 ± 0.4 3.0 ± 0.5 2.8 ± 0.3

1.7 1.9 2.0

3.4 3.9 3.7

Table 4 Statistical comparisons between each habitat at Rainbow (a), Menez Gwen (b) and 9°47′ N-EPR (c). a: KW test : χ2 = 44; p-value b0.05 WRS test A1 A3 B

Table 3 Mean, minimum and maximum δ13C values measured in the calcite layer of B. azoricus and B. thermophilus shells from the different habitats of the three studied sites. δ13C (‰VPDB) Vent field

Rainbow Menez Gwen 9°47′N

Habitat

N

A1 A3 B Cage Site White Flame Center Periphery

32 24 31 53 31 12 12

Mean ± SD

Min

Max

3.4 2.8 2.8 3.8 3.3 2.5 2.3

2.8 2.5 2.3 3.0 2.6 2.2 1.7

4.1 3.3 3.5 4.5 4.3 3.3 2.9

± ± ± ± ± ± ±

0.3 0.2 0.3 0.4 0.5 0.4 0.3

A3 b0.05 – –

CS – –

WF b0.05 –

C – –

P N0.05 –

B b0.05 N0.05 –

b: MW test : p-value b0.05 CS WF

from different vent fields on this basis, we calculated the difference ΔC = δ13Cshell − δ13CDIC (Table 2). On the EPR, the δ13CDIC values measured in seawater at V-vent site (−0.2‰) are close to values measured in seawater (between −0.4‰ and −0.5‰) at Bio-9 and in the mussel habitat at this site (− 0.3‰). These carbon isotopic compositions are also in good agreement with the values measured in seawater by Fisher et al. (1994) and Proskurowski et al. (2008b) in different sites from this area (−0.4‰ to −0.5‰). The mean carbon isotopic composition in the north Atlantic deep-waters is higher, with δ13CDIC ranging between 0.5 and 0.8‰ (Kroopnick, 1985; Wisshak et al., 2009; Lang et al., 2012). In order to account for the differences induced on shell isotopic composition by the different isotopic composition of seawater DIC between the two ridges, we used a median δ13CDIC of −0.35‰ for the EPR and 0.65‰ for the MAR. Shells from 9°47′N and Menez Gwen display the most important fractionation with ΔC values of 2.8 ± 0.3‰ and 3.0 ± 0.5‰, respectively, whereas B. azoricus shells from Rainbow display a lower fractionation on average (2.4 ± 0.4‰) (Table 2). At habitat scale, differences were observed among different assemblages of a vent field on the MAR. At Rainbow, shells from the site A1 (3.4 ± 0.3‰) exhibit significant higher δ13C values than the shells from A3 (2.8 ± 0.2‰) and B (2.8 ± 0.3‰) (Tables 3 and 4). Shells from the sites A3 and B have similar δ13C mean values (Table 4). At Menez Gwen, shells collected at Cage Site have significant higher δ13C values than shells from White Flame (δ13CCS = 3.8 ± 0.4‰ and δ13CWF = 3.3 ± 0.5‰) (Tables 3 and 4). Shells from White Flame are more similar to those from the A1 group from Rainbow. At 9°47′N, we did not observe differences between individual pools in the same mussel bed: shells from the central part of the mussel bed cannot be distinguished from the shells located at the periphery using their carbon isotope compositions (δ13CC = 2.5 ± 0.4 and δ13CP = 2.3 ± 0.3) (Tables 3 and 4). Inter-individual variability is still observed among shells from a single micro-habitat (Fig. 4). For shells from Rainbow, at the site A1, δ13CA1-1 is significantly higher than δ13CA1-2 and δ13CA1-3 (Table 5). Conversely, A1-2 and A1-3 shells have similar δ13C mean values (Table 5). At the site A3, all shells have similar δ13C values (Table 5). At site B, δ13CB-1 is significantly lower than δ13CB-3 (Table 5). Shell B-2 has an intermediate composition which is not different from B-1 and B-3 (Table 5). For the Menez Gwen vent field, at Cage Site, δ13CCS-2 is significantly higher than δ13CCS-3 (Table 5). At White Flame, δ13CWF-1 is significantly lower than δ13CWF-2 and δ13CWF-3 (Table 5 and Fig. 4).

A1 – – –

c: MW test : p-value N0.05 C P

3.2. Isotopic composition and growth rate B. azoricus shells from Rainbow revealed large variability in the increment width and present an ontogenic growth rate decrease (stationarity tests between increment width variation and time, p-values b 0.05 and r b 0 for all shells, except for the shell A1-2) (Fig. 5). Significant differences in mean growth are observed between shells, with mussels from A3 (increment width of 60 ± 31 μm) having a higher daily growth rate than mussels from A1 (41 ± 22 μm) and B (41 ± 21 μm) (KW test between increment width on shells from A1, A3 and B, chi-squared = 385, pvalue b 0.05; WRS tests, p-values N 0.05 between A1 and B only) (Fig. 6). Additionally, shells from A3 exhibit wider variability in the increment width (varied mostly between 37 and 77 μm based on quartiles 1 and 3), than shells from A1 (24 to 53 μm) and B (25 to 53 μm) (Fig. 6). Similarly to Bathymodiolus brevior (Schöne and Giere, 2005) and contrarily to B. thermophilus (Nedoncelle et al., 2013), each B. azoricus shell from Rainbow displayed up to 5 growth breaks which are characterized by major growth lines extending from the upper calcitic layer to the lower nacreous layer (Fig. 3). They are randomly distributed with variable number of increment or shell length between two successive major growth lines and are not associated with δ13C changes (Nedoncelle, 2013). High-resolution δ13C profiles of the shells from Rainbow (i.e. sampled every 1 mm) display the same mean δ13C value than low-resolution profiles (every 5 mm) (MW tests, p-values N 0.05), even though the variation range is slightly enlarged in the former case. At the shell scale, isotopic profiles are relatively stable, only interrupted by unfrequent positive or negative anomalies and in few cases steep increase or decrease (Fig. 5). Surprisingly, these anomalies are not observed in all shells and their number differs among shells from a same site. They are neither related to particular shell deformation (major growth lines) (Fig. 5). At individual scale, the variations of the carbon isotopic composition are not significantly correlated with growth rate fluctuations (Spearman tests between mean increment width and δ13C value measured every 1 mm from the ventral margin to the hinge, p-values N 0.05). However, at the vent field scale, when these parameters are averaged over the shell length, there is a negative relationship between these two parameters (p-value b 0.05) (Fig. 6). Shells from A1 location exhibit the higher δ13C and lower mean increment width values, whereas shells from A3 have lower δ13C and higher mean increment width. Ssite B is intermediate between these two cases (Fig. 6).

4. Discussion 4.1. Shell carbon isotopes as potential signature of chemosynthetic pathway The isotopic measurements performed in this study are in good agreement with previous works on B. thermophilus from 9°50′N

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Fig. 4. Box plots of δ13Cshell. Minimum, 1st quartile, median, 3rd quartile and maximum values are represented for each individual, from Rainbow (white color), Menez Gwen (light gray color) and 9°47′N V-vent (dark gray color). The dotted vertical lines distinguish different sample pools. Median δ13CDIC, present as horizontal black lines, are equal to 0.65‰ on the MAR and −0.35‰ on the EPR. CS: Cage Site; WF: White Flame; C: Center; P: Periphery.

(Bio-vent: 2.1 to 4.0‰) and Galápagos spreading center (Rose Garden : 2.2 to 2.9‰), and B. azoricus from Rainbow (2.4 to 3.3‰), Menez Gwen (3.0 to 4.4‰) and Lucky Strike (3.5 to 4.7‰) (Lietard and Pierre, 2009; Lartaud et al., 2011a). These authors also observed differences between the two species and between the vent fields on the MAR with shells from Menez Gwen and Lucky Strike characterized by heavier isotopic composition than shells from Rainbow. The isotopic composition of ambient DIC is generally considered as the main controlling factor for the interpretation of observed δ13C of bivalve shells (Arthur et al., 1983; Surge et al., 2001; Lorrain et al., 2004). Although seawater DIC is generally considered as the predominant contributor to the shell carbonate, methane oxidation and its contribution to pore-water DIC sometimes result in low δ13C values of precipitated carbonates. This is well described at cold seeps where sediment pore fluids are characterized by highly depleted 13 C DIC sources. Bathymodiolus species inhabiting these habitats (as Bathymodiolus childressi and Bathymodiolus boomerang) have a lower δ13Cshell signature (− 7.3 to − 3.6‰ and − 9.4 to 2.7‰ respectively) than species living in hydrothermal vents such as Bathymodiolus septemdierum (6.2‰), B. brevior (1.9 to 3.7‰), Bathymodiolus elongata (2.0 to 3.7‰), B. thermophilus and B. azoricus (Schöne and Giere, 2005; Naraoka et al., 2008; Lietard and Pierre, 2009; Lartaud et al., 2011a). This was also considered to generate the lower values measured in fossil B. aff. azoricus shells from Ghost City (− 2.6 to 0.6‰). With respect to carbonate formation conditions, the mussel environment in these alkaline and low-temperature vents could strongly differ from the environment associated to high-temperature on the MAR (Lartaud et al., 2011a). In the current study, significant differences in δ13CDIC have been recorded between shells from the MAR and EPR. The difference in seawater DIC isotopic composition between oceanic deep waters is important between these two areas and is likely to contribute to these differences. Conversely, the contribution of vent fluids is unlikely significantly influencing the isotopic composition of the shell. Though CO2 and methane have 13C depleted isotopic compositions in the end-member

fluids (δ13CO2 = −9.1 to −6.8‰ at Menez Gwen, − 4 to 1‰ at Rainbow and − 4.4 to − 3.7‰ at 9°47′N on the EPR). The mussel habitat from these high-temperature vent fields is characterized by a large dilution rate of the hydrothermal fluid in seawater (Le Bris and Duperron, 2010). For instance, for a maximum habitat temperature of 15 °C and end-member fluid temperatures of 284 °C at Menez Gwen, 365 °C at Rainbow (Charlou et al., 2000; 2002) and 371 °C at 9°47′N-EPR (Von Damm et al., 2000), the fluid mix surrounding shells always exceed 96% seawater. Temperature data obtained in the immediate mussels environment, furthermore, reveal that these micro-habitats are frequently depleted of hydrothermal contribution (Sarradin et al. (1999) and Desbruyères et al. (2001) for the MAR and Le Bris et al. (2006), Le Bris and Duperron (2010) and Moore et al. (2009) for the EPR), continuous records confirm large period of time at near-ambient seawater temperature (Johnson et al., 1994; Contreira-Pereira et al., 2013). The environmental DIC composition for these mussels is thus likely to remain close to the seawater value, as illustrated by our measurements at Bio-9. More important is the fact that whatever the difference in seawater DIC isotopic composition between EPR and MAR deep waters, shells from all vent fields appear richer in 13C than predicted from seawater DIC incorporation in the lattice. The equilibrium fractionation measured in abiotic mineral synthesis between calcite and bicarbonate ranges between −0.1‰ at 5 °C (Rubinson and Clayton, 1969; Emrich et al., 1970) and + 1.0‰ at 10–40 °C (Romanek et al., 1992). The difference measured between abiotic mineral synthesis and biomaterials is attributed to kinetic or metabolic effects (Beirne et al., 2012). For all the studied shells, the fractionation between skeleton and bicarbonate seawater is higher than the predicted equilibrium fractionation (Table 2). Only a 13C-enriched internal DIC could therefore explain the unexpectedly high values obtained for shells in this study, possibly indicating a metabolic signature. Indeed, the carbonate pool used by the deep-sea bivalves for shell mineralization can have two different origins: environmental DIC (seawater and carbon derived from oxidized methane) and metabolic DIC (respired carbon and carbon used by the symbionts) (McConnaughey

Table 5 Statistical comparisons between each individuals from Rainbow (a: site A1; b: site A3 and c: site B) and Menez Gwen (d: Cage Site; e: White Flame). a: KW test : x2 = 15; p-value b0.05

b: KW test : x2 = 4; p-value N0.05

c: KW test : x2 = 15; p-value b0.05

d: KW test : x2 = 18; p-value b0.05

e: KW test : x2 = 18; p-value b0.05

WRS test

A1-1

A1-2

A1-3

WRS test

A3-1

A3-2

A3-3

WRS test

B-1

B-2

B-3

WRS test

CS-1

CS-2

CS-3

WRS test

WF-1

WF-2

WF-3

A1-1 A1-2 A1-3

– – –

b0.05 – –

b0.05 N0.05 –

A3-1 A3-2 A3-3

– – –

N0.05 – –

N0.05 N0.05 –

B-1 B-2 B-3

– – –

N0.05 – –

b0.05 N0.05 –

CS-1 CS-2 CS-3

– – –

N0.05 – –

N0.05 b0.05 –

WF-1 WF-2 WF-3

– – –

b0.05 – –

b0.05 N0.05 –

42

K. Nedoncelle et al. / Chemical Geology 387 (2014) 35–46

Fig. 5. Microincrement width variability and carbon isotope profiles from the hinge (left) to the ventral margin (right) of shells from the sites A1 (a), A3 (b) and B (c) of Rainbow vent field. δ13C is represented by black lines with full circles dots. The evolution of microincrement width along the shell is presented by a light gray line, and the dark gray line is the average over a period of 15 increments. Vertical black dotted lines represent major growth lines. Black arrow indicates the direction of growth.

and Gillikin, 2008; Lartaud et al., 2010a; Lartaud et al., 2010b). A higher respiration rate was shown to induce more metabolically-derived CO2 to be incorporated in the shell, leading to a decrease in the shell δ13C compared to seawater, as respired CO2 is 13C depleted (Krantz et al., 1987; McConnaughey, 1989b; Gillikin et al., 2007). However, only a small fraction of respired carbon is expected to be transferred to the shells (b15% of respired C in bivalves shells) (McConnaughey and Gillikin, 2008). Respiration should have a minor importance on the final δ13C composition of hydrothermal shells, and in any case could not explain their heavier 13C content. In contrast, symbiosis can

have a reverse effect on the 13C isotopic composition of the shell. In symbiotic corals and bivalves, it is recognized that higher skeletal δ13C can results from photosynthetic activity of the associated algae which preferably consumes the light isotopic fraction (McConnaughey, 1989b; McConnaughey and Gillikin, 2008). Uptake of lighter DIC concentrates 13 C in the extrapallial liquid and consequently induces a 13Cenrichment in the carbonate shell. Such an isotopic fractionation of carbon might similarly occur with chemoautotrophic bacteria using CO2 as carbon source. As indicated by the 13C depleted mussel soft tissues signature (Fisher, 1995; Dubilier et al., 1998; Colaço et al., 2002; Fiala-Médioni et al., 2002), SOx symbionts incorporate preferentially light CO2 (12C) (Robinson and Cavanaugh, 1995). As a result, the heavy δ13C composition found in B. azoricus and B. thermophilus shells is a result of chemosynthetic pathway as these two species host SOx symbionts in their gills (Fig. 7). The role of chemosynthesis was previously proposed to explain δ13C differences between shells from shallow and deep waters (Rio et al., 1986; 1988), and the presence of methanotroph instead of thiotroph symbionts was suspected to modify the shell carbon composition (Rio et al., 1992). But to date, this feature could not be tested accurately, particularly for shells hosting the both symbionts. 4.2. Dual symbioses and variation in the 13C fractionation

Fig. 6. Mean ± SD carbon isotopic values as a function of increment width for the 9 shells from Rainbow. Gray triangles: site A1, black circles: site A3 and white diamonds: site B.

Substantial differences are exhibited among vent fields from the MAR, with higher δ13Cshell values at Menez Gwen than at Rainbow despite a similar seawater DIC composition, suggesting variable degree of metabolism-driven fractionation. One possible influence could rely on the relative contribution of oxidized CH4 from hydrothermal fluids to the environmental DIC on these vent fields. At Menez Gwen and Rainbow, δ13CH4 values of − 19.6 to − 18.8‰ and − 18.2 to − 13‰,

K. Nedoncelle et al. / Chemical Geology 387 (2014) 35–46

43

Fig. 7. Processes potentially influencing δ13C composition during the transfer of carbon from the environment to the shell of Bathymodiolus. + and − indicate, respectively, an enrichment or depletion of 13C. Large arrows represent major carbon fluxes whereas doted arrows represent minor fluxes. SOx: sulfide-oxidizing bacteria; MOx: methane-oxidizing bacteria.

respectively, were measured in end-member fluids (Charlou et al., 2000; Lein et al., 2000; 2002; Proskurowski et al., 2008a; 2010). Considering the important dilution of the fluid in seawater and the partial biological use of methane during the mixing process before reaching the seafloor, which can reach up to 60% in the diffuse fluid supplying mussel beds (Le Bris and Duperron, 2010), the contribution of oxidized methane to environmental DIC should not exceed micromolar concentrations. In these vent mussel habitat, methane oxidation cannot explain a substantial 13C depletion of DIC. In consequence, the influence of methane and CO2 from the hydrothermal fluids on the environmental DIC isotopic composition is unlikely to explain the 13C enrichment between Menez Gwen and Rainbow shells, and rather suggest a metabolic influence. In contrast to SOx, MOx symbionts use methane as carbon source and should not induce significant δ13C change in the extrapallial fluid (Fig. 7). As a result, mussels relying mostly on methanotrophic pathway would have an isotopic composition closer to seawater DIC than mussels dominated by a sulfide-oxidizing symbiosis. The variable predominance in the importance of these two pathways along the MAR was discussed in previous studies using complementary approaches. The depletion of free sulfide together with the high methane concentration in the mussel environment distinguishes Rainbow from Menez Gwen where both electron donors are available and should favor methanotrophy over chemoautotrophy in the carbon fixation process (Le Bris and Duperron, 2010). Despite the possibility for SOx symbionts to use hydrogen at this vent field, a higher contribution of MOx symbionts in the carbon fixation process, as compared to SOx symbionts, is also supported from their higher abundance in the gills of B. azoricus at Rainbow, whereas they are similar in abundance in Menez Gwen mussels (Duperron et al., 2006; Halary et al., 2008). These considerations are further supported by the isotopic composition of tissues of mussels from the MAR reflecting variable contribution of SOx and MOx symbionts to the host organic carbon, which was shown to be correlated with the relative enrichment of sulfide and methane in the end-member fluids

(Kennicutt et al., 1992; Southward et al., 2001; Colaço et al., 2002; Duperron, 2010). There is thus a bundle of evidence that the depletion of bioavailable sulfide for the symbionts in Rainbow environments should result in a stronger MOx signature in all individuals contributing to the lower 13C fractionation values. Conversely, Menez Gwen and 9°47′N shells are characterized by larger 13C fractionation, suggesting a higher dependence on chemoautotrophy using sulfide as an energy resource. This consideration would also be valid for shells from Lucky Strike which have similar or higher isotopic carbon composition than Menez Gwen shells (Lietard and Pierre, 2009) consistent with the fact that Lucky Strike mussels harbor a symbiotic immunity dominated by SOx bacteria, and supported by chemical measurements showing that Lucky Strike mussel habitats are richer in sulfide and depleted in methane compared to Rainbow and Menez Gwen (Le Bris and Duperron, 2010). The differences observed between shells from distinct habitats can also be considered in the light of this assumption. At Rainbow, A1 mussels still have a larger contribution than A3 and B mussels suggesting that SOx symbiont activity might still be significant. Despite the absence of free sulfide in the habitat, these symbionts are present in the gills to significant abundance (Duperron et al., 2006). As recently demonstrated by Petersen et al. (2011) for another H2-rich vent field, SOx symbionts of MAR, Bathymodiolus species can use H2 to fix CO2. Hydrogen is highly concentrated in the hydrothermal fluid of Rainbow (10–16 mM) (Charlou et al., 2010) and, despite the absence of measurement so far, it may be available in the mussel habitat (Le Bris and Duperron, 2010). We can thus hypothesize that H2 use by SOx is reflected in variable δ13C among and within assemblages at this vent field. Within this hypothesis, the decrease in isotopic 13C content in shells from A3 and B would reflect a decrease in H2 use as energy source by these individuals with respect to methane. In contrast, individuals from A1 rather display a higher SOx signature. The latter group of individual is located close to a small active chimney and their micro-environment is possibly

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influenced by the plume of this small high temperature vent (Fig. 2a), while the other appears to have settled on diffusing porous substrates. Further studies involving in situ sensing are needed to investigate whether these differences in the spatial organization of venting and local fluid source composition could discriminate habitats where H2 is available from those where this compound is fully depleted, as a result of biotic and abiotic oxidation processes. At Menez Gwen the highest δ13C values in shells from Cage Site suggest that these organisms rely more on their SOx symbionts than mussels from White Flame, which highlight a larger contribution from methanotrophy. This hypothesis is consistent with the model simulation of Marcon et al. (2013) suggesting a higher CH4 uptake by mussels at White Flame than at Cage Site (25–715 mol·yr− 1 versus 11– 302 mol·yr−1 respectively). At Menez Gwen, the discrepancy between individual δ13Cshell compositions could also reflect micro-habitat variability. Indeed, important differences have been documented in the symbiont abundance and tissue isotopic composition even at small spatial scale (Trask and Van Dover, 1999; Colaço et al., 2002; Fiala-Médioni et al., 2002; Duperron et al., 2006) and recently attributed to flexibility of symbiosis in response to vent chemistry fluctuations (Halary et al., 2008; Riou et al., 2008). 4.3. Relationship between isotopic variability and shell growth rate Except for some minor δ13C anomalies, isotopic profiles are stable over the shell length. Each data correspond to an averaged isotope composition over a period of 22 days for B. azoricus and 17 days for B. thermophilus, corresponding to a mean daily shell growth rate of 46 μm for B. azoricus and 58 μm for B. thermophilus Even averaged, these stable values contrast with the known variability of hydrothermal habitats (Le Bris et al., 2006). Rather than a stable micro-environment, this observation suggests biological buffering processes over the lifetime of individuals. This would indicate that the energy allocated to the shell biomineralization does not solely reflect the energy available for the organisms. Other parameters such as pH or O2 limitation can also have an impact on the shell growth dynamics. Another buffering factor is related to ontogenic development. The weak but significant negative relationship between δ13C and growth rate indicates a vital effect influence. Usually, two hypotheses sustain this vital effect: the kinetic model (1989b; McConnaughey, 1989a) and the carbonate model (Adkins et al., 2003). The kinetic model predicts + that hydration (CO2 + H2O → H2CO3 → HCO− 3 + H ) and hydroxylation 13 ) induce low δ C in the extrapallial fluid and (CO2 + OH− → HCO− 3 DIC thus low δ13C shell carbonate. This fractionation effect is preserved in the shell when carbonate precipitation from DIC is faster than equilibrium with extrapallial fluid (McConnaughey, 2003). Consequently, a fast growth rate leads to low δ13Cshell values. The carbonate model is driven by an active transport of the Ca2+ ions into the extrapallial cavity. This active transport is accompanied with H+ expulsion leading to extrapallial compared to fluid alkalinization which increases proportion of CO2− 3 2− is more depleted in 13C HCO− 3 . However, at isotopic equilibrium, CO3 than HCO− 3 (Shanahan et al., 2005). Thus, a fast shell growth rate increases the Ca2+ demand and induces internal δ13CDIC and δ13Cshell depletion. However, this vital effect still remains low, as shell δ13C's values are higher than the equilibrium fractionation with seawater, and is not the driving factor explaining the shell δ13C's compositions. The steep δ13C anomalies that are unfrequently observed on some shells, together with the major growth breaks, are other interesting features to examine. Considering that Bathymodiolus shell length is proportional to the age of the individual (Schöne and Giere, 2005; Nedoncelle et al., 2013), these major δ13C anomalies are not related to the B. azoricus ontogenic trend, as they are not present at the same distance from the umbo. However, they are often observed during low growth rate periods. Steep temperature increase, decrease in pH or reduction of oxygen content of the surrounding water could exceed the physiological tolerance of the mussels and then induce a growth slowdown by

allocating their available energy for the maintaining of its metabolism. Similarly, a reduction of energetic compounds in the mussels' environment could have an impact on their growth rate, as it has been previously suggested by transplant experiments (Smith et al., 2000; Berguist et al., 2004; Raulfs et al., 2004). Indeed, mollusks' shell formation needs more than one third of the total energy reserve (Wilbur and Saleuddin, 1983), this stock being used as potential sources for important energy request. Although some authors have suggested a link between major growth line formation and significant variation in hydrothermal vent activity (Roux and Fatton, 1985; Schein et al., 1991), the presence of major growth lines is not related to any significant change in stable carbon isotopes ratio profiles for B. azoricus. Schöne and Giere (2005) found the same in B. brevior shells from the North Fiji Basin. These shell features could rather be due to metabolic activity such as reproduction than environmental variability. So far nothing is known about the relative efficiency of the methanotrophic pathway on the growth rate compared to thiotrophy. Methane is both a source of carbon and of energy whereas H2S and H2 are only energetical compounds (Pimenov et al., 2002; Petersen et al., 2011). Thiotroph bacteria use CO2 as carbon source. Whether or not conditions favorable to MOx bacteria could allocate more energy for shell growth than SOx bacteria will deserve further investigation in the future, if we are to fully constraint the environmental influences on growth for these species. 5. Conclusion The carbon isotope analysis of the shell of symbiotic deep-sea hydrothermal vent mussels reveals marked chemosynthetic signature and might constitute a useful tool to investigate chemosynthesis pathways in population from hardly accessible habitats or dead assemblages. The large δ13C range observed in shells of two Bathymodiolus species from the Atlantic and Pacific ridges highlights a combination of influence factors, with background seawater DIC and chemosynthesis pathways being the most important ones. Shells appear much richer in 13C than predicted from seawater DIC incorporation in the lattice, likely reflecting the influence of chemoautotrophic CO2 fixation by the symbionts in the isotopic composition of B. thermophilus and B. azoricus shells. Furthermore, the results suggest that the different chemosynthetic pathways that can be supported in dual-symbiosis species are reflected in the isotopic composition, with lower δ13Cshell values for organisms mainly relying on methanotrophy and higher δ13Cshell values for individual mainly or exclusively relying on sulfide-oxidizing symbiosis. Despite flexibility in the use of chemosynthesis resources reflected by isotopic differences between individuals, the inter-habitat variability does not seem to affect B. azoricus' mean growth rate except during major perturbations. The identification of a strong inter-habitat difference in δ13Cshell highlights the interest in using shell carbon isotopes for the characterization of actual and fossil chemosynthetic-based fauna, habitats, and discriminate the large diversity of fluids sustaining chemosynthetic assemblages especially on serpentinite-hosted vents (Le Bris and Duperron, 2010; Lartaud et al., 2011b; Ohara et al., 2012). Acknowledgments We are grateful to the captains and crews of the R/Vl'Atalante and Meteor, as well as the Nautile, Victor 6000 and Quest 4000 operation groups. The chief scientists of the research expeditions MoMARDREAM (J. Dyment, CNRS), MESCAL2010 (co-chief scientists N. Le Bris, F. Lallier, UPMC) and MenezMAR (N. Dubillier, MARUM) and the scientific parties are gratefully acknowledged for their support and help during the cruises. We also acknowledge C. Pierre for the use of the mass spectrometer for isotopic analyses of the water samples and O. Falaize for the technical assistance. We are also indebted to the “Biomineralizations and sedimentary environments” Laboratory for its kind hosting for these analytical works. The study benefited from the joint support of

K. Nedoncelle et al. / Chemical Geology 387 (2014) 35–46

the Fondation TOTAL and UPMC to the chair “Extreme environment, biodiversity and global change” and from the financial support of the CNRS Institute of Ecology and Environment (INEE) (SE2010), to the UMR8222 LECOB for participation to the cruises and equipments, and from Ifremer for the vessel and submersible operations as part of the French Research fleet. K. Nedoncelle's PhD grant has been supported by MESR and UPMC via the Doctoral School “Science de l'Environnement d'Ile de France” ED129. References Adkins, J.F., Boyle, E.A., Curry, W.B., Lutringer, A., 2003. Stable isotopes in deep-sea corals and a new mechanism for “vital effects”. Geochim. Cosmochim. Acta 67 (6), 1129–1143. Arthur, M.A., Williams, D.F., Jones, D.S., 1983. Seasonal temperature-salinity changes and thermocline in the mid-Atlantic Bight as recorded by the isotopic composition of bivalves. Geology 11, 655–659. Beirne, E.C.,Wanamaker, A.D.,Feindel, S.C., 2012. Experimental validation of environmental controls on the δ13C of Arctica islandica (ocean quahog) shell carbonate. Geochim. Cosmochim. Acta 84, 395–409. Berguist, D.C., Fleckenstein, C., Szalai, E.B., Knisel, J., Fisher, C.R., 2004. Environment drives physiological variability in the cold seep mussel Bathymodiolus childressi. Limnol. Oceanogr. 49 (3), 706–715. Cavanaugh, C.M.,Wirsen, C.O.,Jannasch, H.W., 1992. Evidence for methylotrophic symbionts in a hydrothermal vent mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge. Appl. Environ. Microbiol. 58, 3799–3803. Charlou, J.L., Donval, J.P., Douville, E., Jean-Baptiste, P., Radford-Knoery, J., Fouquet, Y., Dapoigny, A., Stievenard, M., 2000. Compared geochemical signatures and the evolution of Menez Gwen (37°50′N) and Lucky Strike (37°17′N) hydrothermal fluids, south of the Azores Triple Junction on the Mid-Atlantic Ridge. Chem. Geol. 171, 49–75. Charlou, J.,Donval, J.P.,Fouquet, Y.,Jean-Baptiste, P.,Holm, N.G., 2002. Geochemistry of high H2 and CH4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36°14′N, MAR). Chem. Geol. 191, 345–359. Charlou, J.L.,Donval, J.P.,Konn, C.,Ondréas, H.,Fouquet, Y., 2010. High production and fluxes of H2 and CH4 and evidence of abiotic hydrocarbon synthesis by serpentinization in ultramafic-hosted hydrothermal systems on the Mid-Atantic Ridge. In: Rona, P.A., Devey, C.W., Dyment, J., Murton, B.J. (Eds.), Diversity of Hydrothermal Systems on Slow Spreading Ocean Ridges. American Geophysical Union, Washington, pp. 265–296. Colaço, A.,Dehairs, F.,Desbruyères, D.,Le Bris, N.,Sarradin, P.M., 2002. δ13C signature of hydrothermal mussels is related with the end-member fluid concentrations of H2S and CH4 at the Mid-Atlantic Ridge hydrothermal vent fields. Cah. Biol. Mar. 43, 259–262. Colaço, A., Prieto, C., Martins, A., Figueiredo, M., Lafon, V., Monteiro, M., Bandarra, N.M., 2009. Seasonal variations in lipid composition of the hydrothermal vent mussel Bathymodiolus azoricus from the Menez Gwen vent field. Mar. Environ. Res. 67, 146–152. Contreira-Pereira, L., Yücel, M., Omanovic, D., Brulport, J.P., Le Bris, N., 2013. Compact autonomous voltammetric sensor for sulfide monitoring in deep sea vent habitats. Deep Sea Res. I 80, 47–57. Craig, H., 1957. Isotopic standards for carbon and oxygen and correction factors for massspectrometric analysis of carbon dioxide. Geochim. Cosmochim. Acta 12, 133–149. Cravo, A., Foster, P., Almeida, C., Company, R., Cosson, R.P. and Bebianno, M.J., 2007. Metals in the shell of Bathymodiolus azoricus from a hydrothermal vent site on the Mid-Atlantic Ridge. Environ. Int. 33, 609–615. De Busserolles, F., Sarrazin, J., Gauthier, O., Gélinas, Y., Fabri, M.C., Sarradin, P.M., Desbruyères, D., 2009. Are spatial variations in the diets of hydrothermal fauna linked to local environmental conditions? Deep-Sea Res. II 56, 1649–1664. Desbruyères, D., Biscoito, M., Caprais, J.C., Colaço, A., Comtet, T., Crassous, P., Fouquet, Y., Khripounoff, A., Le Bris, N., Olu, K., Riso, R., Sarradin, P.M., Segonzac, M., Vangriesheim, A., 2001. Variations in deep-sea hydrothermal vent communities on the Mid-Atlantic Ridge near the Azores plateau. Deep-Sea Res. I Oceanogr. Res. Pap. 48, 1325–1346. Douville, E., Charlou, J.L., Oelkers, E.H., Bienvenu, P., Colon, C.F.J., Donval, J.P., Fouquet, Y., Prieur, D., Appriou, P., 2002. The Rainbow vent fluids (36°14′N, MAR): the influence of ultramafic rocks and phase separation on trace metal content in Mid-Atlantic Ridge hydrothermal fluids. Chem. Geol. 184, 37–48. Dubilier, N.,Windoffer, R.,Giere, O., 1998. Ultrastructure and stable carbon isotope composition of the hydrothermal vent mussels Bathymodiolus brevior and B. sp. affinis brevior from the North Fiji Basin, western Pacific. Mar. Ecol. Prog. Ser. 165, 187–193. Dubilier, N., Bergin, C., Lott, C., 2008. Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nat. Rev. Microbiol. 6, 725–740. Duperron, S., 2010. The diversity of deep-sea mussels and their bacterial symbioses. In: Kiel, S. (Ed.), The Vent and Seep Biota: Aspects from Microbes to Ecosystems, London, pp. 137–167. Duperron, S., Bergin, C., Zielinski, F., Blazejak, A., Pernthaler, A., McKiness, Z.P., DeChaine, E., Cavanaugh, C.M., Dubilier, N., 2006. A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia: Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge. Environ. Microbiol. 8, 1441–1447. Duperron, S.,Halary, S.,Lorion, J.,Sibuet, M.,Gaill, F., 2008. Unexpected co-occurence of six bacterial symbionts in the gills of the cold seep mussel Idas sp. (Bivalvia: Mytilidae). Environ. Microbiol. 10, 433–445.

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Emrich, K., Ehhalt, D.H., Vogel, J.C., 1970. Carbon isotope fractionation during the precipitation of calcium carbonate. Earth Planet. Sci. Lett. 8, 363–371. Fiala-Médioni, A., Métivier, C., Herry, A., Le Pennec, M., 1986. Ultrastructure of the gill filament of an hydrothermal vent mytilid Bathymodiolus sp. Mar. Biol. 92, 65–72. Fiala-Médioni, A., McKiness, Z.P., Dando, P., Boulegue, J., Mariotti, A., Alayse-Danet, A.M., Robinson, J.J.,Cavanaugh, C.M., 2002. Ultrastructural, biochemical, and immunological characterization of two populations of the mytilid mussel Bathymodiolus azoricus from the Mid-Atlantic Ridge: evidence for a dual symbiosis. Mar. Biol. 141, 1035–1043. Fisher, C.R., 1990. Chemoautotrophic and methanotrophic symbioses in marine invertebrates. Rev. Aquat. Sci. 2, 399–613. Fisher, C.R., 1995. Toward an appreciation of hydrothermal-vent animals: their environment, physiological ecology, and tissue stable isotope values. In: Humphris, S.E., Zierenberg, R.A., Mullineaux, L.S., Thomson, R.E. (Eds.), Seafloor Hydrothermal Systems: Physical, Chemical, Biological, and Geological InteractionsGeophysical Monograph Series. AGU, Washington, D C, pp. 297–316. Fisher, C.R.,Brooks, J.M.,Vodenichar, J.S.,Zande, J.M.,Childress, J.J.,Burke, R.A., 1993. The cooccurrence of methanotrophic and chemoautotrophic sulfur-oxidizing bacterial symbionts in a deep-sea mussel. Mar. Ecol. 14, 277–289. Fisher, C.R., Childress, J.J., Macko, S.A., Brooks, J.M., 1994. Nutritional interactions in Galápagos Rift hydrothermal vent communities: inferences from stable carbon and nitrogen isotope analyses. Mar. Ecol. Prog. Ser. 103, 45–55. Fisher, C.R., Takai, K., Le Bris, N., 2007. Hydrothermal vent ecosystems. Oceanography 20 (1), 14–23. Gillikin, D.P., Lorrain, A., Meng, L., Dehairs, F., 2007. A large metabolic carbon contribution to the δ13C record in marine aragonitic bivalve shells. Geochim. Cosmochim. Acta 71, 2936–2946. Halary, S.,Riou, V.,Gaill, F.,Boudier, T.,Duperron, S., 2008. 3D FISH for the quantification of methane- and sulphur-oxidizing endosymbionts in bacteriocytes of the hydrothermal vent mussel Bathymodiolus azoricus. ISME J. 2, 284–292. Johnson, K.S., Childress, J.J., Beehler, C.L., Sakamoto, C.M., 1994. Biogeochemistry of hydrothermal vent mussel communities: the deep-sea analogue to the intertidal zone. Deep-Sea Res. I Oceanogr. Res. Pap. 41 (7), 993–1011. Kennicutt, M.C., Burke, R.A., MacDonald, I.R., Brooks, J.M., Denoux, G.J., Macko, S.A., 1992. Stable isotope partitioning in seep and vent organisms: chemical and ecological significance. Chem. Geol. 101, 293–310. Krantz, D.E., Williams, D.F., Jones, D.S., 1987. Ecological and paleoenvironmental information using stable isotope profiles from living and fossil molluscs. Palaeogeogr. Palaeoclimatol. Palaeoecol. 58, 249–266. Kroopnick, P.M., 1985. The distribution of 13C of CO2 in the world oceans. Deep-Sea Res. A Oceanogr. Res. Pap. 32 (1), 57–84. Lang, S.Q., Früh-Green, G.L., Bernasconi, S.M., Lilley, M., Proskurowski, G., Méhay, S., Butterfield, D.A., 2012. Microbial utilization of abiogenic carbon and hydrogen in a serpentinite-hosted system. Geochim. Cosmochim. Acta 92, 82–99. Lartaud, F.,de Rafelis, M.,Oliver, G.,Krylova, E.,Dyment, J.,Ildefonse, B.,Thibaud, R.,Gene, P., Hoisé, E.,Meistertzheim, A.L.,Fouquet, Y.,Gaill, F.,Le Bris, N., 2010a. Fossil clams from a serpentinite-hosted sedimented vent field near the active smoker complex Rainbow, MAR, 36°13′N: insight into the biogeography of vent fauna. Geochem. Geophys. Geosyst. 11, 1–17. Lartaud, F., Emmanuel, L., De Rafelis, M., Pouvreau, S., Renard, M., 2010b. Influence of food supply on the δ13C signature of mollusc shells: implications for palaeoenvironmental reconstitutions. Geo-Mar. Lett. 30, 23–34. Lartaud, F., Little, C.T.S., de Rafelis, M., Bayon, G., Dyment, J., Ildefonse, B., Gressier, V., Fouquet, Y.,Gaill, F.,Le Bris, N., 2011a. Fossil evidence for serpentinization fluids fueling chemosynthetic assemblages. PNAS 108 (19), 7698–7703. Lartaud, F., de Rafelis, M., Little, C.T.S., Dyment, J., Bayon, G., Ildefonse, B., Le Bris, N., 2011b. Ultramafic hydrothermal systems on the Rainbow abyssal hill: a wide variety of active and fossil chemosynthetic habitats. Int. Res. InterRidge News 20, 32–36. Le Bris, N., Duperron, S., 2010. Chemosynthetic communities and biogeochemical energy pathways along the Mid-Atlantic Ridge: the case of Bathymodiolus azoricus, diversity of hydrothermal systems on slow spreading ocean ridges. In: Rona, P.A. (Ed.), Geophysical Monograph Series. AGU, Washington, D C, pp. 409–429. Le Bris, N.,Govenar, B., Le Gall, C., Fisher, C.R., 2006. Variability of physico-chemical conditions in 9°50′N EPR diffuse flow vent habitats. Mar. Chem. 98, 167–182. Lein, A.Y.,Grichuk, D.V., Gurvich, E.G., Bogdanov, Y.A., 2000. A new type of hydrogen- and methane-rich hydrothermal solutions in the Rift zone of the Mid-Atlantic Ridge. Dokl. Earth Sci. 375 (9), 1304–1391. Lietard, C., Pierre, C., 2009. Isotopic signatures (δ18O and δ13C) of bivalve shells from cold seeps and hydrothermal vents. Geobios 42, 209–219. Lorrain, A., Paulet, Y.M., Chauvaud, L., Dunbar, R., Mucciarone, D., Fontugne, M., 2004. δ13C variation in scallop shells: increasing metabolic carbon contribution with body size? Geochim. Cosmochim. Acta 68 (17), 3509–3519. Lutz, R.A., Kennish, M.J.,Pooleyl, A.S.,Fritz, L.W., 1994. Calcium carbonate dissolution rates in hydrothermal vent fields of the Guaymas Basin. J. Mar. Res. 52, 969–982. Marcon, Y., Sahling, H., Borowski, C., dos Santos Ferreira, C., Thal, J., Bohrmann, G., 2013. Megafaunal distribution and assessment of total methane and sulfide consumption by mussel beds at Menez Gwen hydrothermal vent, based on georeferenced photomosaics. Deep-Sea Res. I 75, 93–109. McConnaughey, T., 1989a. 13C and 18O isotopic disequilibrium in biological carbonates: II. In vitro simulation of kinetic isotope effects. Geochim. Cosmochim. Acta 53, 163–171. McConnaughey, T., 1989b. 13C and 18O isotopic disequilibrium in biological carbonates: I. Patterns. Geochim. Cosmochim. Acta 53, 151–162. McConnaughey, T.A., 2003. Sub-equilibrium oxygen-18 and carbon-13 levels in biological carbonates: carbonate and kinetic models. Coral Reefs 22, 316–327. McConnaughey, T.A., Gillikin, D.P., 2008. Carbon isotopes in mollusk shell carbonates. Geo-Mar. Lett. 28, 287–299.

46

K. Nedoncelle et al. / Chemical Geology 387 (2014) 35–46

Moore, T.S., Shank, T.M., Nuzzio, D.B., Luther, G.W., 2009. Time-series chemical and temperature habitat characterization of diffuse flow hydrothermal sites at 9°50′N East Pacific Rise. Deep-Sea Res. II 56, 1616–1621. Mutvei, H., Westermark, T., Dunca, E., Carell, B., Forberg, S., Bignert, A., 1994. Methods for the study of environmental changes using the structural and chemical information in molluscan shells. Bull. Inst. Océanogr. Monaco 13, 163–186. Naraoka, H., Naito, T., Yamanaka, T., Tsunogai, U., Fujikura, K., 2008. A multi-isotope study of deep-sea mussels at three different hydrothermal vent sites in the northwestern Pacific. Chem. Geol. 255, 25–32. Nedoncelle, K., 2013. Bivalve growth processes in extreme environment: a case-study of two symbiotic species Bathymodiolus azoricus and Bathymodiolus thermophilus in contrasted hydrothermal environments, Ph.D thesis. mem. Université Pierre et Marie Curie, Banyuls-sur-mer, p. 232 pp. Nedoncelle, K., Lartaud, F., De Rafelis, M., Boulila, S., Le Bris, N., 2013. A new method for high-resolution bivalve growth rate studies in hydrothermal environments. Mar. Biol. 160 (6), 1427–1439. Ohara, Y., Reagan, M.K., Fujikura, K., Watanabe, H., Michibayashi, K., Ishii, T., Stern, R.J., Pujana, I., Martinez, F., Girard, G., Ribeiro, J., Brounce, M., Komori, N., Kino, M., 2012. A serpentinite-hosted ecosystem in the Southern Mariana Forearc. PNAS 109 (8), 2831–2835. Paull, C.K.,Jull, A.J.T.,Toolin, L.J.,Linick, T., 1985. Stable isotope evidence for chemosynthesis in an abyssal seep community. Lett. Nat. 317, 709–711. Petersen, J.M., Zielinski, F.U., Pape, T., Seifert, R., Moraru, C., Amann, R., Hourdez, S., Girguis, P.R.,Wankel, S.D.,Barbe, V.,Pelletier, E.,Fink, D.,Borowski, C.,Bach, W.,Dubilier, N., 2011. Hydrogen is an energy source for hydrothermal vent symbioses. Nature 476, 176–180. Pierre, C., Vangriesheim, A., Laube-Lenfant, E., 1994. Variability of water masses and of organic production-regeneration systems as related to eutrophic, mesotrophic and oligotrophic conditions in the northeast Atlantic ocean. J. Mar. Syst. 5, 159–170. Pimenov, N.V.,Kaliuzhnaya, M.G.,Khmelenina, V.N.,Mityushina, L.L.,Trotsenko, Y.A., 2002. Utilization of methane and carbon dioxide by symbiotrophic bacteria in gills of Mytilidae (Bathymodiolus) from the Rainbow and Logachev hydrothermal fields on the Mid-Atlantic Ridge. Microbiology 71, 587–594. Proskurowski, G.,Lilley, M.D.,Seewald, J.S.,Früh-Green, G.L.,Olson, E.J.,Lupton, J.E.,Sylva, S.P., Kelley, D.S., 2008a. Abiogenic hydrocarbon production at lost city hydrothermal field. Science 319 (604), 604–607. Proskurowski, G., Lilley, M.D., Olson, E.J., 2008b. Stable isotopic evidence in support of active microbial methane cycling in low-temperature diffuse flow vents at 9°50′N East Pacific Rise. Geochim. Cosmochim. Acta 72, 2005–2023. Rau, G.,Hedges, J.I., 1979. Carbon-13 depletion in a hydrothermal vent mussel: suggestion of a chemosynthetic food source. Science 203 (4381), 648–649. Raulfs, E.C., Macko, S.A., Van Dover, C.L., 2004. Tissue and symbiont condition of mussels (Bathymodiolus thermophilus) exposed to varying levels of hydrothermal activity. J. Mar. Biol. Assoc. U. K. 84, 229–234. Rio, M., Roux, M., Renard, M., Duvault, Y.H., Davanzo, F., Clauser, S., 1986. Carbon isotopic fractionnation of bivalvia depending on chemo-autotrophic primary production in abyssal and littoral environment. C. R. Acad. Sci. Paris 303 (2), 1553–1556. Rio, M., Renard, M., Roux, M., Clauser, S., Davanzo, F., Herrerra Duvault, Y., 1988. Composition chimique et isotopique des tests de bivaves des sources hydrothermales océaniques. Bull. Soc. Geol. Fr. 8, 151–159. Rio, M., Roux, M., Renard, M., Schein, E., 1992. Chemical and isotopic features of present day bivalve shells from hydrothermal vents or cold seeps. Palaios 7 (4), 351–360. Riou, V., Halary, S., Duperron, S., Bouillon, S., Elskens, M., Bettencourt, R., Serrão Santos, R., Dehairs, F.,Colaço, A., 2008. Influence of CH4 and H2S availability on symbiont distribution, carbon assimilation and transfer in the dual symbiotic vent mussel Bathymodiolus azoricus. Biogeosciences 5, 1681–1691. Riou, V., Colaço, A., Bouillon, S., Khripounoff, A., Dando, P., Mangion, P., Chevalier, E., Korntheuer, M.,Serrão Santos, R.,Dehairs, F., 2010. Mixotrophy in the deep sea: a dual endosymbiotic hydrothermal mytilid assimilates dissolved and particulate organic matter. Mar. Ecol. Prog. Ser. 405, 187–201. Robinson, J.J., Cavanaugh, C.M., 1995. Expression of form I and form II ribulose-1,5bisphosphate carboxylase/oxygenase (Rubsico) in chemoautotrophic symbioses:

implications for the interpretation of stable carbon isotope ratios. Limnol. Oceanogr. 40, 1496–1502. Romanek, C.S.,Grossman, E.L.,Morse, J.W., 1992. Carbon isotopic fractionation in synthetic aragonite and calcite: effects of temperature and precipitation rate. Geochim. Cosmochim. Acta 56, 419–430. Roux, M., Fatton, E., 1985. Clam growth and thermal spring activity recorded by shells at 21°N. Bull. Biogeogr. Soc. Washington 6, 211–221. Rubinson, M., Clayton, R.N., 1969. Carbon-13 fractionation between aragonite and calcite. Geochim. Cosmochim. Acta 33, 997–1002. Sadler, J., Carré, M., Azzoug, M., Schauer, A.J., Ledesma, J., Cardenas, F., Chase, B., Bentaleb, I., Muller, S., Mandeng, M., Rohling, E.J., Sachs, J.P., 2012. Reconstructing past upwelling intensity and the seasonal dynamics of primary productivity along the Peruvian coastline from mollusk shell stable isotopes. Geochem. Geophys. Geosyst. 13 (1), 1525–2027. Sarradin, P.M.,Caprais, J.C., Riso, R.,Kerouel, R.,Aminot, A., 1999. Chemical environment of the hydrothermal mussel communities in the Lucky Strike and Menez Gwen vent fields, Mid-Atlantic Ridge. Cah. Biol. Mar. 40, 93–104. Schein, E., Roux, M., Barbin, V., Chiesi, F., Renard, M., Rio, M., 1991. Enregistrement des paramètres écologiques par la coquille des bivalves: approche pluridisciplinaire. Bull. Soc. Géol. Fr. 162 (4), 687–698. Schöne, B.R.,Giere, O., 2005. Growth increments and stable isotope variation in shells of the deep-sea hydrothermal vent bivalve mollusk Bathymodiolus brevior from the North Fiji Basin, Pacific Ocean. Deep-Sea Res. I Oceanogr. Res. Pap. 52, 1896–1910. Shanahan, T.M., Pigati, J.S., Dettman, D.L., Quade, J., 2005. Isotopic variability in the aragonite shells of freshwater gastropods living in springs with nearly constant temperature and isotopic composition. Geochim. Cosmochim. Acta 69 (16), 3949–3966. Smith, E.B., Scott, K.M., Nix, E.R., Korte, C., Fisher, C.R., 2000. Growth and condition of seep mussels (Bathymodiolus childressi) at a Gulf of Mexico Brine Pool. Ecology 81 (9), 2392–2403. Southward, E.C., Gebruk, A., Kennedy, H., Southward, A.J., Chevaldonne, P., 2001. Different energy sources for three symbiont-dependent bivalve molluscs at the Logatchev hydrothermal site (Mid Atlantic-Ridge). J. Mar. Biol. Assoc. U. K. 81, 655–661. Surge, D., Lohmann, K.C., Dettman, D.L., 2001. Controls on isotopic chemistry of the American oyster, Crassostrea virginica: implications for growth patterns. Palaeogeogr. Palaeoclimatol. Palaeoecol. 172, 283–296. Tivey, M.K., 2007. Generation of seafloor hydrothermal vent fluids and associated mineral deposits. Oceanography 20 (1), 50–65. Trask, J.L., Van Dover, C.L., 1999. Site-specific and ontogenetic variations in nutrition of mussels (Bathymodiolus sp.) from the Lucky Strike hydrothermal vent field, Mid-Atlantic Ridge. Limnol. Oceanogr. 44, 334–343. Van Dover, C.L., 2000. The Ecology of Deep-sea Hydrothermal Vents. Princeton, New Jersey, pp. 1–424. Von Damm, K., 2000. Chemistry of hydrothermal vent fluids from 9°10'N, East Pacific rise: “Time zero”, the immediate posteruptive period. J. Geophys. Res. 105 (B5), 11203–11222. Von Damm, K.L.,Lilley, M.D., 2004. Diffuse flow hydrothermal fluids from 9°50'N East Pacific Rise: Origin, evolution, and biogeochemical controls. In: Wilcock, W.S., DeLong, E.F., Kelley, D.S., Baross, J.A., Cary, S.C. (Eds.), The Subseafloor Biosphere at MidOcean Ridges. Geophysical Monograph Series. vol. 144. American Geophysical Union, Washington, DC, pp. 245–268. Wilbur, K.M., Saleuddin, A.S.M., 1983. Shell formation. In: Saleuddin, A.S.M., Wilbur, K.M. (Eds.), The Mollusca. Academic Press, London, pp. 236–279. Wisshak, M.,Lopez Correa, M.,Gofas, S.,Salas, C.,Taviani, M.,Jakobsen, J.,Freiwald, A., 2009. Shell architecture, element composition, and stable isotope signature of the giant deep-sea oyster Neopycnodonte zibrowii sp. n. from the NE Atlantic. Deep-Sea Res. I 56, 374–407.