# Mg/Ca from mussel shells rather than δ18w O as a promising temperature proxy for hydrothermal vent ecosystems

V. Mouchi, K. Nedoncelle, O. Bruguier, Z. Garmirian, N. Le Bris, F. Lartaud

► To cite this version:

V. Mouchi, K. Nedoncelle, O. Bruguier, Z. Garmirian, N. Le Bris, et al.. Mg/Ca from mussel shells rather than δ18w O as a promising temperature proxy for hydrothermal vent ecosystems. Deep Sea Research Part I: Oceanographic Research Papers, 2025, 219, pp.104485. 10.1016/j.dsr.2025.104485. hal-04996044

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# Mg/Ca from mussel shells rather than δ¹⁸O as a promising temperature proxy for hydrothermal vent ecosystems

V. Mouchi<sup>a,b</sup>, K. Nedoncelle<sup>c</sup>, O. Bruguier<sup>d</sup>, Z. Garmirian<sup>c</sup>, N. Le Bris<sup>c,e</sup>, F. Lartaud<sup>c,*</sup>

## ARTICLE INFO

**Keywords:**

Deep-sea hydrothermal habitat

*Bathymodiolus* shells

Magnesium-calcium ratio

Oxygen stable isotopes

Paleothermometer

## ABSTRACT

Minor and trace metal fluctuations in biogenic carbonates have been demonstrated to be important potential environmental tracers in coastal areas, but remained poorly studied in deep-sea environments. For the first time, this study assesses the use of Mg/Ca signal as a thermometer proxy in hydrothermal vent ecosystems together with the analysis of oxygen stable isotope composition. *Bathymodiolus azoricus* and *B. thermophilus* mussels were collected at three hydrothermal vent fields from the mid-Atlantic ridge (Rainbow, Menez Gwen) and the East Pacific Rise (EPR 9°50N), presenting contrasted temperature and chemical habitat conditions. The variation of Mg/Ca was analysed via LA-ICP-MS and is revealed suitable to reconstruct temperature variations in these ecosystems, presumably due to stable Mg compositions of the seawater surrounding hydrothermal systems. Temperature anomalies inferred from Mg/Ca ratios can be good tracers of fluid pulses. Important fluid emissions appear however to generate major growth cessation in shell mineralization. Temperatures inferred from Isotope Ratio Mass Spectrometry (IRMS) δ¹⁸O analyses systematically underestimated the measured values in the environment. The isotopic disequilibrium is likely due to pH fluctuations in the mussel habitat, in the vicinity of vent fluid discharges, and/or interactions with the symbiotic chemosynthetic bacteria. Those results will first benefit to the ecological study of deep-sea mussels, but also provide a promising contribution for the study of the environmental dynamic in hydrothermal systems at short (daily) to long-term (pluri-annual) scales, recorded in the calcite material of bivalves.

## 1. Introduction

As they build carbonate structures from ions sampled from the surrounding environment, mollusc shells are used as a natural recorder of past and present climate and environmental parameters (Dodd, 1965; Klein et al., 1996; Huyghe et al., 2015). The shells are formed by incremental growth, preventing the destruction of previously secreted parts, which allows the evaluation of the environmental variations throughout the life of an organism (Schöne, 2008). Historically, this type of study started with the measurements of oxygen isotope ratios (δ¹⁸O; Urey, 1947), which are commonly used as a proxy for seasonal temperature reconstructions in a variety of environments such as freshwater (Dettman et al., 1999; Goodwin et al., 2019) and coastal (Klein et al., 1996; Lartaud et al., 2010a; Welsh et al., 2011) settings. However,

several limitations have been identified for the use of this proxy. First, δ¹⁸O in shells, not only changes with temperature, but also with the seawater δ¹⁸O composition (δ¹⁸O<sub>w</sub>), which covaries with salinity, and is not properly constrained particularly in coastal environments (Rohling, 2000). Second, strong disequilibrium between seawater and biogenic carbonates has been observed in numerous taxa (Aharon, 1991; Daëron et al., 2019). Although these disequilibria, called ‘vital effects’ (i.e., resulting from kinetic and physiological impacts during biomineralization), appear limited in mollusc biominerals (Aharon, 1991; Welsh et al., 2011), strong isotopic anomalies have been recently highlighted in juvenile oyster shells, likely due to kinetic effects during this period of rapid growth (Huyghe et al., 2020), and for deep-sea oysters (Wishak et al., 2009; Huyghe et al., 2022).

As alternative proxies for temperature, elemental ratios (i.e., Mg/Ca

<footnote><sup>a</sup> Sorbonne Université, CNRS, UMR 7144, Station Biologique de Roscoff, Place Georges Teissier, 29680, Roscoff, France</footnote>

<footnote><sup>b</sup> Current address: CNRS UMR 6566 CReAAH (Centre de Recherche en Archéologie, Archéosciences, Histoire), Université de Rennes, Campus Beaulieu, 263 avenue Général Leclerc, bât. 24-25 -CS74205, 35042, Rennes Cedex, France</footnote>

<footnote><sup>c</sup> Sorbonne Université, CNRS, Laboratoire d'Ecogéochimie des Environnements Benthiques, LECOB, Observatoire Océanologique, Banyuls-sur-mer, F-66650, France</footnote>

<footnote><sup>d</sup> Géosciences Montpellier, CNRS UMR 5243, Université Montpellier, CC60, F-34095, Montpellier, CEDEX 05, France</footnote>

<footnote><sup>e</sup> Sorbonne Université, MNHN, CNRS, SU, EPHE-PSL, UA, Institut de Systématique, Évolution, Biodiversité (ISYEB), 12 rue Buffon, F-75005, Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: franck.lartaud@obs-banyuls.fr (F. Lartaud).</footnote>
in calcite and Sr/Ca in aragonite) have been investigated and salinity
fluctuations appear to have a weak impact, if any (Vander Putten et al.,
2000; Mouchi et al., 2013). However, differences in Mg uptake from
natural sites or aquarium-based rearing experiments have been reported
for mytilids (Klein et al., 1996; Vander Putten et al., 2000; Freitas et al.,
2008; Wanamaker et al., 2008; Tanaka et al., 2019), oysters (Surge and
Lohmann, 2008; Mouchi et al., 2013; Tynan et al., 2017) and pectinids
(Freitas et al., 2012). In coastal waters, although some
Mg/Ca-temperature relationships appear very similar depending on the
environmental settings (Mouchi et al., 2018), it has been reported that
Mg/Ca variations in seawater caused by rapid salinity changes and Mg
supply from continental rock alteration can mask the temperature effect
on the Mg uptake in shell carbonates (Dodd, 1965; Wanamaker et al.,
2008). Vital effects may also impact elemental uptake (Carré et al.,
2006; Schöne et al., 2011; Mouchi et al., 2013).

In deep-sea environments, few studies were dedicated to the analyses
of metal concentrations in calcifying materials. Most focus has been on
investigations in cold-water coral skeletons, where Mg/Ca ratio has been
demonstrated to be an unreliable paleothermometer proxy
(Lopez-Correa et al., 2010; Robinson et al., 2014). Although potentially
in the vicinity of hydrothermal systems, those corals are not directly
associated with vent fluids, in contrast to chemosynthetic bivalve spe-
cies such as hydrothermal mussels. The geochemical proxies studied in
bivalve shells from hydrothermal vents focused on stable isotopes, such
as the δ¹³C to reconstruct chemosynthetic pathways (Nedoncelle et al.,
2014) or carbon sources (Naraoka et al., 2008; Lietard and Pierre, 2009;
Lartaud et al., 2010b, 2011), and the use of minor and trace elements for
the characterization of heavy metals present in the mixing zone (Kádár
et al., 2005; Cravo et al., 2008), or the study of metal detoxification
processes (Demina et al., 2013). The studies thus focused on trace metals
typically associated with hydrothermal systems (e.g., Fe, Mn, Zn, Cu,
Cd), and other classical elements used in shallow-water biological ar-
chives were not considered, particularly Mg/Ca as end-member hydro-
thermal fluids are severely depleted in magnesium (Corliss et al., 1979;
Charlou et al., 2002). Although no precise calibrations were performed,
previous works from Rio et al. (1988) suggest that the Mg/Ca and Sr/Ca
ratios in hydrothermal bivalve shells are consistent with those of the
ambient seawater. Shirai et al. (2008) studied the distribution of Mg/Ca
in *Bathymodiolus* shells in depth but they focused on the inner aragonite
nacreous layer, which is generally not used for environmental recon-
struction in shallow-water species. Indeed, sclerochemistry in bivalve
shells is mostly performed on the outer calcite prismatic layer, an area
associated with higher growth rates leading to better time-resolution
sampling.

Mytilids from the genus *Bathymodiolus* are among the most successful colonizers of vent environments and often dominate the biomass of hydrothermal communities (Van Dover, 2000). *Bathymodiolus* habitat is characterized by fluctuation of the environmental parameters, including pH, oxygen and temperature, resulting from hydrothermal fluid mixing with the background cold-seawater (Le Bris and Duperron, 2010; Le Bris et al., 2019). The temperatures range from strictly deep seawater temperature (i.e., 2–4 °C) to fluid-related (reaching 10–15 °C), associated to the influence of tides on the deep-sea current regime and fluid vent flux (Johnson et al., 1994; Tivey et al., 2002; Nedoncelle et al., 2015; Lelievre et al., 2017). This range seems to be maintained for long time periods, only disrupted by occasional anomalies (Scheirer et al., 2006; Khripounoff et al., 2008; Mullineaux et al., 2012).

The purpose of this paper is to investigate the potential of using Mg/
Ca and δ¹⁸O in deep-sea mussel shells from hydrothermal vents.
Particularly, the study focuses on the variations of these proxies during
growth, to investigate the record of the vent dynamics. Using Bathy-
modiolus azoricus (von Cosel et al., 1999) and B. thermophilus (Kenk and
Wilson, 1985) growing at five contrasting temperature vent habitats
from three sites, the evolution of Mg/Ca was measured by LA-ICP-MS
and δ¹⁸O by IRMS and compared to the temperature monitored in the
mussel habitat.

## 2. Methods

2.1. *Sampling area*

Mussel shells were collected at three hydrothermal vent sites from the Mid-Atlantic Ridge (MAR) and the East Pacific Rise (EPR): Rainbow (36°13.8′N-33°54.1′W, 2275 m), Menez Gwen (37°50.6′N-31°31.2′W, 815 m) and EPR 9°50′N (09°47.3′N-104°17.0′W, 2513m). Rainbow end-member fluids formed in ultramafic basement are characterized by high methane and hydrogen contents but also high metal concentrations. Menez Gwen fluids are also characterized by methane and hydrogen gases but depleted in metals (Charlou et al., 2002). The average temperature in the mussel habitat is higher at Menez Gwen (10.9 °C) than at Rainbow (3.7 °C), due to the greater depth of the latter (Desbruyères et al., 2001). EPR vent fluids exhibit important free sulphide but low methane and hydrogen concentrations.

Live *B. azoricus* from Rainbow were collected during the MoMAR-DREAM cruise (CNRS, R/V L'Atalante, August-September 2008) using the Victor 6000 ROV and then dissected onboard. Two areas of collection were selected as described in Nedoncelle et al. (2014): Z1 zone, which corresponds to mussels living at the basis of a sulphide structure about 1 m-high in the immediate vicinity of a small spire-type active chimney; and Z3 zone, which harbours a higher density of mussels and is located further away from the chimney on the top of the rocky structure at ~1 m above Z1 (Fig. 1).

At Menez Gwen, live *B. azoricus* shells were collected during the MenezMAR cruise (MARUM, R/V *Meteor*, September-October 2010) using the Quest 4000 ROV. Mussels were sampled at Cage Site (CS), from a small discretely distributed mussel patch at the top of a hydrothermal area presenting some active diffuse vents, and along the flank of the White Flame (WF) site fully colonized by mussels and dominated by a large active white smoker (Fig. 1).

The live *B. thermophilus* from EPR 9°50N were collected during the MESCAL-1 cruise (UPMC, R/V *L'Atalante*, April-May 2010) with the Nautilus submersible in a ~50 m² mussel bed at V-Vent site (MES), a diffuse vent area on the basaltic seafloor (Fig. 1) (see Nedoncelle et al., 2013 for more details).

## 2.2. Shell preparation and observation

*Bathymodiolus* shells are formed of an aragonite lower (inner) layer and a calcite upper (outer) layer (Carter, 1990; Nedoncelle et al., 2013). Geochemical analyses were performed on the calcite upper layer, since calcite is less sensitive to alteration and dissolution than aragonite in those environments, and preserves the pristine signal from diagenetic alterations (Lutz et al., 1994). Shells were sectioned along the maximum growth axis and mounted on epoxy blocks. Sections were ground flat using 180, 400 and 800 grit powder with distilled water and then polished using Al₂O₃ powder at 3, 1 and 0.3 µm. A Mutvei treatment (Schöne et al., 2005) was used to reveal growth structures (Fig. 2A). Major growth lines were identified as growth lines present both in the upper calcitic and lower aragonitic layers, often accompanied with shell distortion on the external surface (Schöne and Giere, 2005). Part of the data were from Nedoncelle et al. (2014).

During the MenezMAR cruise, shells from Cage Site at Menez Gwen
were stained with calcsein following the protocol described in Nedon-
celle et al. (2013) and recovered after 10 days. Despite the success of
staining (100 % of the shells revealed the calcsein line), no growth was
observed during the 10 days mark and recapture period (Supplementary
Fig. S1), in contrast to the B. thermophilus used in Nedoncelle et al.
(2013).

2.3. LA-ICP-MS analyses

Four shells from Rainbow (1 for Zone 1 and 3 for Zone 3, with sizes
ranging from 34 to 52 mm), four shells from Menez Gwen vent sites (2
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Sampling areas for the three hydrothermal vent sites. At Rainbow, shells were collected from two micro-habitats: Z1, where mussels lived close to the active chimney (in black), and Z3, corresponding to a denser mussel patch ~1m above (© Ifremer - MoMARDREAM). At Menez Gwen, shells originated from two locations: White Flame (WF) and Cage Site (CS). For CS, the shells collected are the ones below the red autonomous probe (© Marum - MenezMAR). At EPR 9°50N, shells were collected in the V-Vent area (here called MES), around the yellow autonomous probe (© Ifremer - Mescal-1). White scale bar is 10 cm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Photographs of cross section of a *B. azoricus* shell (specimen Z3-116), at the ventral margin highlighting the details of the microstructures and growth increments revelation under Mutvei treatment (A), and the position of the LA-ICP-MS samplings (B). d.o.g. is direction of growth. Red arrows highlight the position of major growth lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

for Cage Site, previously stained with calcein, see above, and 2 for White Flame, with shell sizes from 46 to 84 mm), and two shells from EPR 9°50N (sizes of 154 and 170 mm) were analysed for Mg abundances.

Before elemental analyses, the sections were repolished to remove the Mutvei solution and prevent any influence of this treatment on the chemical analyses. The shell sections were analysed by Laser Ablation-
Inductively Coupled-Plasma-Mass Spectrometry (LA-ICP-MS) with a Compex102 193 nm excimer laser (Lambda Physik) coupled to a sector field ICP-MS (Thermofinnigan Element XR) at Montpellier University (AETE-ISO platform of the OSU OREME). Measured elements were ²⁴Mg and ⁴³Ca as the internal standard to control the ablated volume. The laser was operated at a repetition rate of 7 Hz and an energy density of 12 J cm⁻². Each analysis was done as a single spot using a spot size of 51 µm (Fig. 2B) after a pre-ablation time of 1 s for surface cleaning and corresponds to a total acquisition time of 180 s with the first 120 s as background acquisition (gas blank). Data were normalized using the ⁴³Ca signal (internal standard) according to Longerich et al. (1996) and calibrated using NIST SRM 612 glass reference material (values after Pearce et al., 1997). Reproducibility of the values in the shell samples was also tested using the USGS MACS-3 carbonate reference material. Both reference materials were analysed every 10 measurements. Precision based on the analysis of the reference materials was approximately ±5 %.

## 2.4. Temperature reconstruction from Mg/Ca

Values of Mg/Ca were converted into temperatures using the equation from Vander Putten et al. (2000), established on the shell of the shallow water mussel *Mytilus edulis*:

$$ \text{Mg/Ca (mmol. mol}^{-1}\text{)} = 0.70 * \text{T(°C)} - 0.63 \quad (\text{r} = 0.91) $$

This equation was chosen among the different equations existing for bivalve shells as it was established with a mytilid species, as *Bathymodiolus* (Mikkelsen et al., 2006), with a similar shell microstructure (i. e., fibrous prismatic calcite layer) in the area investigated (Carter, 1980; Machado et al., 2009; Génio et al., 2012; Checa et al., 2014). Additionally, in the course of this study, the Mg/Ca measurement was performed with LA-ICP-MS, as in Vander Putten et al. (2000). This contrasts with the other temperature-Mg/Ca relationships published for *M. edulis* which were acquired by ICP-AES analyses (Klein et al., 1996; Wanamaker et al., 2008).

## 2.5. Oxygen isotope ratios

The remaining section of the shells was used for oxygen isotopes analysis. When possible we tried to use the same specimen already analysed for Mg/Ca. As some of them were broken during handling, we had to include additional shells. Shells were placed in a peroxide solution (H₂O₂ 3.4 %) during 6–10 h at 60 °C to remove the periostracum and then rinsed with demineralized water before sampling the calcite outer layer for the oxygen isotopes (δ¹⁸O) analysis (see Nedoncelle et al., 2014). Shells were then drilled every 1 mm from the ventral margin to the hinge area along the maximum growth axis, with a Dremel device equipped with a 0.3 mm drill bit. The nacreous aragonite underlayer was carefully avoided to preclude any mineralogical effect on the isotopic values as aragonite is enriched in ¹⁸O by 0.6 ‰ compared to calcite (Tarutani et al., 1969).

The collected calcium carbonate powders were acidified in 100 % H₃PO₄ at 90 °C under vacuum. The CO₂ produced was collected and analysed using a VG Instruments Isoprime mass spectrometer. Isotopic data are reported in the conventional delta (δ) notation relative to the Vienna Pee Dee Belemnite (V-PDB). The standard deviation for δ¹⁸O values was ±0.1 ‰.

Seawater samples directly above the mussel bed were additionally collected during the dives for mussel collection at EPR 9°50N (MES) and Rainbow (Z1 and Z3). 20 mL of *in situ* water were sampled in airtight titanium syringes and analysed following the method of Pierre et al. (1994). The analyses were performed at the Institut d'Écologie et des Sciences de l'Environnement de Paris at Grignon. Seawater oxygen isotope compositions (δ¹⁸Ow) were obtained after CO₂·H₂O equilibration using an Isoprime mass spectrometer coupled to a Gilson X222

Micromass Aquaprep. Standard error for δ¹⁸Ow was 0.15 ‰ and values expressed relative to SMOW reference.

## 2.6. Temperature reconstruction from δ¹⁸O

Based on the thermodynamic model for synthetic and biogenic carbonates, temperatures can be estimated from shell δ¹⁸O and δ¹⁸Ow measured in each habitat. We used the equation of Kim and O'Neil (1997):

$$ 1000 \times \ln(\alpha) = 18.03 \times (10^3 \times T^{-1}) - 32.42 $$

where α represents the isotope fractionation factor:

$$ \alpha = (1000 + \delta^{18}\text{O}_{\text{shell}}) / (1000 + \delta^{18}\text{O}_{\text{w}}) $$

In this last equation, both δ¹⁸Oshell and δ¹⁸Ow are expressed in ‰ SMOW. δ¹⁸Oshell were converted from V-PDB to SMOW according to the equation of Coplen et al. (1983).

As no δ¹⁸Ow were measured from habitats at Menez Gwen, and considering its proximity with Rainbow, we used the mean δ¹⁸Ow value of Rainbow for temperature reconstructions from shells of Menez Gwen.

## 2.7. Temperature probes

*In situ* temperature measurements in mussel habitats were acquired at discrete locations in Rainbow (on both Z1 and Z3 areas), based on continuous records over 5 min for each analysis using a glass Ag/AgCl electrode coupled to a thermocouple (MICREL, France) (Fig. 1). Discrete measurements were also acquired at Menez Gwen (White Flame) by a temperature probe fixed to the sampling nozzle of the Kiel In Situ Pumping System (KIPS) (Meier et al., 2016; raw data pers. comm. C. Vidoudez). At Menez Gwen (Cage Site) and EPR 9°50N (V-Vent), autonomous probes (S2T6000, NKE) were used above the collected mussels (Fig. 1). The autonomous probes were deployed during 10 days at Menez Gwen (i.e., during the staining experiment) on the mussel bed studied and recovered at the time of mussel sampling, with an acquisition every 5 min during this period. The deployment protocol of autonomous probes at EPR 9°50 was described in Nedoncelle et al. (2015). The probes monitored the habitat conditions during 10 days with a frequency of 2 measurements per hour.

## 2.8. Statistical analysis

Comparisons of isotopic and Mg compositions between shells were performed using Matlab (R2022a; www.mathworks.com) by applying the Kruskal and Wallis test (KW) followed by a post hoc test to discriminate the outlying groups (Tukey's honestly significant difference procedure; HSD). 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 HSD test determines which sample is different from the others. Before using this non-parametric test, the non-respect of the normality and homoscedasticity conditions were checked using the Kolmogorov-Smirnov and Levene tests, respectively. Shell Mg/Ca were compared using the correlation test of Spearman.

# 3. Results

## 3.1. Mg/Ca analysis

The Mg/Ca values range from 1.8 to 11.1 mmol mol⁻¹ (average of 3.1 ± 1.1 mmol mol⁻¹) in shells from Rainbow, from 3.3 to 19.5 mmol mol⁻¹ (average of 6.8 ± 3.4 mmol mol⁻¹) in shells from Menez Gwen, and from 2.8 to 4.5 mmol mol⁻¹ (average of 3.4 ± 0.4 mmol mol⁻¹) in shells from EPR 9°50N (Fig. 3). We observe highly significant differences in shell Mg/Ca between sites (Kruskal and Wallis test, p < 0.01). Similarly, we note significant differences between habitats within both
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Shell Mg/Ca and corresponding estimated temperatures. For each habitat, recorded temperature ranges are indicated by the red dashed lines and the average temperature in red dotted lines (this study; EPR: Nedoncelle et al., 2015). The historical temperature ranges, measured at each hydrothermal site, are indicated by the blue dashed lines (Rainbow: Desbruyères et al., 2001; Sarrazin et al., 2020; Menez Gwen: Colaço et al., 1998; Sarradin et al., 1999; Desbruyères et al., 2001; Sarrazin et al., 2020; EPR 9°50N: Mills et al., 2007; Moore et al., 2009; Luther et al., 2012). The red arrows correspond to the positions of major growth lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

Menez Gwen and Rainbow (Kruskal and Wallis test, *p* < 0.01).

The temperatures for Rainbow, estimated from Mg/Ca in the shells, range from 3.5 to 16.7 °C (average of 5.3 ± 1.5 °C). These calculated temperatures are quite stable (variation of ~1 °C) with the exceptions of one very localized peak at 10.1 °C in the shell Z3-116 and two thin peaks above 12 °C in the shell Z3-131 (Fig. 3). For each shell, these values are close similar to temperatures measured in the mussel habitat, showing

an average of 5.6 ± 0.9 °C in Z1 mussel habitat and 4.8 ± 0.4 °C in Z3 habitat (Table 1, Fig. 3).

The estimated temperatures from the Menez Gwen shells range from 5.6 to 28.8 °C (average of 10.7 ± 4.8 °C) (Fig. 3). The two shells from Cage Site exhibit the same pattern with a slight variability in the estimated temperatures (between 8.4 and 13.5 °C) before a strong increase at the end of the deployment period (close to the shell ventral margin), with temperatures over 25 °C. We observe a similar pattern at White Flame for shell WF-24, which shows a relatively stable estimated temperature (between 7.0 and 8.9 °C) before an increase up to 16.4 °C at the end of the profile. However, shell WF-20 does not exhibit such an increase of estimated temperature, which remains between 5.6 and 8.0 °C across the transect.

For EPR 9°50N, both shells exhibit the same relatively stable estimated temperatures, ranging from 4.9 to 6.9 °C and from 4.9 to 7.4 °C, for MES6 and MES10, respectively.

The shell Z3-131, from Rainbow vent site, displays two major growth anomalies, close similar to the major growth lines (based on the description of major growth line made by Schöne and Giere (2005) in *Bathymodiolus brevior* and by Nedoncelle et al. (2014) in *B. azoricus* shells). These growth breaks are associated with the two peaks of Mg/Ca (Fig. 3). For both, the Mg/Ca values increased just before the occurrence of the major growth line (following the direction of growth), reaching the maximum values in the major growth line, and fall to the baseline values after. Two major growth lines were also detected in Z3-116 and one of these (M1, see Fig. 2) corresponds to a Mg/Ca peak (Fig. 3).

At Menez Gwen, the two shells from Cage Site, as well as shell specimen WF24 from White Flame, displayed a significant increase in Mg/Ca at the end of the profile (Fig. 3), but no major growth line was clearly observed. Nevertheless, the staining experiment revealed an absence of mineralization during the last 10 days of life (Supplementary Fig. S1), likely suggesting the formation of a growth stop (i.e., a major growth line) at this period. Thus Mg/Ca increase would anticipate the growth line formation as observed for Z3-116 and Z3-131 (Fig. 3). No major growth lines were detected in the shells from EPR 9°50N.

## 3.2. Oxygen stable isotope ratios

The measured δ¹⁸O<sub>w</sub> at Rainbow range from 0.05‰ (SMOW) at Z3 to 0.27‰ (SMOW) at Z1. Due to the lack of data in mussel beds at Menez Gwen and its proximity to Rainbow vent field, we assume a mean value of 0.16‰ (SMOW) for mussel habitats at Menez Gwen. The measurements in water surrounding mussels from EPR 9°50N gives a δ¹⁸O<sub>w</sub> value of -0.44‰ (SMOW).

Shells from Rainbow present δ¹⁸O values between -1.37 and 4.12‰ (VPDB), and exhibit significant differences between (*p* < 0.01) and within habitats (Z1-7 different from Z1-1 and Z1-4: *p* < 0.01; Z3-131 different from A3-LIV4 and Z3-118: *p* < 0.01). Shells from Menez Gwen range from 2.52 to 3.91‰. A significant difference between shells between (*p* < 0.01) and within each habitat is also observed (WF-24 different from WF-20 and WF-22: *p* < 0.01; CS-4 different from CS-7: *p* < 0.01). δ¹⁸O of shells from EPR 9°50N range from 2.21 to 4.27‰, and show no significant difference between shells from this site (*p* = 0.57). We also note a significant difference between sites (*p* < 0.01).

Reconstructed temperatures from each shell are presented in Fig. 4. The estimated temperatures are mostly lower than those measured on site (Fig. 4; Table 2). At Rainbow, two shells from Zone 1 present estimated temperatures lower than the measured *in situ* temperature range (mean estimates at 2.3 °C and 2.9 °C respectively, while mean measured temperature is 5.6 °C), along with some values below 0 °C, and only one shell (Z1-7) harbors reconstructed values in agreement with measured temperatures (mean estimated temperature: 5.2 °C; Fig. 4). For the Zone 3 of Rainbow, except for two values from one shell (A3-LIV4) which give estimated temperatures within the range of measured temperatures, the rest of reconstructed temperatures are below the minimum recorded temperature, with some negative values. All estimated temperatures
<table><caption>**Table 1**
Reconstructed temperatures from shell Mg/Ca compared to measured temperatures on study sites.</caption><thead><tr><th rowspan="2">Site</th><th rowspan="2">Habitat</th><th rowspan="2">Sample</th><th colspan="2">Estimated temperature (°C)</th><th colspan="2">Measured temperature (°C), this study</th><th colspan="2">Measured temperature (°C), literature</th></tr><tr><th>mean ± SD</th><th>min - max</th><th>mean ± SD</th><th>min - max</th><th>min - max</th><th>references</th></tr></thead><tbody><tr><td rowspan="4">Rainbow</td><td>Z1</td><td>Z1-4</td><td>4.4 ± 1.5</td><td>3.6-8.1</td><td>5.6 ± 0.9</td><td>4.1-8.1</td><td rowspan="4">3.1-39.7</td><td rowspan="4">Desbruyères et al. (2001)<br>Sarrazin et al. (2020)</td></tr><tr><td rowspan="3">Z3</td><td>Z3-116</td><td>4.8 ± 1.5</td><td>3.8-10.1</td><td rowspan="3">4.8 ± 0.4</td><td rowspan="3">4.0-5.8</td></tr><tr><td>Z3-118</td><td>4.7 ± 0.6</td><td>3.5-5.8</td></tr><tr><td>Z3-131</td><td>5.5 ± 1.6</td><td>4.0-16.7</td></tr><tr><td rowspan="4">Menez Gwen</td><td rowspan="2">Cage Site</td><td>CS-6</td><td>14.0 ± 5.3</td><td>9.1-27.2</td><td rowspan="2">9.9 ± 0.2</td><td rowspan="2">9.3-11.4</td><td rowspan="4">7.2-26.0</td><td rowspan="4">Colaço et al. (1998)<br>Sarradin et al. (1999)<br>Desbruyères et al. (2001)<br>Meier et al. (2016)<br>Sarrazin et al. (2020)<br>Mills et al. (2007)<br>Moore et al. (2009)<br>Luther et al. (2012)</td></tr><tr><td>CS-9</td><td>12.3 ± 5.0</td><td>8.4-28.8</td></tr><tr><td rowspan="2">White Flame</td><td>WF-20</td><td>6.8 ± 0.6</td><td>5.6-8.0</td><td rowspan="2">10.4 ± 3.1</td><td rowspan="2">8.4-20.4</td></tr><tr><td>WF-24</td><td>8.9 ± 2.4</td><td>7.0-16.4</td></tr><tr><td rowspan="2">EPR 9°50N</td><td rowspan="2">V-vent</td><td>MES-6</td><td>5.8 ± 0.6</td><td>4.9-6.8</td><td rowspan="2">6.3 ± 2.1</td><td rowspan="2">2.0-9.9</td><td rowspan="2">2.0-15.0</td><td rowspan="2"></td></tr><tr><td>MES-10</td><td>5.8 ± 0.7</td><td>4.9-7.4</td></tr></tbody></table>

from shells of Menez Gwen are considerably less than the minimum measured on site, with occasionally negative reconstructed temperatures at Cage Site.

Only one value of estimated temperatures from shells of EPR 9°50N is above 0 °C. All others are significant below the measured temperatures.

## 4. Discussion

### 4.1. Mg/Ca of bivalve shells as a possible thermometer proxy in hydrothermal settings

Within an error of ~1 °C, the skeletal Mg/Ca ratios in *B. azoricus* and *B. thermophilus* allow a reliable reconstruction of the average temperatures measured in the mussel habitat of the investigated vent sites, from the Atlantic and Pacific ridges (Table 1; Fig. 3). Although established with a shallow-water mussel species, the equation from Vander Putten et al. (2000) appears suitable to estimate temperatures using Mg/Ca ratios of deep-sea mussel shells. This is presumably due to (i) similar mineralogical and microstructural shell organisation of the two mytilid groups (Génio et al., 2012; Checa et al., 2014) and (ii) the specificity of geochemical conditions in these ecosystems. Far from the main continental inputs of Mg, the mussels used a stable supply of Mg from seawater to form their shells as the hydrothermal fluids are depleted in Mg (Charlou et al., 2002). Based on this assertion, the distribution coefficient of Mg²⁺ into the calcite material is strongly influenced by temperature changes. Thus, the difference observed between shells from the vent sites rely mainly on the different temperature conditions.

As reported for shallow-water mussel species (Lorens and Bender, 1980), the Mg/Ca ratio does not change in relation to growth variations in *Bathymodiolus* shells. An ontogenic trend in Mg/Ca was however described for different bivalve species (Freitas et al., 2005; Schöne et al., 2011; Mouchi et al., 2013). This could explain why the specimen Z3-131, the smallest shell from Rainbow, exhibits the highest Mg/Ca values among the shells on this site (Fig. 3). But this ‘age effect’ should have a lower influence in regard to temperature as specimen CS9 from Menez Gwen is in the same size range than the specimens from Rainbow, with clearly higher Mg/Ca ratios, and shells from EPR 9°50N exhibit similar values to shells from Rainbow while three times bigger. A better ontogenic growth profile for this species is however needed as growth rates can change according to the local environmental conditions (Nedoncelle et al., 2013).

Except for rare peaks, the reconstructed temperatures are quite stable at Rainbow (within ~1 °C variation), which is consistent with the measurements done at this site. At Menez Gwen - Cage Site habitat, before the large increase at the end of both shell profiles close to the ventral margin, that might be related to a specific process (see below), the estimated temperatures show more variability, with ~5 °C range. This variability is higher compared to our measurements done at this site

during 10 days, but close to the temperature changes observed on long-term monitoring at Lucky Strike, the closest known site to Menez Gwen, and could refer to the lunar and two lunar periodicities identified there (Sarrazin et al., 2014). The lunar-related temperature dynamic is typically observed in *Bathymodiolius* sp. habitats (Khripounoff et al., 2008; Nedoncelle et al., 2015). Similarly, at White Flame, the reconstructed temperatures (i.e., 5.6-8.9 °C when final increase of WF-24 shell is avoided) should reflect the environmental periodicity of temperatures. The range of values are however slightly underestimated compared to the discrete measured temperature from this study during the Menez-MAR cruise (8.4-20.4 °C), but are close similar to the minimum recorded from other studies on this vent site (i.e., 7.2°C; Colaço et al., 1998).

Short-term peaks of Mg/Ca are however observed in some shells, and seems to be related to the formation of major growth breaks (called major growth lines in Schöne and Giere, 2005). The corresponding estimated temperatures increase reaches up to 17 °C at Rainbow and 29 °C at Menez Gwen, consistent with the maximum values measured at these sites (Table 1; Sarradin et al., 1999; Sarrazin et al., 2020). The maximum temperature recorded in the major growth lines can be however slightly overestimated due to possible high Mg concentrations in the shell organic matrix (Rosenberg and Hughes, 1991; Takesue et al., 2008; Lartaud et al., 2010b; Pérez-Huerta et al., 2020). Formation of shell organic matrix in biogenic carbonates is rich in sulfur, either as sulphated polysaccharides or S-rich amino acids, and for some species distribution of Mg correlates with S variability (Lorens and Bender, 1980; England et al., 2006; Foster et al., 2008). Some shells (WF-24, CS-6, CS-9, Z1-4) display a rise of Mg/Ca in the most recent shell parts (near collection date). This increase may correspond to the current formation of major growth lines, as we noted an absence of shell growth from calcite labels 10 days prior collection (Supplementary Fig. S1).

Different factors can act on Mg/Ca of bivalve shells, including the physiology of the organism which seems to generate breakdowns in the control of temperature on the incorporation of Mg, as illustrated for the mussel *M. edulis* (Vander Putten et al., 2000) and the oyster *Magallana gigas* (Mouchi et al., 2013). Although these types of breakdowns are not yet identified in deep-sea mussels, we cannot preclude their existence and further studies are required before applying directly the equation of Vander Putten et al. (2000) to reconstruct temperature changes in hydrothermal vent habitats.

### 4.2. Oxygen isotope ratios from hydrothermal vent mussels are unreliable temperature proxies

In contrast to Mg/Ca data, δ¹⁸O-inferred temperatures are underestimated, with values generally near or below 0 °C, which are unrealistic in these settings. Only one shell from Z1 presents reconstructed values close to the temperature measurements. All other shells from the different sites and habitats present reconstructed temperatures significantly lower than the minimum *in situ* temperature (Fig. 4). The
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Shell $\delta^{18}$O and corresponding estimated temperatures. For each habitat, recorded temperature ranges are indicated by the red dashed lines and the average temperature in red dotted lines (this study; EPR: Nedoncelle et al., 2015). The historical temperature ranges measured at each hydrothermal site are indicated by the blue dashed lines (Rainbow: Desbruyères et al., 2001; Sarrazin et al., 2020; Menez Gwen: Colaço et al., 1998; Sarradin et al., 1999; Desbruyères et al., 2001; Sarrazin et al., 2020; EPR 9°50N: Mills et al., 2007; Moore et al., 2009; Luther et al., 2012). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
<table><caption>**Table 2**
Reconstructed temperatures from shell δ¹⁸O compared to measured temperatures on study sites.</caption><thead><tr><th rowspan="2">Site</th><th rowspan="2">Habitat</th><th rowspan="2">Sample</th><th colspan="2">Estimated temperature (°C)</th><th colspan="2">Measured temperature (°C), this study</th><th colspan="2">Measured temperature (°C), literature</th></tr><tr><th>mean ± SD</th><th>min - max</th><th>mean ± SD</th><th>min - max</th><th>min - max</th><th>references</th></tr></thead><tbody><tr><td rowspan="3">Rainbow</td><td rowspan="3">Z1</td><td>Z1-1</td><td>2.3 ± 2.1</td><td>-1.8 - 8.7</td><td rowspan="3">5.6 ± 0.9</td><td rowspan="3">4.1-8.1</td><td rowspan="3">3.1-39.7</td><td rowspan="3">Desbruyères et al. (2001)<br>Sarrazin et al. (2020)</td></tr><tr><td>Z1-4</td><td>2.9 ± 1.9</td><td>-0.4 - 10.1</td></tr><tr><td>Z1-7</td><td>5.2 ± 3.9</td><td>2.8-22.6</td></tr><tr><td rowspan="3">Z3</td><td rowspan="3">Cage Site</td><td>A3-LIV4</td><td>1.5 ± 1.5</td><td>-0.1 - 5.7</td><td rowspan="3">4.8 ± 0.4</td><td rowspan="3">4.0-5.8</td><td rowspan="3">7.2-26.0</td><td rowspan="3">Colaço et al. (1998)<br>Sarradin et al. (1999)<br>Desbruyères et al. (2001)<br>Meier et al. (2016)<br>Sarrazin et al. (2020)</td></tr><tr><td>Z3-118</td><td>0.7 ± 1.1</td><td>-2.0 - 3.9</td></tr><tr><td>Z3-131</td><td>0.1 ± 0.5</td><td>-0.9 - 0.9</td></tr><tr><td rowspan="5">Menez Gwen</td><td rowspan="5">Cage Site</td><td>CS-4</td><td>0.5 ± 0.6</td><td>-0.5 - 1.7</td><td rowspan="5">9.9 ± 0.2</td><td rowspan="5">9.3-11.4</td><td rowspan="5">2.0-15.0</td><td rowspan="5">Mills et al. (2007)<br>Moore et al. (2009)<br>Luther et al. (2012)</td></tr><tr><td>CS-6</td><td>1.0 ± 1.3</td><td>-1.0 - 4.4</td></tr><tr><td>CS-7</td><td>1.5 ± 1.0</td><td>-1.4 - 2.5</td></tr><tr><td>CS-9</td><td>1.1 ± 0.5</td><td>0.7-1.8</td></tr><tr><td>White Flame</td><td>WF-20</td><td>2.1 ± 0.6</td><td>1.0-2.9</td></tr><tr><td rowspan="3">EPR 9°50N</td><td rowspan="3">V-vent</td><td>WF-22</td><td>1.7 ± 0.4</td><td>0.9-2.5</td><td rowspan="3">10.4 ± 3.1</td><td rowspan="3">8.4-20.4</td><td rowspan="3">2.0-15.0</td><td rowspan="3">Mills et al. (2007)<br>Moore et al. (2009)<br>Luther et al. (2012)</td></tr><tr><td>WF-24</td><td>1.2 ± 0.5</td><td>0.4-2.1</td></tr><tr><td>Ca21</td><td>-2.9 ± 3.1</td><td>-5.3 - 3.1</td></tr><tr><td rowspan="2">EPR 9°50N</td><td rowspan="2">V-vent</td><td>Ca22</td><td>-4.0 ± 1.2</td><td>-5.1-2.0</td><td rowspan="2">6.3 ± 2.1</td><td rowspan="2">2.0-9.9</td><td rowspan="2">2.0-15.0</td><td rowspan="2">Mills et al. (2007)<br>Moore et al. (2009)<br>Luther et al. (2012)</td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

measured values of δ¹⁸O<sub>w</sub>, required to convert shell δ¹⁸O into temperatures, may be a source of uncertainty if this parameter fluctuates outside the sampling period. Based on Kim and O'Neil (1997) equation, the δ¹⁸O<sub>w</sub> value expected to reconstruct accurate temperatures from shell δ¹⁸O can be inferred. The expected values at both sites of Rainbow (0.55 and 0.71‰ at Z1 and Z3, respectively) are slightly higher than those measured (0.05‰ and 0.27‰, respectively), but within the known δ¹⁸O<sub>w</sub> from Rainbow, which range linearly from 0‰ (seawater) to ~1‰ (hydrothermal fluid) (Andreani et al., 2014). However, such δ¹⁸O<sub>w</sub> values would correspond to a mixing of >50 % of end-member fluid, leading to a temperature of ~180 °C if we assume temperature of hydrothermal fluids reaching 365 °C at Rainbow (Charlou et al., 2002), which is unlikely for an area suitable for mussels. The expected δ¹⁸O<sub>w</sub> values at Menez Gwen (White Flame and Cage Site habitats) and EPR 9°50N are substantially higher (1.91, 1.97 and 1.74‰, respectively). These values are largely above the current measured or estimated values for this site (i.e., 0.16 and -0.44‰ at Menez Gwen and EPR 9°50N, respectively). They also exceed δ¹⁸O<sub>w</sub> reports from Menez Gwen hydrothermal end-member (1.17 ± 0.06‰) (Jean-Baptiste et al., 1997) but are close to the higher values recorded in the hydrothermal fluids from EPR 9-10°N (0.6-1.9 ‰) (Shanks et al., 1991). Still, the corresponding conditions for such δ¹⁸O<sub>w</sub> values surrounding mussels would reach a mixing with >90 % of end-member, thus a temperature of ~347 °C if we assume temperature of hydrothermal fluids reaching 390 °C at EPR 9°50N (Proskurowski et al., 2008), far above the tolerance limit values of temperature for *Bathymodiolus* observed experimentally (i.e., 25-30°C, Boutet et al., 2009). A wrong estimation of δ¹⁸O<sub>w</sub> cannot therefore explain the discrepancy in reconstructed temperatures from shell δ¹⁸O.

Based on measured temperatures and δ¹⁸O<sub>w</sub> at sampling sites, the expected value of shell δ¹⁸O at equilibrium with the environment was done using the equation of Kim and O'Neil (1997), and compared to the median shell δ¹⁸O values. The resulting difference corresponds to the

oxygen isotope disequilibrium (Table 3). *Bathymodiolus azoricus* from Rainbow are closer to isotopic equilibrium (i.e., mean disequilibrium of 0.28‰ and 0.66‰ for Z1 and Z3 habitats, respectively) than the shells from the other sites (disequilibrium of 1.75 ‰ for *B. azoricus* shells from White Flame and 1.81 ‰ for the ones from Cage Site, and 2.18‰ for *B. thermophilus* from EPR 9°50N). Such disequilibrium could be related to the influence of pH on oxygen isotope composition in biogenic carbonates (Usdowski et al., 1991; Kim and O'Neil, 1997; Zeebe, 1999). Particularly at hydrothermal vents, pH can greatly change with time and location, leading to a range of pH of 6.2-8 in *Bathymodiolus* habitats on the MAR and the EPR (Desbruyères et al., 2001; Kádár et al., 2005; Nedoncelle et al., 2015). Although bivalves can regulate their internal pH, it is estimated that this process is lower compared to other calcifying species such as corals, foraminifera and coralligenous algae, with an up-regulation limited to 0.5 pH unit (Crenshaw and Neff, 1969; Ip et al., 2006; McConnaughey and Gillikin, 2008).

Using the model described by Zeebe (2007), we reconstructed potential oxygen isotope disequilibrium induced by several pH scenarios (Table 4), in order to evaluate the impact of this parameter on shell δ¹⁸O. To do this, we considered salinity to be 34.7 at the EPR and 35 at the MAR vent sites (Haalboom et al., 2020). The oxygen isotope disequilibrium was tested for different pH scenarios and relative to a maximum pH of 8 (i.e., pH of deep seawater, Nedoncelle et al., 2015). At Rainbow, the oxygen isotope disequilibrium estimated in Table 3 can be explained by relatively restricted decrease in pH values compared to deep seawater. A fractionation of 0.2-0.3 at Z1 habitat is consistent with pH values of 7.7 ± 0.3 measured in *Bathymodiolus* habitats at this site (Desbruyères et al., 2001). The disequilibrium observed at Z3 habitat however requires a mean pH of 7.2, which appears low compared to the *Bathymodiolus* environment. A stronger decrease in pH would be necessary to cause the high isotopic disequilibrium observed at both Menez Gwen and EPR 9°50N. Although a pH decrease (values up to 6.7) has been observed at Menez Gwen (Desbruyères et al., 2001) and could

<caption>**Table 3**</caption>

Shell oxygen isotope disequilibrium based from measured temperature and seawater δ¹⁸O. The indicated value of δ¹⁸O disequilibrium is the difference between shell δ¹⁸O and expected shell δ¹⁸O at equilibrium.

<table><thead><tr><th>Site</th><th>Measured mean temperature (°C)</th><th>Measured δ¹⁸O<sub>w</sub> (‰ SMOW)</th><th>Expected shell δ¹⁸O at equilibrium (‰ VPDB)</th><th>Median measured shell δ¹⁸O (‰ VPDB)</th><th>Shell δ¹⁸O disequilibrium (‰ VPDB)</th></tr></thead><tbody><tr><td>Rainbow - Z1</td><td>5.6</td><td>0.27</td><td>2.72</td><td>3.00</td><td>0.28</td></tr><tr><td>Rainbow - Z3</td><td>4.8</td><td>0.05</td><td>2.71</td><td>3.37</td><td>0.66</td></tr><tr><td>Menez Gwen - White Flame</td><td>10.4</td><td>0.16</td><td>1.41</td><td>3.16</td><td>1.75</td></tr><tr><td>Menez Gwen - Cage Site</td><td>9.9</td><td>0.16</td><td>1.53</td><td>3.34</td><td>1.81</td></tr><tr><td>EPR 9°50N</td><td>6.3</td><td>-0.44</td><td>1.83</td><td>4.01</td><td>2.18</td></tr></tbody></table>
<caption>**Table 4**</caption>

Impact of pH on δ¹⁸O disequilibrium. The model of Zeebe (2007) was used to reconstruct the α parameter from the thermodependent equation of Kim and O'Neil (1997), from salinity (S), temperature (T) and various pH scenarios. Theoretical δ¹⁸O from potential pH values were then calculated from the equation using α and the measured δ¹⁸O<sub>w</sub>.

<table><thead><tr><th>Site</th><th>S</th><th>T (°C)</th><th>pH</th><th>α</th><th>Theoretical shell δ¹⁸O (VPDB)</th><th>δ¹⁸O disequilibrium compared to deep seawater pH = 8 (VPDB)</th></tr></thead><tbody><tr><td rowspan="3">Rainbow Z1</td><td rowspan="3">35</td><td rowspan="3">5.6</td><td>8</td><td>1.0355</td><td>4.97</td><td>0.29</td></tr><tr><td>7.7</td><td>1.0358</td><td>5.26</td><td>3.31</td></tr><tr><td>6.2</td><td>1.0389</td><td>8.27</td><td></td></tr><tr><td rowspan="4">Rainbow Z3</td><td rowspan="4">35</td><td rowspan="4">4.8</td><td>8</td><td>1.0358</td><td>5.00</td><td>0.24</td></tr><tr><td>7.7</td><td>1.0360</td><td>5.24</td><td>0.65</td></tr><tr><td>7.2</td><td>1.0364</td><td>5.65</td><td>3.30</td></tr><tr><td>6.2</td><td>1.0392</td><td>8.30</td><td></td></tr><tr><td rowspan="5">Menez</td><td rowspan="5">35</td><td rowspan="5">9.9</td><td>8</td><td>1.0344</td><td>3.83</td><td>0.26</td></tr><tr><td>7.7</td><td>1.0347</td><td>4.09</td><td>1.04</td></tr><tr><td>6.9</td><td>1.0355</td><td>4.87</td><td>1.43</td></tr><tr><td>6.7</td><td>1.0359</td><td>5.25</td><td>3.10</td></tr><tr><td>6.2</td><td>1.0376</td><td>6.93</td><td></td></tr><tr><td rowspan="5">Menez</td><td rowspan="5">35</td><td rowspan="5">10.4</td><td>8</td><td>1.0343</td><td>3.71</td><td>0.26</td></tr><tr><td>7.7</td><td>1.0346</td><td>3.97</td><td>1.03</td></tr><tr><td>6.9</td><td>1.0354</td><td>4.74</td><td>1.42</td></tr><tr><td>6.7</td><td>1.0358</td><td>5.13</td><td>3.09</td></tr><tr><td>6.2</td><td>1.0375</td><td>6.80</td><td></td></tr><tr><td rowspan="5">EPR</td><td rowspan="5">34.7</td><td rowspan="5">6.3</td><td>8</td><td>1.0354</td><td>4.13</td><td>0.24</td></tr><tr><td>7.7</td><td>1.0356</td><td>4.37</td><td>1.05</td></tr><tr><td>6.9</td><td>1.0364</td><td>5.17</td><td>2.04</td></tr><tr><td>6.5</td><td>1.0375</td><td>6.16</td><td>3.23</td></tr><tr><td>6.2</td><td>1.0387</td><td>7.36</td><td></td></tr></tbody></table>

explain a disequilibrium of 1.43 (Table 4), closer but still below to the ones described in Menez Gwen habitats (Table 3), it is unlikely mussels experienced such low pH values continuously. Other discrete measures in *Bathymodiolus* habitats from Menez Gwen exhibited pH values of 6.9 (Kádár et al., 2005), leading to a disequilibrium of ~1 while the current fractionation is > 1.7. Thus another factor would explain the high δ¹⁸O values observed in mussel shells from this site. Similarly, at EPR 50°N continuous measurements show that pH range between 6.9 and 8, leading to a maximum disequilibrium of 1.05 (Table 4). To reach an oxygen isotope disequilibrium >2 as observed in Table 3, pH should decrease to 6.5, which is not consistent with the living conditions of this species there. It is thus unlikely that pH mainly controls shell δ¹⁸O at Menez Gwen and EPR 9°50N.

Another potential influencing factor of stable isotope composition in shells is chemosynthesis, as already shown for δ¹³C of *Bathymodiolus* (Nedoncelle et al., 2014). The sulfur-oxidizing symbionts provide CO₂ fixation in the Calvin-Benson cycle by the use of the RuBisCo enzyme. This enzyme favors light carbon incorporation in the organic tissues (Robinson and Cavanaugh, 1995; Fiala-Médioni et al., 2002), resulting in an enriched ¹³C availability in the extrapallial cavity and thus in the shell. As they use carbon from methane rather than dissolved inorganic carbon from seawater as source of carbon, methanotroph symbionts have lower effect on the ¹³C used for mineralization, leading to produce shells closer to isotopic equilibrium (Nedoncelle et al., 2014). Such effect on δ¹⁸O was however never described for hydrothermal vent shells, although RuBisCo was also shown to induce oxygen isotope fractionation in cyanobacteria, photosynthetic bacteria and high-level plants, in favor of ¹⁶O (Guy et al., 1993; Helman et al., 2005). An offset on oxygen isotopes occurs during microbial oxidation of sulfide (Markovic et al., 2016), which can lead to higher δ¹⁸O values for the water pool used by the host organisms. The sulfur-oxidizing symbiont activity thus might additionally explain the positive disequilibrium measured on δ¹⁸O of shells. Interestingly, contrary to the shells from Rainbow, shells from Menez Gwen (partly) and EPR 9°50N (mostly) have δ¹⁸O values far from the isotopic equilibrium (Table 3), likely corresponding to a higher

proportion of sulfur-oxidizing symbionts. This is consistent with the known repartition of symbionts in these mussel populations. *Bathymodiolus thermophilus* from EPR 9°50N rely only on sulfur-oxidizing symbionts and *B. azoricus*, which can harbor a dual symbiosis of sulfur-oxidizing and methanotrophs, exhibit a higher contribution to sulfo-oxidizing bacteria at Menez Gwen compared to Rainbow (Duperron et al., 2006; Halary et al., 2008; Nedoncelle et al., 2014).

## 5. Conclusion

Investigation of the Mg/Ca-temperature relationship in hydrothermal mussel shells reveals this proxy as a promising paleotemperature tracer for deep-sea species. This is the first suitability report of this proxy in deep-sea ecosystems, and although limited to hydrothermal vents, numerous applications of Mg/Ca analyses can be listed both with ecological and geological perspectives. Estimation of temperatures based on shell material will provide complementary tools to the direct measurements made by *in situ* probes, useful for the reconstruction of temperature changes at hydrothermal vents both over short-term (daily) and long-term (pluri-annual) scales. Although this relationship needs to be investigated deeper in details, deep-sea mussels, as they inhabit various ecosystems (e.g., hydrothermal vents, cold seeps) seem to be a promising archive of long-term trends in deep-seawater temperatures. As such, they would give important information for oceanographic current modifications, as well as monitoring of fluid emissions.

On the contrary, δ¹⁸O provides under-estimated temperatures. Although more study should be undertaken to explain this observation, pH variations on site may explain a substantial part of the isotopic disequilibrium. Still, we hypothesize that the type of symbiotic chemo-synthetic bacteria has a strong impact on the oxygen isotope disequilibrium of their host's shell.

Finally, the results also show great benefits in the ecological study of vent mussels, particularly to determine temperature variations in the habitat and identify the response of biodiversity to thermal anomalies. This will give clues to better describe the ecological features of those species living in inaccessible habitats.

### CRediT authorship contribution statement

**V. Mouchi:** Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Formal analysis. **K. Nedoncelle:** Writing – review & editing, Methodology, Investigation, Formal analysis. **O. Bruguier:** Writing – review & editing, Validation, Methodology, Formal analysis. **Z. Garmirian:** Writing – review & editing, Investigation. **N. Le Bris:** Writing – review & editing, Supervision, Funding acquisition. **F. Lartaud:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nadine Le Bris reports financial support was provided by TOTAL Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

We kindly thank captain and crew of the R/V L'Atalante and R/V Meteor, as well as the Nautilus submersible, ROV Victor 6000 and Quest 4000 operation groups. The chief scientists of the research expeditions MoMARDREAM (J. Dyment, CNRS) and MenezMAR (N. Dubillier, MARUM), and the scientific parties are gratefully acknowledged for
their support and help during the cruises. The study benefited from the joint support of Fondation TOTAL and UPMC to the chair "Biodiversity, extreme marine environment and global change" (PI N. Le Bris) and from the financial support of the CNRS and the Institute of Ecology and Environment (INEE) to the UMR8222 LECOB for participation to the cruises and equipments. K Nedoncelle's PhD grant was funded by the French Ministry of Higher Education, Research and Innovation through the Doctoral School 'Sciences de l'Environnement d'Ile de France - ED129'. We also acknowledge L. Contreira Pereira for deployment of the temperature autonomous sensors at Menez Gwen - Cage Site and Charles Vidoudez for providing discrete temperature measurements at Menez Gwen - White Flame. Finally, we acknowledge the two anonymous reviewers who contributed to improve the manuscript.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.dsr.2025.104485.

## Data availability

Data will be made available on request.

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