# Exploring diet shifts and ecology in modern sharks using calcium isotopes and trace metal records of their teeth

Alexandre Assemat, Sylvain Adnet, Kani Bayez, Auguste Hassler, Florent Arnaud-godet, Frederik H Mollen, Catherine Girard, Jeremy E Martin

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Alexandre Assemat, Sylvain Adnet, Kani Bayez, Auguste Hassler, Florent Arnaud-godet, et al. Exploring diet shifts and ecology in modern sharks using calcium isotopes and trace metal records of their teeth. Journal of Fish Biology, 2022, 10.1111/jfb.15211. hal-03821763

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# Exploring diet shifts and ecology in modern sharks using calcium isotopes and trace metal records of their teeth

Alexandre Assemat<fnref n="1" /> ID

Sylvain Adnet<fnref n="1" /> ID

Kani Bayez<fnref n="2" />

Auguste Hassler<fnref n="3" /> ID

Florent Arnaud-Godet<fnref n="3" />

Frederik H. Mollen<fnref n="4" /> ID

Catherine Girard<fnref n="1" /> ID

Jeremy E. Martin<fnref n="3" /> ID

**Correspondence**

Alexandre Assemat, Université Montpellier, Campus Triolet, Institut des sciences de l'évolution de Montpellier (ISEM), CC 064, Place E. Bataillon, 34095 Montpellier cedex 05, France.

Email: alexandre.assemat@umontpellier.fr

**Funding information**

INSU INTERRVIE CNRS; LabEx CEMEB

**Abstract**

Sharks occupy all living environments of the marine realm as well as some freshwater systems. They display varied and flexible feeding behaviours, but understanding their diet remains challenging due to their elusive ecology and the invasiveness of stomach content analyses in regard of their threatened status. As a potential alternative, we discuss the variability in δ⁴⁴/⁴²Ca values recorded in the tooth enamel of size-graded individuals belonging to three species of large sharks with distinct diets (Isurus oxyrinchus, Hexanchus griseus and Carcharodon carcharias). The preliminary results highlight shifts in diet linked to ontogeny (I. oxyrinchus and H. griseus) and spatial distribution (C. carcharias) characterizing feeding behaviour in these species at individual and population level. These outcomes agree with the results of traditional stomach analyses supporting that nontraditional stable isotopes thus represent new perspectives for the study of modern and extinct shark ecology. In addition, for the first time, the Sr/Ca elemental ratios measured in H. griseus reflect sexual differences that could be interpreted in terms of spatial segregation or physiological heterogeneities.

**KEYWORDS**

alkaline metals, calcium, ecology, elasmobranchs, ontogeny, trophic level

## 1 | INTRODUCTION

As marine apex predators, large sharks exert strong top-down control on the food web (Ferretti et al., 2010; Hussey et al., 2014). They are principally opportunistic predators displaying behaviours such as active predation or scavenging (Domeier, 2009). Understanding their feeding behaviours is a key to estimating the trophic interactions in both extinct and present marine ecosystems and an opportunity to increase conservation efforts as well. However, the feeding ecology of numerous shark species remains poorly known due to their scarcity in the wild, their threatened conservation status and their elusive behaviour during their life spans (Hussey et al., 2012). Feeding preferences in sharks have

been estimated for years on the basis of stomach contents from captured or dead specimens (Cortés, 1999; Ebert, 1994; Simpfendorfer et al., 2001a). This method offers a snapshot of the short-term diet of individuals that allows the last meal of a specimen to be characterized (Baker et al., 2014).

In recent decades, the use of stable isotopic analyses (SIA) on soft and hard tissues using nitrogen (δ¹⁵N) and carbon (δ¹³C) isotopes has been proposed to investigate the ecology and feeding behaviours of shark species (Barría et al., 2018; Cree et al., 1999; Estrada et al., 2006; Kast et al., 2016, 2022; Tierney et al., 2008; Vennemann et al., 2001). These analyses offer the opportunity to estimate the diet over periods of time from months to years to complement previous data from

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2022 The Authors. *Journal of Fish Biology* published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.

<footnote n="1">¹ISEM, Université Montpellier CNRS EPHE IRD, Montpellier, France</footnote>

<footnote n="2">²University of Lyon, Lyon, France</footnote>

<footnote n="3">³Laboratoire de Géologie de Lyon: Terre, Planète, Environnement, Ecole Normale Supérieure de Lyon, Lyon, France</footnote>

<footnote n="4">⁴Elasmobranch Research Belgium, Bonheiden, Belgium</footnote>
stomach content analyses (Reum et al., 2020). However, the nitrogen isotopic composition of seawater, and by extension in the food-web baseline, is subject to strong geographical variability due to the spatial variations of nutrient supply and terrigenous inputs (Holmden et al., 1997; Matich et al., 2021; Schmitt et al., 2001) that complicate the comparison of specimens from distant areas (Jennings & van der Molen, 2015; Pethybridge et al., 2018), which is problematic for highly mobile organisms such as sharks. Furthermore, the carbon isotopic composition of biological tissues is also subject to variations linked to photosynthetic pathways in the trophic baseline (Matich et al., 2021), allometry or tissue-dependent fractionation processes (Angilletta et al., 2004). Alternatively, even if biopurification of trace elements contained in mineralized tissues (e.g., strontium or barium concentrations normalized to calcium) allow to infer trophic levels in terrestrial ecosystems (Balter, 2004; Peek & Clementz, 2012; Tacail et al., 2020), trace element behaviour in marine ecosystems appears to follow different processes that do not reflect trophic ecology (Peek & Clementz, 2012).

Calcium isotopes (expressed below as δ[^44/42]Ca), measured in mineralized tissues (e.g., tooth bioapatite), represent a novel addition to the SIA toolkit for reconstructing trophic organization in marine ecosystems (Akhtar et al., 2020; Clementz et al., 2003; Martin et al., 2015; Skulan & DePaolo, 1999). Among bony and cartilaginous fishes, the circulation of water through the gills is considered to act as a stable buffer of calcium,

with observed variations in δ[^44/42]Ca values reflecting dietary inputs (Clementz et al., 2003; Martin et al., 2015; Tacail et al., 2020). As a general rule, the observed decrease in δ[^44/42]Ca values across the marine food web has been hypothesized to arise from a depletion of heavy calcium ([^44]Ca) in mineralized tissues from the bottom to the top of the food web (Figure 1) (Martin et al., 2015; Skulan & DePaolo, 1999). This is estimated to result from the partition of lighter against heavier isotopes from the feeding bowl (or dietary source) during physiological assimilation of the Ca (e.g., during bone mineralization and/or urine excretion). Current knowledge on the physiological processes behind Ca isotopic fractionation is limited to experiments and box models conducted on mammals (e.g., Morgan et al., 2012; Eisenhauer et al., 2019; Tacail et al., 2020; Hassler et al., 2021), while no experimental studies on elasmobranchs have been published yet. Nevertheless, teeth from elasmobranchs with filter-feeding behaviours have the highest δ[^44/42]Ca values (~-0.1 to -0.3‰) (Akhtar et al., 2020; Martin et al., 2015; Skulan & DePaolo, 1999), whereas top predators ingesting the mineralized tissue of smaller predators (by swallowing other elasmobranchs and/or bony fishes) have the lowest δ[^44/42]Ca values (~-0.7 to -1‰). However, because most sharks are opportunistic predators, feeding differently on both live food and carrion during their life span, the intraspecies range of δ[^44/42]Ca values can be wide (Figure 1), which blurs the delimitation between the groups. Since the publication of Martin et

<figure><img src="image_2.png" /><figcaption>**FIGURE 1** Global trophic level of elasmobranchs species using δ[^44/42]Ca isotopes (see Supporting Information Table S1) from the literature (Akhtar et al., 2020; Martin et al., 2015). Relative size between species and their global shapes are provided. Boxes represent trophic levels based on Martin et al. (2015). Squares, this paper; triangles, data from Martin et al. (2015); circles, data from Akhtar et al. (2020); rhombuses, combined data from Martin et al. (2015) and Akhtar et al. (2020)</figcaption></figure>
al. (2015), a large range of elasmobranchs Ca stable isotopes is now available (Akhtar et al., 2020) to enrich the comprehension of elasmobranch ecology (Figure 1). Here, we can observe that even if the completion of the trophic spectrum has smoothed the distinction between the four groups of consumers based on prey type that are present in the literature (i.e., plankton-feeders, primary, secondary and tertiary consumers), these groups have been recognized through δ⁴⁴/⁴²Ca analyses of elasmobranch bioapatite. Elasmobranchs display a wide range of trophic position at the species level regardless to the order showing that the trophic signal highlighted by calcium SIA does not rely on phylogeny (Figure 1). Moreover, physiological differences between fishes and mammals (e.g., involving tooth replacement, seawater consumption or lactation, respectively) prevent a direct comparison of δ⁴⁴/⁴²Ca values between these two groups and trophic food-web inferences should be discussed considering these physiological differences (Martin et al., 2015). With these assets and limitations in mind, the replacement and loss of elasmobranch teeth represent an opportunity for analysing calcium isotopes, allowing a more comprehensive understanding of shark ecology based on a larger sampling from freshly captured specimens and those from public collections (including fossil material).

As an overview, Figure 1 allows the comparison of the trophic positions of numerous species of elasmobranchs. It shows that the trophic signal retrieved from calcium SIA does not rely on phylogeny, as each elasmobranch order displays a wide range of trophic position at the species level. Well-known plankton feeders such as the Giant manta ray *Mobula birostris* and the Megamouth shark *Megachasma pelagios* are characterized by particularly high δ⁴⁴/⁴²Ca values. It is noticeable that the filter-feeder basking shark (*Cetorhinus maximus*) displays variations in δ⁴⁴/⁴²Ca values that fall in the lower part of the plankton-feeders spectrum. This could be explained by the nonselective consumption of small fish induced by its foraging behaviour, which drives Ca isotopic compositions to more negative values. The tiger shark (*Galeocerdo cuvier*) is known to be a top predator in its environment, displaying a wide range of prey and behaviours from active predation on small fish to mammal scavenging (Simpfendorfer et al., 2001b). The δ⁴⁴/⁴²Ca values presented in the literature for this species are high for an apex predator. The regurgitation of marine mammal bones by tiger sharks after digestion of mammal soft tissues (a common behaviour in sharks; Brunnschweiler et al., 2005; Randall & Cea, 2011) can limit the contribution of mammals to the sum of dietary Ca intakes, which could drive average diet δ⁴⁴/⁴²Ca values closer to the isotopic composition of the fish pole. The opportunistic aspect of its feeding behaviour coupled with a wide range of prey, including mammal flesh, could explain its medium Ca isotope values. A broader analysis of G. cuvier calcium stable isotopes could assess more precisely its feeding behaviour and trophic position. The wide range of trophic position exhibited by the great hammerhead shark (*Sphyrna mokarran*) could highlight shifts in trophic position linked to ontogeny as sharks from the *Sphyrna* genus hunt in shallow waters, which allows access to a large range of prey. Even if adult hammerhead sharks mainly feed on elasmobranchs, this should be supported by low tooth δ⁴⁴/⁴²Ca values. Juveniles seem to incorporate a high percentage of invertebrates (Torres-Rojas et al., 2010), which could significantly drive the Ca isotope values toward δ⁴⁴Ca-depleted values. However,

the lack of size information for literature specimens has precluded most precise inferences on the variations in *Sphyrna mokarran* δ⁴⁴/⁴²Ca values so far. Akhtar et al. (2020) discussed the low Ca isotope values in the bull shark *Carcharhinus leucas* and assigned its high trophic level to an overestimation linked to its incursions in freshwater. This behaviour could generate an environmental bias by inducing a depletion in heavy calcium within the organism caused by the more δ⁴⁴Ca-depleted isotopic compositions of riverine waters passing through the gills (Holmden et al., 2012).

For many shark species, ontogeny may impact the way they behave and feed, influencing the size of prey they can catch and the distribution of habitat they occupy (Ebert, 2002a; Ellis & Musick, 2007; Lucifora et al., 2009; Vögler et al., 2009). This change in predatory behaviour is often marked by an increase in the trophic position as testified by analyses from stomach contents (Vögler et al., 2009). The diets of *Hexanchus griseus*, *Isurus oxyrinchus* and *Carcharodon carcharias* have been studied for years for ecological purposes and for this reason they represent key taxa for isotopic analyses. Their feeding behaviours have been principally estimated on the basis of stomach contents (Cortés, 1999; Ebert, 1994; Simpfendorfer et al., 2001a). Based on this evidence, the shortfin mako shark (*I. oxyrinchus*) is estimated to be a highly piscivorous species from pelagic habitats, preying mainly on teleosts and elasmobranchs (Cliff et al., 1990; Maia et al., 2006; Stevens, 2008). The bluntnose sixgill shark lives in deep waters and displays a feeding behaviour that is known to change with growth (Celona et al., 2005; Ebert, 1994). While small specimens <120 cm of total length (TL) feed mainly on primary consumers like cephalopods and small bony fishes, larger specimens start to incorporate bigger prey like large osteichthyes or other elasmobranchs as well. Specimens over 2 m TL present another shift in diet as they start to incorporate mammals in their diet. The great white shark is known as one of the major apex predators of marine ecosystems. It displays a wide range of prey linked to an opportunistic feeding behaviour. However, prey distribution impacts diet preferences among populations of great white sharks. South African great white sharks prey preferentially on fur seal colonies (Hammerschlag et al., 2006; Martin et al., 2005). However, as the Mediterranean Sea no longer presents pinniped colonies, nowadays Mediterranean great white sharks mainly predate on large bony fishes like tuna, with which it shares migratory behaviours (Kabasakal, 2016).

Here we refine our understanding of Ca isotope variability in elasmobranchs and investigate the relationship between body size, ecology and tooth δ⁴⁴/⁴²Ca values and trace metal concentrations (Sr, Ba and Mg) among three species of large sharks presenting distinctive ecologies and feeding behaviours: the shortfin mako shark *I. oxyrinchus*, the great white shark *C. carcharias* and the bluntnose sixgill shark *H. griseus*.

## 2 | MATERIALS AND METHODS

### 2.1 | Sample collection

Tooth fragments were sampled from 34 complete tooth crowns of 34 small to large individuals belonging to three large shark species:
<table><caption>TABLE 1 Calcium isotope values of teeth coupled with biometric and ecological data from elasmobranchs analysed in this study</caption><thead><tr><th rowspan="2">Collection name</th><th rowspan="2">Lab sample</th><th rowspan="2">Taxon</th><th rowspan="2">Tooth CH (mm) (cm)</th><th rowspan="2">TL (kg)</th><th rowspan="2">Weight (kg)</th><th rowspan="2">Weight (kg) estimated<sup>a</sup></th><th rowspan="2">Sex</th><th rowspan="2">Provenance</th><th rowspan="2">Sample weight (mg)</th><th rowspan="2">Sample d44/42Ca (%)</th><th rowspan="2">Lyon</th><th rowspan="2">2SD</th><th rowspan="2">d43/42 (%)</th><th rowspan="2">ICP Ca Lyon</th><th rowspan="2">2SD Replicates (%)<sup>1915a</sup></th><th rowspan="2">Ca (%)</th><th rowspan="2">P (%)</th><th rowspan="2">Sr (ppm)</th><th rowspan="2">Ba (ppm)</th><th rowspan="2">Mg (ppm)</th><th rowspan="2">Sr/Ca (mMol/mol)</th><th rowspan="2">Ba/Ca (mMol/mol)</th><th rowspan="2">Mg/Ca (mMol/mol)</th><th rowspan="2">References</th></tr><tr><th>TL (kg)</th><th>Weight (kg)</th><th>Weight (kg) estimated<sup>a</sup></th><th>Sex</th><th>Provenance</th><th>Sample weight (mg)</th><th>Sample d44/42Ca (%)</th><th>Lyon</th><th>2SD</th><th>d43/42 (%)</th><th>ICP Ca Lyon</th><th>2SD Replicates (%)<sup>1915a</sup></th><th>Ca (%)</th><th>P (%)</th><th>Sr (ppm)</th><th>Ba (ppm)</th><th>Mg (ppm)</th><th>Sr/Ca (mMol/mol)</th><th>Ba/Ca (mMol/mol)</th><th>Mg/Ca (mMol/mol)</th><th>References</th></tr></thead><tbody><tr><td>n°1 Kabasakal, & Gredikoglu, S.O., 2008</td><td>aa43</td><td>Carcharodon carcharias</td><td>-</td><td>125</td><td>-</td><td>21.66</td><td>M</td><td>MED</td><td>1.09</td><td>-0.82</td><td>0.17</td><td>-0.39</td><td>0.07</td><td>4</td><td>-0.3</td><td>18.0</td><td>7.8</td><td>1392.4</td><td>169.8</td><td>623.1</td><td>3.55</td><td>0.276</td><td>5.720</td><td>This study</td></tr><tr><td>ERB0932</td><td>aa46</td><td>Carcharodon carcharias</td><td>-</td><td>212</td><td>55</td><td>108.48</td><td>F</td><td>SW IND</td><td>0.97</td><td>-0.51</td><td>0.05</td><td>-0.28</td><td>0.08</td><td>4</td><td>0.01</td><td>14.9</td><td>6.7</td><td>615.0</td><td>26.6</td><td>468.2</td><td>1.89</td><td>0.052</td><td>5.176</td><td>This study</td></tr><tr><td>ERB0495</td><td>aa44</td><td>Carcharodon carcharias</td><td>-</td><td>266</td><td>133</td><td>216.72</td><td>M</td><td>SW IND</td><td>1.19</td><td>-0.54</td><td>0.12</td><td>-0.27</td><td>0.09</td><td>3</td><td>-0.02</td><td>14.5</td><td>7.6</td><td>638.4</td><td>1.6</td><td>292.9</td><td>2.02</td><td>0.003</td><td>3.338</td><td>This study</td></tr><tr><td>ERB0496</td><td>aa45</td><td>Carcharodon carcharias</td><td>-</td><td>343.8</td><td>280</td><td>473.97</td><td>F</td><td>SW IND</td><td>1.27</td><td>-0.55</td><td>0.11</td><td>-0.26</td><td>0.05</td><td>3</td><td>-0.03</td><td>14.3</td><td>7.5</td><td>713.5</td><td>1.0</td><td>757.6</td><td>2.29</td><td>0.002</td><td>8.746</td><td>This study</td></tr><tr><td>REC 0610 M</td><td>-</td><td>Carcharodon carcharias</td><td>-</td><td>300</td><td>-</td><td>312.78</td><td>-</td><td>MED</td><td>-</td><td>-0.74</td><td>0.07</td><td>-0.38</td><td>0.08</td><td>3</td><td>-0.22</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>REC 0812 M</td><td>-</td><td>Carcharodon carcharias</td><td>-</td><td>400</td><td>-</td><td>752.14</td><td>-</td><td>MED</td><td>-</td><td>-0.78</td><td>0.12</td><td>-0.45</td><td>0.06</td><td>4</td><td>-0.26</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>REC567M</td><td>aa58</td><td>Isurus oxyrinchus</td><td>9.38</td><td>109.9<sup>a</sup></td><td>-</td><td>9.63</td><td>-</td><td>MED</td><td>0.89</td><td>-0.57</td><td>0.1</td><td>-0.29</td><td>0.04</td><td>4</td><td>-0.05</td><td>24.1</td><td>12.6</td><td>984.9</td><td>-</td><td>831.7</td><td>1.87</td><td>-</td><td>5.686</td><td>This study</td></tr><tr><td>REC564M</td><td>aa51</td><td>Isurus oxyrinchus</td><td>10.51</td><td>121.2<sup>a</sup></td><td>-</td><td>13.22</td><td>-</td><td>MED</td><td>1.19</td><td>-0.55</td><td>0.15</td><td>-0.28</td><td>0.09</td><td>4</td><td>-0.03</td><td>15.4</td><td>8.2</td><td>728.1</td><td>0.6</td><td>1329.3</td><td>2.16</td><td>0.001</td><td>14.196</td><td>This study</td></tr><tr><td>REC182M</td><td>aa53</td><td>Isurus oxyrinchus</td><td>16.85</td><td>184.5<sup>a</sup></td><td>-</td><td>47.06</td><td>-</td><td>MED</td><td>1.06</td><td>-0.58</td><td>0.13</td><td>-0.30</td><td>0.07</td><td>4</td><td>-0.06</td><td>17.4</td><td>9.1</td><td>621.7</td><td>0.7</td><td>516.6</td><td>1.64</td><td>0.001</td><td>4.904</td><td>This study</td></tr><tr><td>REC565M</td><td>aa54</td><td>Isurus oxyrinchus</td><td>19.04</td><td>206.4<sup>a</sup></td><td>-</td><td>66.26</td><td>-</td><td>MED</td><td>0.94</td><td>-0.7</td><td>0.13</td><td>-0.32</td><td>0.08</td><td>3</td><td>-0.18</td><td>18.9</td><td>9.8</td><td>744.5</td><td>8.3</td><td>575.2</td><td>1.80</td><td>0.013</td><td>5.019</td><td>This study</td></tr><tr><td>REC563M</td><td>aa59</td><td>Isurus oxyrinchus</td><td>20.74</td><td>223.3<sup>a</sup></td><td>-</td><td>84.26</td><td>-</td><td>MED</td><td>1.25</td><td>-0.77</td><td>0.07</td><td>-0.37</td><td>0.04</td><td>4</td><td>-0.25</td><td>18.1</td><td>9.3</td><td>709.2</td><td>2.0</td><td>461.5</td><td>1.79</td><td>0.003</td><td>4.202</td><td>This study</td></tr><tr><td>REC10-11 M</td><td>aa56</td><td>Isurus oxyrinchus</td><td>21.69</td><td>232.8<sup>a</sup></td><td>-</td><td>93.75</td><td>-</td><td>MED</td><td>0.92</td><td>-0.64</td><td>0.08</td><td>-0.34</td><td>0.01</td><td>3</td><td>-0.12</td><td>17.6</td><td>9.2</td><td>817.3</td><td>-</td><td>501.7</td><td>2.13</td><td>-</td><td>4.705</td><td>This study</td></tr><tr><td>REC160M</td><td>aa50</td><td>Isurus oxyrinchus</td><td>24.25</td><td>258.4<sup>a</sup></td><td>-</td><td>131.05</td><td>-</td><td>MED</td><td>1.15</td><td>-0.68</td><td>0.07</td><td>-0.34</td><td>0.06</td><td>3</td><td>-0.16</td><td>17.0</td><td>8.5</td><td>1333.4</td><td>6.8</td><td>599.4</td><td>3.59</td><td>0.012</td><td>5.821</td><td>This study</td></tr><tr><td>REC1330M</td><td>aa52</td><td>Isurus oxyrinchus</td><td>25.6</td><td>271.9<sup>a</sup></td><td>-</td><td>153.81</td><td>-</td><td>MED</td><td>1.03</td><td>-0.75</td><td>0.13</td><td>-0.37</td><td>0.08</td><td>4</td><td>-0.23</td><td>14.3</td><td>7.6</td><td>648.6</td><td>0.3</td><td>878.3</td><td>2.08</td><td>0.001</td><td>10.153</td><td>This study</td></tr><tr><td>REC566M</td><td>aa57</td><td>Isurus oxyrinchus</td><td>26.75</td><td>283.4<sup>a</sup></td><td>-</td><td>173.44</td><td>-</td><td>MED</td><td>0.91</td><td>-0.65</td><td>0.06</td><td>-0.34</td><td>0.08</td><td>4</td><td>-0.13</td><td>13.0</td><td>7.6</td><td>674.4</td><td>1.4</td><td>3595.3</td><td>2.37</td><td>0.003</td><td>45.510</td><td>This study</td></tr><tr><td>REC381M</td><td>aa55</td><td>Isurus oxyrinchus</td><td>29.4</td><td>309.8<sup>a</sup></td><td>-</td><td>228.59</td><td>-</td><td>MED</td><td>1.2</td><td>-0.68</td><td>0.09</td><td>-0.36</td><td>0.03</td><td>4</td><td>-0.16</td><td>17.6</td><td>9.1</td><td>919.4</td><td>2.7</td><td>515.4</td><td>2.39</td><td>0.004</td><td>4.825</td><td>This study</td></tr><tr><td>REC201 M</td><td>201 M</td><td>Hexanchus griseus</td><td>-</td><td>58</td><td>-</td><td>1.03</td><td>-</td><td>MED</td><td>2.89</td><td>-0.50</td><td>0.15</td><td>-0.22</td><td>0.08</td><td>3</td><td>0.02</td><td>13.5</td><td>8.0</td><td>945.0</td><td>1.0</td><td>1865.0</td><td>3.21</td><td>0.002</td><td>22.840</td><td>This study</td></tr><tr><td>REC201 M</td><td>201 Mb</td><td>Hexanchus griseus</td><td>-</td><td>58</td><td>-</td><td>1.03</td><td>-</td><td>MED</td><td>1.27</td><td>-0.54</td><td>0.06</td><td>-0.25</td><td>0.09</td><td>3</td><td>-0.02</td><td>23.0</td><td>13.7</td><td>3593.0</td><td>1286.0</td><td>4549.0</td><td>7.16</td><td>1.635</td><td>32.680</td><td>This study</td></tr><tr><td>REC201 M</td><td>-</td><td>Hexanchus griseus</td><td>-</td><td>58</td><td>-</td><td>1.03</td><td>-</td><td>MED</td><td>-</td><td>-0.66</td><td>0.06</td><td>-0.37</td><td>0.08</td><td>3</td><td>-0.14</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>REC172M</td><td>172</td><td>Hexanchus griseus</td><td>-</td><td>72</td><td>-</td><td>2.03</td><td>-</td><td>MED</td><td>0.68</td><td>-0.62</td><td>0.10</td><td>-0.32</td><td>0.10</td><td>4</td><td>-0.10</td><td>24.0</td><td>13.0</td><td>1180.0</td><td>3.0</td><td>2191.0</td><td>2.25</td><td>0.0037</td><td>15.080</td><td>This study</td></tr><tr><td>REC173M</td><td>173</td><td>Hexanchus griseus</td><td>-</td><td>75</td><td>-</td><td>2.30</td><td>F</td><td>MED</td><td>0.69</td><td>-0.57</td><td>0.03</td><td>-0.27</td><td>0.05</td><td>3</td><td>-0.05</td><td>8.9</td><td>5.2</td><td>1091.0</td><td>3.0</td><td>2068.0</td><td>5.59</td><td>0.0098</td><td>38.170</td><td>This study</td></tr><tr><td>REC174M</td><td>174</td><td>Hexanchus griseus</td><td>-</td><td>80</td><td>-</td><td>2.81</td><td>M</td><td>MED</td><td>1.56</td><td>-0.54</td><td>0.18</td><td>-0.27</td><td>0.10</td><td>3</td><td>-0.02</td><td>22.7</td><td>13.0</td><td>1290.0</td><td>4.0</td><td>2995.0</td><td>2.60</td><td>0.0051</td><td>21.780</td><td>This study</td></tr></tbody></table>
<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Collection name</th><th>Lab sample</th><th>Taxon</th><th>Tooth CH (mm) (cm)</th><th>TL</th><th>Weight (kg)</th><th>Weight (kg) estimated<sup>a</sup></th><th>Sex</th><th>Provenance</th><th>Sample weight (mg)</th><th>d44/42Ca (%)</th><th>ICP</th><th>2SD</th><th>d43/42 (%)</th><th>d44/42Ca</th><th>Ca (%)</th><th>P (%)</th><th>Sr (ppm)</th><th>Ba (ppm)</th><th>Mg (ppm)</th><th>Sr/Ca (mMol/mol)</th><th>Ba/Ca (mMol/mol)</th><th>Mg/Ca (mMol/mol)</th><th>References</th></tr></thead><tbody><tr><td>REC166M</td><td>166</td><td>Hexanchus griseus</td><td>-</td><td>117</td><td>-</td><td>9.17</td><td>F</td><td>MED</td><td>0.72</td><td>-0.58</td><td>0.02</td><td>-0.28</td><td>0.01</td><td>3</td><td>-0.06</td><td>26.0</td><td>14.7</td><td>3475.0</td><td>5.0</td><td>3081.0</td><td>6.10</td><td>0.0056</td><td>19.510</td><td>This study</td></tr><tr><td>REC171M</td><td>171</td><td>Hexanchus griseus</td><td>-</td><td>120</td><td>-</td><td>9.92</td><td>M</td><td>MED</td><td>1.55</td><td>-0.52</td><td>0.09</td><td>-0.21</td><td>0.11</td><td>3</td><td>0.00</td><td>34.1</td><td>19.0</td><td>2120.0</td><td>3.0</td><td>2743.0</td><td>2.84</td><td>0.0026</td><td>13.260</td><td>This study</td></tr><tr><td>REC1236</td><td>1236</td><td>Hexanchus griseus</td><td>-</td><td>126</td><td>-</td><td>11.54</td><td>-</td><td>MED</td><td>1.43</td><td>-0.56</td><td>0.10</td><td>-0.26</td><td>0.08</td><td>3</td><td>-0.04</td><td>21.6</td><td>12.1</td><td>1278.0</td><td>4.0</td><td>2353.0</td><td>2.71</td><td>0.0054</td><td>17.960</td><td>This study</td></tr><tr><td>REC164M</td><td>164</td><td>Hexanchus griseus</td><td>-</td><td>160</td><td>-</td><td>24.27</td><td>M</td><td>MED</td><td>1.64</td><td>-0.54</td><td>0.04</td><td>-0.26</td><td>0.06</td><td>3</td><td>-0.02</td><td>24.9</td><td>13.9</td><td>1386.0</td><td>2.0</td><td>1885.0</td><td>2.55</td><td>0.0023</td><td>12.490</td><td>This study</td></tr><tr><td>REC1478</td><td>1478</td><td>Hexanchus griseus</td><td>-</td><td>190</td><td>-</td><td>41.41</td><td>F</td><td>MED</td><td>2.47</td><td>-0.56</td><td>0.07</td><td>-0.26</td><td>0.18</td><td>3</td><td>-0.04</td><td>11.4</td><td>6.6</td><td>1417.0</td><td>3.0</td><td>1206.0</td><td>5.69</td><td>0.0077</td><td>17.470</td><td>This study</td></tr><tr><td>REC163M</td><td>163</td><td>Hexanchus griseus</td><td>-</td><td>191</td><td>-</td><td>42.09</td><td>F</td><td>MED</td><td>1.22</td><td>-0.62</td><td>0.07</td><td>-0.30</td><td>0.06</td><td>3</td><td>-0.10</td><td>17.4</td><td>10.0</td><td>2372.0</td><td>2.0</td><td>4222.0</td><td>6.22</td><td>0.0033</td><td>39.940</td><td>This study</td></tr><tr><td>REC175M</td><td>175</td><td>Hexanchus griseus</td><td>-</td><td>195</td><td>-</td><td>44.90</td><td>M</td><td>NE ATL</td><td>0.69</td><td>-0.60</td><td>0.03</td><td>-0.29</td><td>0.04</td><td>2</td><td>-0.08</td><td>26.8</td><td>14.6</td><td>1572.0</td><td>2.0</td><td>1588.0</td><td>2.68</td><td>0.0022</td><td>9.760</td><td>This study</td></tr><tr><td>REC203M</td><td>-</td><td>Hexanchus griseus</td><td>-</td><td>200</td><td>-</td><td>48.57</td><td>-</td><td>MED</td><td>-</td><td>-0.68</td><td>0.13</td><td>-0.33</td><td>0.11</td><td>3</td><td>-0.16</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Martin et al., 2015</td></tr><tr><td>REC162M</td><td>162</td><td>Hexanchus griseus</td><td>-</td><td>228</td><td>-</td><td>73.01</td><td>M</td><td>MED</td><td>1.27</td><td>-0.50</td><td>0.04</td><td>-0.23</td><td>0.04</td><td>3</td><td>0.02</td><td>23.4</td><td>13.0</td><td>1368.0</td><td>2.0</td><td>1975.0</td><td>2.68</td><td>0.0025</td><td>13.930</td><td>This study</td></tr><tr><td>REC1332M</td><td>1332</td><td>Hexanchus griseus</td><td>-</td><td>300</td><td>-</td><td>171.41</td><td>M</td><td>MED</td><td>2.61</td><td>-0.60</td><td>0.05</td><td>-0.28</td><td>0.06</td><td>2</td><td>-0.08</td><td>26.9</td><td>14.9</td><td>1602.0</td><td>3.0</td><td>1443.0</td><td>2.72</td><td>0.0033</td><td>8.840</td><td>This study</td></tr><tr><td>REC204M</td><td>204</td><td>Hexanchus griseus</td><td>-</td><td>320</td><td>-</td><td>209.52</td><td>-</td><td>MED</td><td>2.87</td><td>-0.42</td><td>0.13</td><td>-0.16</td><td>0.12</td><td>2</td><td>0.10</td><td>17.9</td><td>10.2</td><td>1091.0</td><td>6.0</td><td>1451.0</td><td>2.79</td><td>0.0098</td><td>13.370</td><td>This study</td></tr><tr><td>REC758M</td><td>758</td><td>Hexanchus griseus</td><td>-</td><td>463</td><td>-</td><td>660.93</td><td>F</td><td>NE ATL</td><td>1.85</td><td>-0.59</td><td>0.04</td><td>-0.29</td><td>0.04</td><td>3</td><td>-0.07</td><td>25.9</td><td>14.1</td><td>3404.0</td><td>8.0</td><td>1906.0</td><td>6.02</td><td>0.009</td><td>12.160</td><td>This study</td></tr><tr><td>-</td><td>SRM1486</td><td>Standard</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-1.01</td><td>0.11</td><td>-0.49</td><td>0.12</td><td>28</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>This study</td></tr><tr><td>-</td><td>IAPSO</td><td>Standard</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>0.37</td><td>0.13</td><td>0.25</td><td>0.04</td><td>6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>This study</td></tr></tbody></table>

Abbreviations: M, male; F, female.

aTotal length estimation following Shimada (2002).

bWeight estimation following Froese et al. (2014).
18 teeth/18 individuals were collected for *H. griseus* (bluntnose sixgill shark), 10/10 for *I. oxyrinchus* (shortfin mako shark) and 6/6 for *C. carcharias* (great white shark). Specimens were collected within the collections of the University of Montpellier, the University of Lyon, Elasmobranch Research Belgium (F.H.M., personal collection) and the Hakan Kabasakal collection. Among *C. carcharias* specimens, three specimens came from the Mediterranean Sea (two from Lion Gulf, southern France; one from Turkey; Kabasakal, & Gedikoğlu, 2008) and three from the Indian Ocean (South Africa). Two specimens of *H. griseus* came from the Atlantic Ocean (western France) while 16 specimens came from the Mediterranean Sea (Lion Gulf, southern France). *I. oxyrinchus* material consisted of 10 lower lateral teeth belonging to the historical collections of the University of Montpellier and fished from the Mediterranean Sea, although no precise geographical information of which area was available. The total length of every specimen of *H. griseus* and *C. carcharias* was measured on the fresh body. Sizes of *I. oxyrinchus* specimens were estimated using crown-high (CH)-based regression [TL = 16.205 + 9.987CH (anterior); Shimada, 2002]. Except for the three South African *C. carcharias* for which the specimen's body mass was known, the weight of other individuals was estimated using Bayesian approach (Froese *et al.*, 2014) for estimating length-weight relationships available for each species in FishBase (Froese & Pauly, 2000): *Isurus*: $W = TL^{3.03} \times 0.00646 \times 10^{-3}$; *Carcharodon*: $W = TL^{3.05} \times 0.00871 \times 10^{-3}$; *Hexanchus*: $W = TL^{3.11} \times 0.00339 \times 10^{-3} (R^2 > 0.5)$.

Enameloid fragments weighing about 1–3 mg were collected using a sharp blade under a binocular microscope. Dentine was removed manually to keep only enameloid, as calcium incorporation is known to differ between these tissues (Akhtar *et al.*, 2020; Martin *et al.*, 2015). Sample information is recorded in Table 1. Specimens from Akhtar *et al.* (2020) and Martin *et al.* (2015) were used to perform a global estimation of the trophic position of elasmobranch species (Figure 1) and data from Akhtar *et al.* (2020) were converted from 915a to ICP Lyon as ($X_{ICP_Lyon} = X_{915a} - 0.52$).

## 2.2 | Chemical analyses

Chemical purifications and analyses were conducted on the facilities at Laboratoire de Géologie de Lyon, France. Each sample was dissolved in 1 ml of concentrated HNO₃ and heated until complete dissolution and then evaporated.

All samples were retaken in 1 ml of 0.05 N HNO₃ for concentration analyses for Ca, P, Sr, Ba and Mg, which were measured from one aliquot of 500 µl for each sample. The concentration analysis was conducted on an iCAP Pro ICP-AES inductively coupled plasma atomic emission spectrometer (ICPAES; Thermo Fischer). Blanks were regularly monitored and bone powder standard NIST SRM 1486 bone meal was treated as a sample and measured three times, yielding an average value within one standard deviation of 247 ± 1.3 ppm of Sr, 30 ± 0.3% of Ca, 14.9 ± 0.4% of P and 4987 ± 178 ppm of Mg in agreement with the certified value.

Calcium purification followed Tacail *et al.* (2014). Two steps of elution were performed using nitric and hydrochloric acids. The first elution was performed through a cationic resin AG50W-X12 to remove matrix elements that could interfere with the calcium isotopic measurements. The second elution step was performed using a Sr-specific resin (Sr-spec Eichrom) that allowed strontium to be separated from calcium. Procedural blanks (0.1 µg) and standard samples with known concentrations of calcium were added to the analyses to assess the congruence of the results.

Ca isotopes from the purified fraction of calcium were measured using a Thermo Neptune Plus Multiple Collector-Inductively Coupled Plasma- Mass Spectrometre (MC-ICP-MS), Laboratoire de Géologie de Lyon equipped with an Aridus for sample introduction (see Tacail *et al.*, 2014 for details). All samples were diluted in 0.05 M HNO₃ before uptake to a concentration of 1.5 ppm. Measurements were conducted at medium or high resolution, with each analysis consisting of 40 measurements of 4.2 s integrations on ⁴²Ca⁺, ⁴³Ca⁺ and ⁴⁴Ca⁺ ions in static mode. Signals at 43.5 m/z, corresponding to ⁸⁷Sr²⁺, were measured for correction of Sr double charge interferences on Ca isotopes (⁸⁸Sr²⁺ on ⁴⁴Ca⁺, ⁸⁶Sr²⁺ on ⁴³Ca⁺ and ⁸⁴Sr²⁺ on ⁴²Ca⁺). Each analysis was preceded by a washout pumping in 0.5 M HNO₃. A second washout pumping was carried out in 0.05 M HNO₃ and measured as the blank. Delta values were obtained using the standard bracketing method with the Lyon ICP Ca standard, referred to as ICP Ca Lyon (Tacail *et al.*, 2014), and issued from a Specpure calcium plasma standard solution (Alfa Aesar). Standard reference material (SRM) 1486 and International association for the physical sciences of the ocean (IAPSO, Ocean scientific international ltd, OSIL) solutions were used as secondary standards. For each analytical session of samples, SRM 1486 was measured in turn. This sequence was repeated at least twice to check that a given sample could be replicated.

The analysis of the blanks revealed that they present about 150 ng of Ca which is far less than the less concentrate samples that display values around 200 µg of Ca. This difference in the concentration of samples versus blanks allows us to consider that Ca pollution in the blanks is negligible. The standard values are $\delta^{44/42}\text{Ca}_{\text{ICP-Ca}}_{\text{Lyon}} = -1.01$ with a two times standard deviation (2SD) of $\pm 0.11\%$ ($n = 28$) for SRM1486 and $\delta^{44/42}\text{Ca}_{\text{ICP-Ca}}_{\text{Lyon}} = +0.37 \pm 0.14\%$ (2sd, $n = 6$) for IAPSO seawater. These values are congruent with the standard values given by Tacail *et al.* (2014) and Martin *et al.* (2015). The calculated linear regression for mass-dependent fractionation is given in Supporting Information Figure S1. The regression slope is 0.5464, which falls in the range of general agreement with the 0.5067 slope predicted by the linear approximation of exponential mass-dependent fractionation.

# 3 | RESULTS

## 3.1 | Ca isotope composition of elasmobranch enameloid

Size and weight in the three species of elasmobranches covariate in the same proportions (Kendall test: $T = 0.925$, $z = 7.774$, $P = 7.55 \times 10^{-15}$).
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Trends of δ<sup>44/42</sup>Ca values with size (total length [TL]) and ecology among three species of large elasmobranchs. Blue, *Carcharodon carcharias*; MED, Mediterranean Sea; SAF, South Africa; orange, *Hexanchus griseus*; triangles, Mediterranean; polygon, Atlantic; green, *Isurus oxyrinchus*. Vertical traits display two times standard deviation (2SD). Comparative species (black points) are derived from Martin *et al.* (2015). Boxes represent trophic levels based on Martin *et al.* (2015)</figcaption></figure>

As body length and weight evolve the same way, δ<sup>44/42</sup>Ca values and trace element concentrations will be expressed below against body length. Here we express the δ<sup>44/42</sup>Ca values relative to the ICP-Ca Lyon standard (Martin *et al.*, 2015, 2017) and all values are converted relative to SRM915a (Table 1). The three species analysed in this paper present δ<sup>44/42</sup>Ca values ranging from -0.42 to -0.84‰ (Figure 2).

Specimens belonging to the shortfin mako shark *I. oxyrinchus* displayed a decrease in δ<sup>44/42</sup>Ca values from -0.54 to -0.77‰ coinciding with an increase in body length and mass in this species. Specimens with total length between 109 and 200 cm presented a median δ<sup>44/42</sup>Ca value of -0.57 ± 0.04‰ (2sd, *n* = 3) while specimens over 200 cm presented a median value of -0.72 ± 0.1‰ (2sd, *n* = 7).

In the Mediterranean bluntnose sixgill shark *H. griseus*, we observed variation ranging from -0.34 to -0.68 ‰ in relation to total body length (TL) (Figure 2). The smallest *Hexanchus* specimens (<1.2 m TL) presented a median δ<sup>44/42</sup>Ca value of -0.54 ± 0.11‰ (2sd, *n* = 8). The specimens between 1.2 and 2 m TL presented a median δ<sup>44/42</sup>Ca value of -0.56 ± 0.07‰ (2sd, *n* = 5) while specimens over 2 m TL displayed a median δ<sup>44/42</sup>Ca value of -0.50 ± 0.10‰ (2sd,

*n* = 3). The two specimens from the Atlantic Ocean that measured 1.95 and 4.63 m presented values of about -0.60‰.

The great white shark *C. carcharias* δ<sup>44/42</sup>Ca values presented large differences between specimens from the Mediterranean Sea and specimens from the Indian Ocean. For similar length, oceanic-dwelling specimens presented values that were 0.20-0.30‰ lower than Mediterranean specimens. All the South African specimens presented very high values, ranging from -0.51 to -0.55‰ ± 0.03 (2sd, *n* = 3), compared to the Mediterranean specimen values, which ranged from -0.74 to -0.82‰ ± 0.09 (2sd, *n* = 3).

**3.2 | Alkaline earth metal concentrations in elasmobranch enameloid**

Measurements of Sr/Ca, Mg/Ca and Ba/Ca elemental ratios in our analysis present fluctuations between and among the species (*n* = 28). The graphs of co-evolution of Sr/Ca, Mg/Ca and Ba/Ca are given in Supporting Information Figure S2. The Sr/Ca ratio appears to be correlated to Mg/Ca
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Evolution of Sr/Ca (mMol.Mol⁻¹) with size (total length [TL]) among three analysed species of large elasmobranchs (blue, *Carcharodon carcharias*; orange, *Hexanchus griseus*; green, *Isurus oxyrinchus*) with a focus on sexual segregation of *H. griseus* specimens (triangle, Mediterranean specimens; hourglass, Atlantic specimens). The right grey boxplot represents a literature compilation of 59 other sharks (e.g., *Carcharhinus*, *Galeocerdo*, *Sphyrna*, *Lamna*, *Carcharias*) and 49 individuals of *Carcharhinus leucas* (encountered at the Fiji Shark Reef Marine Reserve) for which Sr/Ca ratios from teeth enameloid are available in the literatur (Akhtar et al., 2020 and Kocsis et al., 2015, respectively. Data from the literature are presented in Supporting Information Table S1). Blue banner, global open-ocean seawater Sr/Ca range; light blue, global shelves neritic and river-influenced areas Sr/Ca range, according to Lebrato et al. (2020)</figcaption></figure>

(Kendall T = 0.46, P value = 0.0002) and slightly correlated with Ba/Ca (T = 0.31, P value = 0.019). However, Mg/Ca and Ba/Ca appear not to be correlated (T = 0.04, P value = 0.79). These variations are not related to the size of the specimen or to its diet or location, as Mediterranean specimens and Atlantic ones display similar values for these ratios (Supporting Information Table S1). Strontium and magnesium concentrations appear to be higher in *H. griseus* specimens than in *C. carcharias* and *I. oxyrinchus*. We also observed significant differences among Sr/Ca ratios between males (n = 6) and females (n = 5) in *H. griseus* (Wilcoxon t = -25.116, P value = 2.16 x 10⁻⁰⁶) (Figure 3). However, these differences are not clearly observed in Mg/Ca and Ba/Ca ratios. The Sr/Ca ratios displayed by females of *H. griseus* appear to be dramatically higher than those of every other elasmobranchs analysed. The Sr/Ca values measured in *H. griseus* females are about twice those of the *H. griseus* males as well as all the

specimens of *I. oxyrinchus* and *C. carcharias* as well as *Carcharhinus leucas* from Kocsis et al., 2015 (n = 49) and several elasmobranch genera (n = 59, e.g., *Carcharhinus*, *Galeocerdo*, *Sphyrna*, *Lamna*, *Carcharias*, *Hemipristis*, *Negaprion*, *Prionace*, *Somniosus*) from Akhtar et al., 2020 (Figure 3 and Supporting Information Table S1).

## 4 | DISCUSSION

### 4.1 | Intraspecific variation of calcium isotopes in three elasmobranchs

The three species of large sharks *H. griseus*, *I. oxyrinchus* and *C. carcharias* are very elusive and threatened in the oceans. The development of SIA based on nontraditional elements could bring new
<figure><img src="image_6.png" /></figure>

clues to the understanding of their ecology. Even if the statistical power of such a constrained assemblage of specimens remains low, the δ<sup>44/42</sup>Ca analyses permit to underline the contribution of calcium isotopes to diet reconstruction of elasmobranchs. Contrary to carbon and nitrogen isotopes, the calcium isotopic composition of seawater is homogeneous, which allows potential geographical bias to be avoided (Fantle & Tipper, 2014; Gussone et al., 2020). Moreover, Ca isotopic fractionation in elasmobranch bioapatite is largely controlled by dietary input (Akhtar et al., 2020; Martin et al., 2015).

As expected from stomach contents, the shortfin mako (l. oxyrinchus) δ<sup>44/42</sup>Ca values tend to decrease when body length increases, inducing a rise in trophic level with growth (Figure 2). The adult shortfin mako sharks are known to feed on cephalopods and pelagic bony fish populations (Maia et al., 2006; Preti et al., 2012; Stillwell & Kohler, 1982). The δ<sup>44/42</sup>Ca values for this species (≈ −0.55 to −0.75‰) match values for secondary to tertiary consumers (Martin et al., 2015), which is congruent with their stomach content (Stillwell & Kohler, 1982). The decreasing δ<sup>44/42</sup>Ca values with ontogeny present fluctuations but follow a progressive increase in the trophic chain for the biggest specimens. This could be explained by the ability to catch bigger fishes when mako sharks increase in size (Lowe et al., 1996).

The food consumption of bluntnose sixgill sharks (*H. griseus*) dur-
ing their life span is well known, at least for some populations
(Andrews et al., 2009; Ebert, 1994). Stomach contents are known to
change from cephalopods and bony fishes for the smallest individuals
to a majority of bony fishes and elasmobranchs for 2 m long speci-
mens, while the largest specimens tend to feed mainly on large marine
mammals. This supposes a size dependence of the eaten prey related
to the size of the shark, but also an effect of the available feeding
resources and thus spatially related diet. According to carbon and
nitrogen isotope-based analyses on a population of subadult sixgill
sharks, this species was interpreted as a generalist feeder with very
small differences between individuals (Reum et al., 2020), which con-
trasts with stomach contents that suggest a size-based diet
(Ebert, 1994). The calcium isotope values reported here for the sixgill
sharks indicate fluctuations that could be linked with the progressive
incorporation of new prey items in its diet during growth (Figure 2).
As expected, specimens under 2 m TL present δ<sup>44/42</sup>Ca values
(−0.56‰) that correspond to a primary to secondary piscivorous con-
sumer. This is consistent with the ingestion of cephalopods, bony and
cartilaginous prey documented from stomach contents. Such diversity
of prey could explain the fluctuations of δ<sup>44/42</sup>Ca values observed
among these specimens. The largest specimens of sixgill sharks
(TL >2 m) present slightly higher δ<sup>44/42</sup>Ca values (−0.50‰), indicating
a similar to lower trophic level than younger individuals, which is sur-
prising regarding their size and their expected rise in trophic position
with growth. For these large individuals, strong fluctuations in
δ<sup>44/42</sup>Ca values are also reported (from −0.42 to −0.68‰). In this size
range, sixgill sharks are known to incorporate a larger percentage of
marine mammals in their diet. We hypothesize that the relatively high
δ<sup>44/42</sup>Ca values (relevant to their size and stomach contents) associ-
ated with fluctuations of these values in such large specimens could

be linked to the scavenging habits of some specimens as well as the
regurgitation of indigestible parts like bone, a common behaviour in
sharks (Brunnsschweiler et al., 2005; Randall & Cea, 2011). Because
they do not ingest mammal bones that are ⁴⁴Ca depleted compared to
soft tissues, their enamel δ⁴⁴/⁴²Ca values tend to be higher. Alterna-
tively, a mammal-based diet associated with bone regurgitation could
decrease Ca dietary intakes overall when compared to a fish-based diet,
the latter involving the digestion of scales and other mineralized tissues.
This could lead to proportionally increased Ca body intakes from ⁴⁴Ca-
enriched sea water, resulting in higher shark enamel δ⁴⁴/⁴²Ca values.
Supporting these two nonexclusive hypotheses, a large part of the
material reported from the large sixgill shark's stomach from the Medi-
terranean Sea consisted of dolphins (Kabasakal, 2006) and an unidenti-
fiable mishmash of digested tissues (Celona et al., 2005), which could
coincide with the ingestion of marine mammal soft tissues only in large
individuals of *H. griseus*.

The analysis of the great white shark *C. carcharias* specimens
shows that they present two very distinct patterns regarding the
localities they come from rather than variations linked to ontogeny.
As *C. carcharias* are known to dominate the marine trophic chain, it is
not surprising that they display a strong variability in the type of prey
they feed on, incorporating all kinds, from cephalopods to whales
(Grainger *et al.*, 2020; Tricas & McCosker, 1984). However, we
observed that δ<sup>44/42</sup>Ca values were different when considering their
geographic position. Individuals from the western Mediterranean Sea
displayed the lowest δ<sup>44/42</sup>Ca median value of the analysis (−0.78‰,
n = 3), placing them in the tertiary piscivorous group (Figure 2). In
contrast, specimens of the same species from the Indian Ocean
(caught off South Africa) displayed particularly high δ<sup>44/42</sup>Ca values
(−0.53‰, n = 3) (Figure 2). This indicates the existence of notewor-
thy differences in calcium sources for both these populations that
could reflect differences in feeding behaviour between South African
and Mediterranean populations of great white sharks that seem inde-
pendent of their body sizes (Figure 2). Changes in feeding behaviour
could rely on the variations of prey availability between both environ-
ments and/or population prey preferences. The very low δ<sup>44/42</sup>Ca
values displayed by Mediterranean specimens correspond to a high
trophic level that could be linked to a preferential predation on bony
fishes and other large elasmobranchs. This is supported by the occur-
rences and migration synchronicity of great white sharks in the Medi-
terranean area with large populations of tunas that they preferentially
predate (De Maddalena & Heim, 2012; Galaz & De Maddalena, 2004;
Kabasakal, 2016).

Comparatively, the higher δ<sup>44/42</sup>Ca values measured in all South African great white shark individuals coincide with large *H. griseus* isotopic values and could be interpreted as a prevailing consumption of mammals. Large South African great white sharks are known to predate mainly on colonial mammals like fur seals (Martin *et al.*, 2005). As for sixgill sharks, the increased mammal consumption (active predation or scavenging) (Long & Jones, 1996; Dudley *et al.*, 2000; Fallows *et al.*, 2013) could drive their δ<sup>44/42</sup>Ca values toward ⁴⁴Ca-enriched values compared to Mediterranean populations. Moreover, great white sharks are known to regurgitate the bones and
indigestible parts after eating mammals (Martin *et al.*, 2005), which could result in more ⁴⁴Ca-enriched Ca intakes. These results are congruent with the δ⁴⁴/⁴²Ca values (−0.43‰, 2sd = 0.06 ‰, *n* = 3) reported for other South African *Carcharodon* specimens by Akhtar *et al.* (2020).

These behaviours highlight the plasticity of the great white
shark's diet regarding the availability of prey in its environment and/or
the habits of distinct populations. Prey availability, prey density and
prey profitability are sometimes identified as factors influencing the
diet of some large sharks (Simpfendorfer *et al.*, 2001a) and seem to
explain the geographical differences in δ⁴⁴/⁴²Ca values for the great
white shark. Moreover, great white sharks are known to present
demographically isolated populations due to their high degree of site
fidelity (Jorgensen *et al.*, 2009).

Recently, a study reconstructing seawater Ca isotopic composi-
tion through geological time using fossil shark teeth concluded that
fluctuations of δ⁴⁴/⁴²Ca values in elasmobranch enamel must be the
result of numerous factors (Akhtar et al., 2020). Our present results
suggest that the main factors that control the isotopic composition of
calcium are linked to the dietary behaviour of the considered species.
The differences in the isotopic trajectories between *Isurus* and
*Hexanchus* specimens with growth, not only allow to consider trophic
position but also permit to consider the preferential type of prey they
feed on.

## 4.2 | Alkaline earth metal concentrations as a proxy for ecological studies of elasmobranchs

Trace metal elemental ratios (expressed against Ca concentrations), such as Sr/Ca, Mg/Ca and Ba/Ca ratios, have been investigated in marine vertebrates for their relevance in dietary inferences (Peek & Clementz, 2012). However, contrary to calcium isotopes, such elemental ratios show that they seem not to be representative of any dietary pattern across our samples, but rather inform about environmental distribution. Concentrations of elements such as magnesium (Mg), strontium (Sr) and barium (Ba) reflect the dynamic of exchanges between earth, oceans and atmosphere. In terrestrial food webs, these elements represent biogeochemical indicators to study trophic levels as measured from mammal apatite (Balter, 2004; Peek & Clementz, 2012). However, in the marine realm, the contribution of these elements to diet is not well understood due to the buffering effect of seawater. The ratios of these elements expressed against calcium concentration (Mg/Ca, Sr/Ca and Ba/Ca) are known to present some spatial variations in seawater linked to vertical fluxes in the water column or terrigenous inputs in estuaries and coastal areas (Akhtar et al., 2020; Lebrato et al., 2020).

The prevalence of Sr and Mg in *H. griseus* relative to *Isurus* and *Carcharodon* could be explained by its differential occupation of the water column. Unlike the two lamniforms, *H. griseus* spends more time in deep water and in benthic habitats down to 1500 m depth, which are typically rich in some trace metals (Lebrato *et al.*, 2020; Rodríguez-Cabello *et al.*, 2018). For example, Mg and Sr concentrations are

known to increase with depth in the oceans (De Villiers, 1999; Jacquet *et al.*, 2016; Peek & Clementz, 2012), which could explain the high concentration observed in benthic *H. griseus* compared to the pelagic species and the increase in Sr/Ca ratio with growth in *Isurus* (Figure 3) (Sepulveda *et al.*, 2004). Moreover, the unexpected sexual distinction revealed in *H. griseus* Sr/Ca ratios (Figure 3) could reflect differences in the spatial distribution between sexes, as known for other deep water shark species (Coelho & Erzini, 2010). However, estimating the occupation of the water column by males and females of *H. griseus* remains poorly documented and misunderstood (Ebert, 2002b; Grainger *et al.*, 2020; Mili *et al.*, 2021; Moura *et al.*, 2014; Sims, 2006). These sexual differences in Sr/Ca values could also reflect physiological differences between males and females specific to this species of *Hexanchus*. This hypothesis is supported by a lack of difference in sexual variations in other shark species, including our whole dataset of *Isurus* and *Carcharodon* as well as the previously published dataset of Akhtar *et al.* (2020) (e.g., *Carcharhinus*, *Galeocerdo*, *Sphyrna*, *Lamna*, *Carcharias*, *Hemipristis*, *Negaprion*, *Prionace*, *Somniosus*) (Akhtar *et al.*, 2020), which all fall within the Sr/Ca range of *Hexanchus* males. It is noticeable that specimens from *H. griseus* whom sex is unknown and whom Sr/Ca was analysed (*n* = 5), present Sr/Ca values consistent with the two groups based on sex information (Figure 3). The latter ratios in *H. griseus* females show a Sr/Ca ratio of 5.93 (*n* = 5) significantly higher than that of males (2.68, *n* = 6) (Wilcoxon *P* value = 0.004). Even if the process remains unclear, it could result from physiological as well as spatial differences, and could allow us to determine *a posteriori* the sex of undocumented material in historical collections using Sr/Ca ratios. Considering this, among the specimens that do not display sex information in our dataset we speculate that these specimens represent one female and four males. In this paper, Sr/Ca and Mg/Ca ratios are reported to be significantly higher in *H. griseus* than in *I. oxyrinchus* and *C. carcharias* (Figure 3). However, sexual variations of the Mg/Ca ratio appear not to be as clear as reported for Sr/Ca ratios. Concerning the Ba/Ca ratio, even if Ba concentration in seawater is known to increase with depth (Hsieh & Henderson, 2017; Neff, 2002), the absence of a difference in the values we observe between shortfin mako sharks and bluntnose sixgill sharks seems not to segregate pelagic species from more benthic ones. This result indicates that in marine food webs barium is not fractionated from prey by predators (Neff, 2002).

5 | CONCLUSIONS

We conclude that Ca isotope analyses seem promising as a noninva-
sive tool for diet reconstruction in sharks, offering possibilities for the
understanding of changes in the type of prey linked with ontogeny
and/or population distribution in bluntnose sixgill sharks and great
white sharks. Moreover, SIA and trace metal concentrations analyses
open up new perspectives in a large range of biological, ecological and
evolutionary disciplines and will allow a complementary understanding
of marine vertebrate ecology. These proxies coupled with the high
preservation of elasmobranch teeth in the fossil record offer another
<figure><img src="image_7.png" /></figure>

opportunity to investigate the diet of extinct elasmobranch species (Kast et al., 2022; McCormack et al., 2022).

## CONTRIBUTIONS

A.A., J.E.M. and S.A. carried out the analyses and edited the manuscript. A.A., J.E.M., K.B. and F.A.G. performed the data acquisition. All authors drafted the manuscript and gave final approval for publication.

## ACKNOWLEDGEMENTS

We thank Henri Cappetta for providing the osteological specimens of sharks that are presented in this analysis. We also acknowledge Hakan Kabasakal for his valuable help in obtaining enamel samples. We are grateful to Vincent Balter for discussions and encouragement as well as Emmanuelle Albalat for help in the clean laboratory and Philippe Telouk for his support with the instruments. We thank CNRS (INSU INTERRVIE) and ENS de Lyon facilities, which permitted the geochemical analyses. We thank the anonymous reviewers for their help in improving the manuscript.

## FUNDING INFORMATION

The project was supported by the CNRS (INSU INTERRVIE) and LabEx CEMEB (MARCON).

## CONFLICT OF INTEREST

We declare we have no competing interests.

## DATA AVAILABILITY STATEMENT

All data used in this study are available from Table 1 and supporting information files Table S1.

## ORCID

Alexandre Assemat https://orcid.org/0000-0002-8649-0383

Sylvain Adnet https://orcid.org/0000-0001-7188-1560

Auguste Hassler https://orcid.org/0000-0003-3004-4680

Frederik H. Mollen https://orcid.org/0000-0002-9934-1029

Catherine Girard https://orcid.org/0000-0003-3123-8276

Jeremy E. Martin https://orcid.org/0000-0001-9159-645X

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Assemat, A., Adnet, S., Bayez, K., Hassler, A., Arnaud-Godet, F., Mollen, F. H., Girard, C., & Martin, J. E. (2022). Exploring diet shifts and ecology in modern sharks using calcium isotopes and trace metal records of their teeth. *Journal of Fish Biology*, **1**, 13. https://doi.org/10.1111/jfb.15211