Intraskeletal variability in phosphate oxygen isotope composition reveals regional heterothermies in marine vertebrates.

. Strategies used by marine vertebrates to regulate their body temperature can result in local variations, and the knowledge of these regional heterothermies is crucial for better understanding the thermophysiologies of extant and extinct organisms. In order to investigate regional heterothermy in vertebrates, we analysed the oxygen isotope composition of phosphatic skeletal elements (δ 18 O p ) of two poikilothermic (Atlantic bluefin tuna and swordfish) and three homeothermic endotherms (dolphins). We observed a consistent link between δ 18 O p variations and temperature heterogeneities recorded by 20 classical methods. Our δ 18 O p data indicate that: (i) bone hydroxyapatite of the axial skeleton of dolphins mineralize at a warmer temperature than that of the appendicular one, (ii) the skull is the warmest body region in swordfish, and (iii) Atlantic bluefin tuna possesses high body temperature in the skull and visceral mass region. These results demonstrate the possibility of tracking regional heterothermies in extant marine vertebrates using the δ 18 O p , paving the way to direct assessment of thermophysiological specificities of both living and extinct vertebrates. From a paleoenvironmental perspective, the significant 25 observed δ 18 O p variability questions the use of some taxa or random skeletal elements for the reconstruction of paleoceanographic parameters such as seawater temperatur e and δ 18 O. strategies of present-day marine vertebrates for which conventional methods of body temperature measurements are difficult to apply. This also allows to investigate thermophysiologies of extinct vertebrates since the oxygen isotope composition of hydroxyapatite phosphate can be preserved in the fossil record due to its good resistance to chemical processes that take place during burying and fossilization (e.g. Blake et al., 1997; Lécuyer et al., 1999; Kral et al., 2021). However, these results highlight the need to update the existing fractionation equations established


Introduction
Within vertebrates, ectotherms (e.g. crocodylomorphs, snakes, lizards, turtles, lissamphibians, chondrichthyans and 30 osteichthyans) rely on environmental heat sources to reach their optimal functional body temperature (Rodbard, 1953) and use behavioural adaptations to maintain it (Crawshaw and Hammel, 1971;Smith, 1979;Hight and Lowe, 2007). Contrarily, endotherms (birds and mammals) produce their body heat physiologically through metabolic processes (e.g. Cannon and Nedergaard, 2004;Legendre and Davesne, 2020). Some of them, the homeotherms, maintain their organs and nervous system at a nearly constant temperature (within ± 2 °C) by regulating their thermogenesis and thermolysis (Scholander, 1955) while 35 poikilotherms possess deep body temperature which covaries with environmental temperatures (Schmidt-Nielsen et al., 1966;Bennett et al., 1993;Sherwin, 2010;Allali et al., 2013;Nicol, 2017). Maintaining a high and constant temperature throughout the body in non-normothermic conditions is extremely energy-consuming for homeotherms. Consequently, many of them let the temperature of some areas of the body drop to reduce their energy need and limit heat losses (Irving and Hart, 1957;Rommel et al., 1992;Eichhorn et al., 2011). On the other hand, some poikilotherms are able to produce heat locally (Carey, 40 1982;Block, 1986;Dickson and Graham, 2004) to improve visual acuity in cold environment (Block, 1987;Fritsches et al., 2005), swim faster or migrate longer distances (Bernal et al., 2001;Blank et al., 2007;Watanabe et al., 2015). These two strategies lead to temperature heterogeneities called regional heterothermies which can be measured on extant organisms thanks to thermometer reading (Ponganis et al., 2003;Morkel et al., 2012) and thermal imagery (Hampton et al., 1971;Tattersall et al., 2009). However, such methods suffer from several types of limitation. Indeed, in situ temperature 45 measurements require the handling of the animal, leading to stress-induced and thus punctual rises in body temperature (Bouwknecht et al., 2007), whereas the infrared thermography is inefficient underwater. It is also difficult to apply them to large and rare living organisms, and in any case impossible to apply on extinct ones.
A possible way to track intra-individual temperature heterogeneities and thus regional heterothermies of both extant and extinct marine vertebrates could be the use of the oxygen isotope composition of phosphate (δ 18 Op) from bioapatite (the mineral 50 forming the bones, teeth and scales of vertebrates). Indeed, vertebrate δ 18 Op values reflect both the oxygen isotope composition of their body water (δ 18 Obw), stemming from ingested water in osteichthyans or from food for marine mammals (Telfer et al., 1970;Hui, 1981;Ortiz, 2001;Rosen and Worthy, 2018), and their body temperature due to the thermo-dependent oxygen https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License.
isotope fractionation between phosphatic tissues and body fluids (δ 18 Obw) from which they mineralize (Longinelli and Nuti, 1973;Kolodny et al., 1983;Longinelli, 1984;Luz et al., 1984;Lécuyer et al., 2013). Based on these considerations, it is 55 expected that homeothermic endotherms record homogeneous intra-skeletal δ 18 Op values, whereas in poikilothermic endotherms, intraskeletal δ 18 Op variability would highlight regional heterothermies. A few studies have investigated the intraskeletal δ 18 Op variability in some terrestrial and semi-aquatic extant organisms but the relatively reduced number of samples (n < 10 per individual) of these datasets considerably limits the significance of the δ 18 Op variability (Barrick, 1998;Stoskopf et al., 2001;Missell, 2004;Coulson et al., 2008;Clauzel et al., 2020). Some palaeontological studies were focused 60 on the search of regional heterothermies in dinosaurs (Barrick andShowers, 1994, 1995;Barrick et al., 1996Barrick et al., , 1998 but the observed variability in δ 18 Op through the skeleton was difficult to interpret without any present-day isotopic framework and concrete evidence that the isotopic method works for extant animals possessing regional heterothermies.
In this study, we present new δ 18 Op data obtained from cephalic, axial and appendicular skeletal elements to document the δ 18 Op variability in selected marine vertebrates with well-documented regional heterothermies and contrasted thermoregulatory 65 strategies. We compare the obtained δ 18 Op variations with available body temperature measurements obtained from classical methods and, finally, we discuss the possibility of using this proxy as a tool to identify thermoregulatory strategies and regional heterothermies of both extant and extinct marine vertebrates.

2.
Materials and methods

Sampled specimens 70
Five wild specimens belonging to four fully aquatic marine species were studied. They consist of three homeothermic  Table S1), and are curated at the Observatoire des mammifères et oiseaux marins (PELAGIS, France) and at the Museum national d'Histoire naturelle (MNHN, Paris, France), while the swordfish and Atlantic bluefin tuna specimens were obtained from a local fish shop (See supplementary information 1). Between 24 and 44 skeletal elements per specimen covering all body regions were analysed for their δ 18 Op values. About 50 mg of each skeletal element were ground 80 into a fine powder using either a Dremel TM diamond-head drill or a mortar and pestle. The cortical part of the bone and areas with minimal physical degradation were selected during the sampling process.

Oxygen isotope analysis of biogenic apatite phosphate
To measure oxygen isotope ratios of biogenic apatite phosphate by gas mass spectrometry techniques, samples were treated 85 according to the wet chemistry protocol described by Crowson et al. (1991) and slightly modified by Lécuyer et al. (2013).
The protocol consists of the isolation of phosphate ions (PO4 3-) from apatite as silver phosphate crystals (Ag3PO4). The Ag3PO4 crystals were filtered, dried and cleaned. For each sample, five aliquots of 300 ± 20 µg of Ag3PO4 were mixed with 400 ± 50 µg of graphite in silver foil capsules. Oxygen isotope compositions were measured using a high temperature vario PYRO cube TM elemental analyser (EA) equipped with the "purge and trap" technology (Fourel et al., 2011) and interfaced in continuous flow 90 mode to an IsoPrime TM isotopic ratio mass spectrometer (Elementar UK Ltd Cheadle, UK) at the Plateforme d'Ecologie Isotopique du Laboratoire d'Ecologie des Hydrosystèmes Naturels et Anthropisés (LEHNA, UMR5023, Université Claude Bernard Lyon 1, Lyon, France). Pyrolysis of Ag3PO4 was performed at 1450 °C. The measurements were calibrated against two standards: a silver phosphate precipitated from the international standards NBS120c (natural Miocene phosphorite from Florida), and from the NBS127 (barium sulfate precipitated using seawater from Monterey Bay, California, USA). The 95 NBS120c δ 18 Op value was fixed at 21.7 ‰ V-SMOW (Vienna Standard Mean Ocean Water) according to Lécuyer et al.(1993), Chenery et al. (2010) and Halas et al. (2011), and that of NBS127 set at the certified value of 9.3 ‰ V-SMOW (see Hut, 1987;Halas and Szaran, 2001) for correction of instrumental mass fractionation during CO isotopic analysis. Silver phosphate precipitated from standard NBS120c were repeatedly analysed (δ 18 Op = 21.7 ± 0.3 ‰, n = 46) along with the silver phosphate https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License. samples derived from bioapatite to ensure that no isotopic fractionation occurred during the wet chemistry. Data are reported 100 as δ 18 O values normalized to V-SMOW (in ‰ δ units).

Statistical analyses
To increase sample size and statistical power for testing the intraskeletal variability of δ 18 Op values, skeletal elements were grouped into several sets corresponding to different parts of the skeleton. The limit between the axial and appendicular skeleton 105 is set at the articulation between the pectoral girdle and the stylopod for dolphins. For Atlantic bluefin tuna and swordfish, the fin rays and fin spines belonging to the fins were considered as appendicular skeleton. For the Atlantic bluefin tuna, we have distinguished anterior and posterior part of the axial skeleton at the limit between precaudal and caudal vertebrae. Since normality and homoscedasticity of the δ 18 Op values were not validated, we used the non-parametric Mann-Whitney-Wilcoxon to compare median values between two observational series. Statistical tests were performed using R software (R Core Team, 110 2017) and the level of significance was set at p-value < 0.05. All the p-values resulting from the statistical tests are reported in supplementary material, Table S4.

115
The δ Fig.1A for the north Atlantic D. delphis delphis and in Fig.2A and 2B for osteichthyans. The results of the two others Delphinidae studied are available in supplementary material, Fig. S1. Intra-bone homogeneity was measured by paired samples on vertebrae in dolphins and osteichthyans and on fin rays in osteichthyans and is systematically lower than inter-bone variability (Table 1). 120 As expected, the inter-bone δ 18 Op variability is higher in poikilothermic endotherms than homeothermic endotherms (Table   1). For dolphins, δ 18 Op values from the axial skeleton are significantly lower than those of the appendicular ones (p-values < https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License. 0.05; Fig. 1B and supplementary material, Fig. S2). Teeth δ 18 Op values of dolphins are higher than those from axial skeletal (Table 1). Nonetheless, the significance of these differences cannot be tested due to the small number of teeth and skull samples (n = 1 to 3). In T. thynnus, the highest mean value of 21.6 ± 0.2 ‰ (1SD, n = 6) is recorded in the posterior part of the axial 125 skeleton, whereas the lowest values (Table 1)

Sources of intraskeletal δ 18 Op variability 140
The measured intraskeletal δ 18 Op variability may results from two main factors identified as the difference in temperature of bone mineralization across the skeleton as well as changing isotopic compositions of oxygen sources throughout the animal life. We found significant δ 18 Op differences (~ 0.5‰) between axial and appendicular bones in dolphins that possess the same mineralization process, strongly suggesting a dominant temperature control (Fig.1B). By contrast, the differences in δ 18 Op recorded between bones and teeth of dolphins (Table 1; Fig.1B and supplementary materials, Fig. S2) cannot be exclusively 145 https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License. attributed to variable body temperature since these elements mineralize at distinct times during ontogeny and possess different rates of remodelling (Myrick, 1991;Ungar, 2010). For osteichthyans with high metabolic rates such as tunas and billfishes, mineralization timing should affect δ 18 Op minimally because all skeletal elements are remodelled (Rosenthal, 1963;Atkins et al., 2014) and teeth are continuously renewed in tunas. The differences in δ 18 Op values between skeletal elements with comparable timing of mineralization and remodelling rates can therefore be confidently attributed to differences in body 150 temperature (Fig.2C). Besides, studied organisms are nektonic predators that feed on fish and invertebrates (Young and Cockcroft, 1994;Kastelein et al., 2000), which in turn possess δ 18 Obw values similar to that of their surrounding water (Picard et al., 1998;Pucéat et al., 2003) but vary depending on the geographical area where they live. The food being the main source of water in dolphins, the consumption of preys coming from different water masses should cause variations in their δ 18 Obw.
Nevertheless, the seasonal changes in δ 18 Osw of the water masses in which the sampled organisms fed are relatively small 155 (± 0.4 ‰; supplementary material, table S5) and cannot fully explain the inter-bone δ 18 Op variability reported herein in dolphins and osteichthyans. Therefore, the link between δ 18 Op values and the intra-individual body temperature differences previously documented among the studied animals strongly suggest that the recorded isotopic variability is mainly due to differences in mineralization temperature rather than different timing of mineralization. 160

Homeothermic endotherms
Intraskeletal δ 18 Op variability of homeothermic endotherms (mapped in Fig. 1A, and supplementary material, Fig.S1) shows an isotopic enrichment in the appendicular skeleton relative to the axial one. This indicates a lower mineralization temperature 165 in these skeletal regions. This observation is consistent with the thermoregulatory strategies used by cetaceans having a trunk at a nearly constant temperature of 36 ± 2°C (Morrison, 1962;Hampton et al., 1971), in agreement with their high metabolic activity (Williams et al., 2001), a thick layer of blubber (Lockyer, 1986;Hashimoto et al., 2015) and counter-current heat exchangers which limit heat losses at the extremities (Scholander and Schevill, 1955). Counter-current heat exchangers, https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License. defined by a particular spatial arrangement of the cardiovascular system, causes cooling of the blood from the arteries in contact 170 with the veins and results in body temperature proximodistal gradient (Irving and Hart, 1957). The few information available for dolphins mentioned body temperature variation of 9°C in the limbs whereas trunk body temperature remains constant (Tomilin, 1950).
Assuming light seasonal changes in marine mammal δ 18 Obw values throughout the seasons and considering the δ 18 Op values obtained (Table 1), our new isotope data can be used to estimate the temperature differences between limb and trunk in the 175 sampled dolphins using the phosphate-water temperature scale published by Lécuyer et al. (2013): The obtained differences in mineralization temperature are of 2 ± 0.5°C for D. delphis delphis, and 1 ± 0.5°C for C.
commersonii kerguelensis. In other words, our data show that the mineralization temperature of the bone is about 2°C lower in the limbs than in the rest of the skeleton in D. delphis delphis and 1°C in C. commersonii kerguelensis. The estimated 180 temperature differences are lower than those recorded by classical methods. This difference could be explained by the time average recorded in the bones. The time record being long, in the order of several years (Rosenthal, 1963;Riccialdelli et al., 2010;Browning et al., 2014), the estimates inferred from bone δ 18 Op represents long term trend rather than precise temperature at a specific time and probably mitigate these temperature differences.

Poikilothermic endotherms
Locally high body temperatures have been recorded in several species of tunas (Carey and Lawson, 1973;Graham and Dickson, 2001) and billfishes (Carey, 1982) using classical methods. Heat in tunas is generated using their red swimming and extraocular muscles (Guppy et al., 1979) and is retained by counter-current heat exchangers (Block and Finnerty, 1994;Dickson and Graham, 2004). Unlike most teleosts, tunas have red muscles positioned close to the spine, limiting heat transfer 190 from the body to the surrounding aquatic medium . Our δ 18 Op values and their variations across the body are in agreement with the temperature heterogeneities previously measured by other techniques (e.g. Carey and Teal, 1966;Carey et al., 1971Carey et al., , 1984Graham and Dickson, 2001), with in particular the lowest δ 18 Op values measured in the skull https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License. and vertebrae near the visceral mass (Table 1 and Fig. 2C). Estimated temperature heterogeneities of tuna assuming slight seasonal changes in δ 18 Obw are of 2 ± 0.5°C between fins and the visceral mass region and 4 ± 0.5°C between fins and teeth 195 (Fig. 3A). These results are consistent with in situ body temperature measurements which indicate a strong thermal gradient of 4 to 20°C between core temperature and environmental water (Carey and Teal, 1966;Carey et al., 1971;Carey and Lawson, 1973;Carey et al., 1984). However, the absolute temperature differences inferred from the two methods are difficult to compare as for dolphins. The high δ 18 Op variability observed in branchial arches can be explained by variable thermal exchanges between hot blood and cold environmental water. 200 Swordfish has warm brain and eyes through a unique heater organ associated with the rectus eye muscle (Carey, 1982;Block, 1987) linked to a system of counter-current exchangers and buried in a thick adipose mass that stores the heat produced (Block, 1986(Block, , 1991. This mechanism allows the swordfish brain temperature to be 5 °C to 30 °C warmer than the surrounding water while the rest of its body remains close to water temperature (Carey, 1982(Carey, , 1990Schwab, 2002;Stoehr et al., 2018). Our δ 18 Op values and the use of the Eq.(1) indicate that the skull temperature is approximately 7 ± 0.5°C warmer than the rest of the body 205 which is consistent with the global trend provided by in situ temperature measurements (Carey, 1982(Carey, , 1990Fritsches et al., 2005).

Implications for extant and extinct marine vertebrates
The proposed oxygen isotope thermometry complements conventional approaches and thermal imaging methods. The use of 210 oxygen isotopes represents a valuable alternative method to access temperature heterogeneities over the body in marine vertebrates for which logger is difficult to install and operate. Unlike techniques involving surgical implants (Carey and Teal, 1966;Ponganis et al., 2008), isotopic method does not require the handling of living animals, that can punctually increase their body temperature due to stress (Bouwknecht et al., 2007). Moreover, isotopic thermometry provides internal temperature data contrary to infrared thermal imagery that limits information to skin temperature (Tattersall et al., 2009;McCafferty et al., 215 2015). These results open up new perspectives for thermophysiological studies both on extant organisms that are difficult to handle (e.g. whales) or which are rare (abyssal organisms), but also on extinct organisms for which only skeleton is available https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License.
(e.g. Steller's sea cow, extinct cetaceans, ichthyosaurs, plesiosaurs…). Beyond these (paleo-)biological implications, our results also highlight a major issue concerning the use of random skeletal elements of marine vertebrates (e.g. chondrichthyans and osteichthyans or cetacean bones and teeth) for the reconstruction of paleoceanographic parameters based on the oxygen 220 isotope composition of bioapatite (e.g. seawater temperatures and δ 18 Osw values). Intraskeletal variability resulting from regional heterothermies can lead to overestimate seawater temperature or underestimate δ 18 Osw values when applying specific fractionation equations to isolated skeletal elements (Fig. 3A, B). For example, the maximum δ 18 Op difference of 2.8 ‰ measured between two bones of the swordfish can result in an overestimation of 10°C of seawater temperature when applying the phosphate-water temperature scale of Lécuyer et al. (2013) (Fig. 3A). In the same idea, the maximum δ 18 Op difference of 225 1.8 ‰ measured between two bones of the North Atlantic short-beaked common dolphin can result in an δ 18 Osw underestimation of 1.7 ‰ when applying the fractionation equation published by Ciner et al. (2016; Fig. 3B). It is noteworthy that existing fractionation equations available for chondrichthyans and osteichthyans or cetaceans were established mixing various skeletal elements including axial or appendicular bones and teeth (Longinelli and Nuti, 1973;Kolodny et al., 1983;Yoshida and Miyazaki, 1991;Lécuyer et al., 2013;Ciner et al., 2016). In order to perform accurate paleoceanographic 230 reconstructions, existing fractionation equations will therefore need to be updated to take into account regional heterothermies.

Conclusion
Detailed intraskeletal δ 18 Op mapping allows to document regional heterothermies in marine vertebrates. Calculated δ 18 Opderived temperatures are consistent with temperature heterogeneities recorded by classical methods. This opens up new 235 perspectives on the determination of the thermoregulatory strategies of present-day marine vertebrates for which conventional methods of body temperature measurements are difficult to apply. This also allows to investigate thermophysiologies of extinct vertebrates since the oxygen isotope composition of hydroxyapatite phosphate can be preserved in the fossil record due to its good resistance to chemical processes that take place during burying and fossilization (e.g. Blake et al., 1997;Lécuyer et al., 1999;Kral et al., 2021). However, these results highlight the need to update the existing fractionation equations established 240 https://doi.org/10.5194/bg-2022-10 Preprint. Discussion started: 19 January 2022 c Author(s) 2022. CC BY 4.0 License. for chondrichthyans and osteichthyans or cetaceans as they do not take into account the significant intraskeletal δ18Op variability caused by regional heterothermies.

Data accessibility.
Stable oxygen isotope compositions are provided in Excel tables as electronic supplementary materials. Informations about 245 Atlantic bluefin tuna are also mentioned in supplementary materials.
Material analysis were performed by N. Séon, R. Amiot and C. Lécuyer,. The first draft of the manuscript was written by N.
Séon, R. Amiot and P. Vincent, and all authors commented on previous versions of the manuscript. All authors read and 255 approved the final manuscript.

Competing interests.
The authors declare that they have no conflict of interest.