Phil K Cook

Elise Dufour

Marie-Angélique Languille

Cristian Mocuta

Solenn Réguer

Loïc Bertrand

Chemical environment of strontium in archaeological otoliths from Peru: Implication for diagenesis studies

Keywords: marking . . . . . . . . . . . . energy synchrotron µXRF mapping . . . . . . . . . . . . Axial distribution of elements . . . . . . . 4.3.2.3 Biogenic versus post-mortem signal . . . . . 4.4 Intermediate conclusion . . . . . . . . . . . . . Otoliths, Paleoenvironment, Synchrotron micro X-ray fluorescence spectroscopy, Elemental mapping, Strontium otoliths, palaeoenvironmental proxies, synchrotron X-ray absorption spectroscopy, strontium, chemical environment

revealed by SR-µXRF mapping . . . . . . . . . 4.3 Discussion and interpretations . . . . . . . . .

Philip Kenneth COOK: Microfocused X-ray methodologies for the biogeochemical study of archaeological and modern otoliths, Thèse doctorale, 22 septembre 2015 directeur : Loïc BERTRAND encadrantes : Élise DUFOUR Marie-Angélique LANGUILLE R É S U M É Les otolithes, croissances aragonitiques de l'oreille interne des poissons téléostéens, peuvent être utilisés comme traceurs des variations de l'environnement rencontrées par un individu au cours de sa vie. Un ensemble d'otolithes de Sciaenidae et d'Ariidae archéologiques et modernes a été étudié dans le but d'améliorer les méthodes de reconstruction paléo-environnementale utilisant les otolithes et les autres biominéraux carbonatés stratifiés. L'incorporation du strontium, l'élément lié à l'environnement le plus facilement accessible, a été étudiée par analyses ponctuelles et par cartographie de spectroscopie d'absorption des rayons X (XAS). Une approche multivariée de cartographie rapide de l'environnement chimique a été mise en place pour déterminer le mode d'incorporation du Sr sur une superficie de 0,25 mm 2 avec une résolution micrométrique. Ces résultats démontrent pour la première fois avec une résolution latérale micrométrique sur des distances millimétriques que le Sr se substitue aléatoirement au calcium dans le réseau de l'aragonite, indépendamment de la concentration en Sr, de l'âge de l'individu ou de la période géologique. D'autre part, des cartes élémentaires sur des zones atteignant 2,6 mm 2 ont été collectées à des résolutions latérales micrométriques avec plusieurs techniques d'émission de rayons X (émissions provoquées par les particules des rayons X (PIXE) et fluorescence des rayons X par rayonnement synchrotron (SR-µXRF)). Ces cartes permettent l'examen détaillé de l'histoire de la vie d'un individu et de la taphonomie de l'échantillon avec une haute résolution temporelle, tout en identifiant les défauts ou les éléments abiogéniques. Enfin, la diffraction des rayons X par rayonnement synchrotron (SR-XRD) a été utilisée pour cartographier la texture cristalline sur des sections complètes d'otolithes afin d'approfondir notre connaissance de la structure interne et de la croissance des otolithes. Ces développements fournissent des outils précieux pour de futures études des biominéraux, mais plus généralement pour les sciences des matériaux.

La sélection et la mise en oeuvre de ces méthodes ont été réalisées dans le but d'exploiter au maximum leur fort potentiel pour l'étude des biocarbonates stratifiés, tout en tenant compte des approches existantes et en cherchant à en améliorer certains aspects tels que la profondeur d'information, la résolution latérale, la sensibilité, et les dégâts d'irradiation provoqués par les faisceau. Ce travail démontre la stabilité et l'homogénéité de l'incorporation du Sr par substitution aléatoire au Ca dans l'aragonite biogénique des otolithes modernes et archéologiques. Des cartes multi-élémentaires ont été collectées à l'aide de la SR-µXRF dans un temps raisonnable de quelques v heures, et permettent de distinguer une contamination ou des défauts dans l'échantillon, mais également de corréler les cartes obtenues à des observations microscopiques des sections pour fournir une résolution temporelle. Les orientations préférentielles des cristallites composant les sections d'otolithes ont été analysées par la méthode d'acquisition rapide « flyscan », permettant de réduire le temps de mesure à quelques minutes au lieu des quelques heures nécessaires auparavant.

vi A B S T R A C T

Otoliths, aragonitic growths in the inner ear of teleost fishes, can be used as proxies for the water conditions experienced by an individual over its lifetime. A set of archaeological Sciaenidae and Ariidae otoliths and modern counterparts was studied with the objective of improving palaeoenvironmental reconstruction methodologies using otoliths and other incremental carbonate biominerals. The incorporation of strontium, the most accessible environment-related element, was studied by X-ray absorption spectroscopy (XAS) point analyses and mapping. A fast multivariate chemical environment mapping approach was implemented to determine the mode of Sr incorporation over an area of 0.25 mm 2 with micrometric resolution. XAS results demonstrate for the first time with a micrometric lateral resolution over millimetric distances that strontium randomly substitutes for calcium in the aragonite lattice, independent of strontium concentration, or individual or geological age. Elemental maps on areas up to 2.6 mm 2 were produced with micrometric lateral resolution X-ray emission techniques (Particle-induced X-ray emission (PIXE) and synchrotron X-ray fluorescence (SR-µXRF)). These maps permit the detailed examination of an individual's life history and sample taphonomy with a high temporal resolution while also identifying defects or abiogenic elements. Synchrotron X-ray diffraction (SR-XRD) was used to map the crystal texture on complete otolith sections and may deepen understanding of otolith internal structure and growth processes, as well as providing a valuable tool for future studies of biominerals and advanced materials.

The selection and implementation of methods was carried out with a view to maximise the potential contribution to the study of stratified biocarbonates, considering and seeking to complement existing approaches in aspects including information depth, lateral resolution, sensitivity, and beam damage. This work demonstrates the stability and homogeneity of Sr incorporation by random substitution for Ca in biogenic aragonite in both modern and archaeological otoliths. Multielemental maps were collected using SR-µXRF in a reasonable time scale, with the ability to distinguish contamination and defects in the sample, as well as to correlate the maps to microscopic observations of the sections to provide temporal resolution. The preferential orientations of crystallites composing the otolith sections were analysed using the rapid acquisition 'flyscan' method, which reduces measurement time to minutes rather than hours. vii Illustration courtesy Julien Norwood ©2013 Poets say science takes away from the beauty of the stars -mere globs of gas atoms. I, too, can see the stars on a desert night, and feel them. But do I see less or more? Richard Feynman

x A C K N O W L E D G E M E N T S
I would like to express my sincere thanks to my three supervisors, Loïc Bertrand, Elise Dufour, and Marie-Angélique Languille for their advice, insight, and support throughout this work. After many discussions ranging from X-ray physics to archaeology, from statistics to graphic design, here we are. I am grateful to Philippe Béarez (UMR 7209), Nicolas Goepfert (UMR 8096), and Tom Dillehay (Vanderbilt University) for providing the samples I have studied, and to Michel Lemoine and Olivier Tombret (UMR 7209) for preparing them. I would like to thank the people of the groups managing the beamlines and equipment I had the opportunity to use during my thesis for their warm reception and expert advice: DiffAbs, LUCIA, ATOMKI, BAMline, Paleoproxus, and the National Ion MicroProbe Facility. In particular, I would like to thank Cristian Mocuta (DiffAbs) for the time and effort spent in close collaboration.

I thank the members of my comité de thèse, Anne-Marie Flank, Claire Lazareth, and Jean-Denis Vigne, for their advice and encouragement along the way. I thank the members of my jury for their time and attention. I am grateful to Sylvain Ravy for presiding over my defence, Karin Limburg for her immediate and active interest in my work, Ina Reiche for her insightful comments, Claire Lazareth for her exceptionally thorough review, Jean-Denis Vigne for his support of interdisciplinary work, and Jean-Pierre Cuif for his eloquent and flattering analogy.

My colleagues of the last few years have helped me at many points along the way and made my time here more memorable. I would like to thank the current and former members of IPANEMA: Alessandra, Fanny, Felisa, Jérémy, Mathieu, Patricio, Pierre, Regina, Sebastian, Sébastien, Serge, Susanna, and William. Thank you to the members of the Archéozoologie, archéobotanique UMR 7209 for making a physicist feel at home in a museum: Andréa, Antoine, Aurélie, Caroline, Colin, Jérome, Liz, Noémie, and Roz.

To Nataša, quite simply: hvala. Thank you for being my partner throughout the ups and downs of these years, for your understanding, for being there when the light at the end of the tunnel was so dim I lost sight of it, and for helping me grow in those rare moments I wasn't focused on work.

Thank you to my friends -on both sides of the ocean -and to my family for your unconditional support and encouragement.

I dedicate this thesis to the memory of my father, Ken Location of otoliths and labyrinth systems within the fish. . . . . . . . . . . . . . . . . . . . . Figure 2 Structure of the labyrinth system and otosacs . . . Figure 3 Interior face of a whole otolith from a channel flatfish showing light and dark zones constituting annuli in transmitted light. . . . . . . Figure 4 Aragonite unit cell . . . . . . . . . . . . . . . . . . Figure 5 The structure of an otolith from atoms to macroscopic object . . . . . . . . . . . . . . . . . . Figure 6 Otolith sections before and after etching . . . . . . Figure 7 Secondary centres of calcification produce complex forms . . . . . . . . . . . . . . . . . . . . . Figure 8 A whole otolith viewed from multiple angles . . . Figure 9 Simplified phylogeny of species in this work . . . Figure 10 Map of sample origins . . . . . . . . . . . . . . . . Figure 11 Photos of archaeological sites . . . . . . . . . . . . Figure 12 Sectioning planes of typical sagitta . . . . . . . . . Figure 13 Photographs of otoliths as analysed . . . . . . . . Figure 14 Reference pellet mounted for analysis . . . . . . . Figure 15 A unified analytical methodology . . . . . . . . . Figure 16 Examples of previous works using SR-XRF for life history studies. . . . . . . . . . . . . . . . . . . Figure 17 Tungsten XRF signal within and outside of a FIB mark . . . . . . . . . . . . . . . . . . . . . . . . . . Figure 18 Atomic cross-sections . . . . . . . . . . . . . . . . . Figure 19 Excitation of a core electron by an X-ray photon . Figure 20 Regions of a XAS spectrum . . . . . . . . . . . . . Figure 21 Schematic of the components in a XAS experiment. Figure 22 A double crystal monochromator is configured with two mirrors, such that an X-ray of the desired energy (hν 1 ) passes through the exit slit. X-rays of a second, undesired energy (hν 2 ) do not follow the same path and are blocked. . . Figure 23 Experimental setup for µXRF on DiffAbs . . . . . Figure 24 The XRF process . . . . . . . . . . . . . . . . . . . Figure 25 Configuration for derivation of quantitative XRF formula . . . . . . . . . . . . . . . . . . . . . . . . . Figure 26 FF-XRF experimental setup . . . . . . . . . . . . . Figure 27 µPIXE experimental setup . . . . . . . . . . . . . . Map and histogram of . . . . . . . . . . . . . . . Figure 56 Map and histogram of δ . . . . . . . . . . . . . . . Figure 57 Map and histogram of β . . . . . . . . . . . . . . . Figure 58 Map and histogram of β normalised for Sr concentration . . . . . . . . . . . . . . . . . . . . . Figure 59 Correlation of β and Sr concentration . . . . . . . Figure 60 Map and histogram of normalised to β . . . . . Figure 61 Map and histogram of δ normalised to β . . . . . Figure 62 Map and histogram of a (fraction aragonite) . . . Figure 63 Map and histogram of b (fraction strontianite) . . Figure 64 Possible modes of incorporation of Sr . . . . . . . Figure 65 Integrated 'time' profiles of Sr and Ca concentrations . . . . . . . . . . . . . . . . . . . . . . . . . Figure 66 X-ray diffraction pattern of otolith QCD . . . . . . Figure 67 XPAD images after transformation to Cartesian coordinates . . . . . . . . . . . . . . . . . . . . . . . Figure 68 Reflection intensity maps of 012, 021, and 112 peaks of QCD binned by ψ . . . . . . . . . . . . . Figure 69 Reflection intensity maps of 002, 020, and 200 peaks of QCD binned by ψ . . . . . . . . . . . . . Figure 70 XRF and XRD signals measured simultaneously in sample 424 . . . . . . . . . . . . . . . . . . . . . Figure 71 Localisation and identification of non-aragonitic phases on otolith QCD . . . . . . . . . . . . . . . . Figure 72 Spotty appearance of 111 reflection in otolith 424 Figure 73 Map of twinning rate in QCD . . . . . . . . . . . . Figure 74 Orientation of the maximum intensity in ψ of 002, 020, and 200 reflections in QCD . . . . . . . . Figure 75 Intensity on ψ of neighbouring points on QCD . . Figure 76 First attempts to compare elemental and isotopic compositions. . . . . . . . . . . . . . . . . . . Figure 77 Integrating additional techniques to the unified methodology . . . . . . . . . . . . . . . . . . . . . Figure 78 Tender XRF Sr map of an internal zone of otolith 424 . . . . . . . . . . . . . . . . . . . . . . . . . . . Figure 79 Comparison of the information volume of medium-energy and tender X-rays . . . . . . . . .

xviii L I S T O F TA B L E S Table 1

Aragonite lattice parameters determined by various authors . . . . . . . . . . . . . . . . . . . . Table 2 Aragonite, strontianite, calcite, and vaterite lattice parameters . . . . . . . . . . . . . . . . . . . Table 3 Typical concentration ranges of elements found in otoliths . . . . . . . . . . . . . . . . . . . . . . . Table 4 Studies on ancient otoliths and methods used for verification of potential alteration. . . . . . . . Table 5 List of samples studied. . . . . . . . . . . . . . . . Table 6 Sample preparation parameters . . . . . . . . . . . Table 7 General characteristics of beam-based elemental analysis techniques. . . . . . . . . . . . . . . . . . . Table 8 Compilation of SR-XRF studies of otoliths . . . . Table 9 Attenuation length of selected characteristic lines in pure aragonite . . . . . . . . . . . . . . . . in the inner ear of bony fish. They grow in increments from a nucleus which produces a layered, temporally-resolved structure.

Cutting into an otolith reveals annular increments which are often compared to the rings of tree trunks. Over the course of their growth, otoliths incorporate elemental and stable isotopic signatures related to the environment (e.g. water temperature and chemistry) at the time each increment is deposited. These signatures reflect the fish's physiology and living conditions throughout ontogenesis, providing a life history archive. The life history in an otolith is in the form of environmental proxies, indirect representations of environmental variables. The most commonly used environmental proxies in otoliths are strontium elemental concentration and δ 18 O (stable oxygen isotope composition). As life history archives, otoliths are valuable for a variety of purposes in modern specimens, including stock identification and migration tracking. Otoliths can be discovered by archaeologists among the artefacts left by ancient humans. These ancient specimens can be valuable palaeoenvironmental proxies for studies of palaeoenvironment, palaeoecology, or archaeological study of the ancient humans who caught them. Though generally stable in macroscopic morphology, ancient otoliths used as palaeoenvironmental proxies are subject to thousands of years in the environment after fish death, which may produce alteration of elemental and/or isotopic composition, mineral phase, or internal micro-or ultrastructure.

The present work contributes to the relatively small extant body of knowledge on the post-mortem alteration of otoliths. Elemental mapping methodologies using micro-X-ray fluorescence and microproton-induced X-ray emission have been elaborated in order to provide a tool capable of quantifying strontium along with simultaneous measurement of multiple other elements within otolith sections. Techniques with a high lateral resolution, i.e. spatial resolution in the plane of the sample surface, were selected to provide a corresponding temporal resolution equal to or surpassing that of current common techniques. This permits a high degree of reliability in measurements, as well as the ability to identify potential alteration through detection of the intrusion of exogenous elements and displacement of endogenous elements. Previous works have shown that the way in which minor and trace elements are incorporated into otoliths strongly impacts their stability. Herein, incorporation of strontium within the aragonite lattice of ancient otoliths was examined using micro-X-ray absorption spectroscopy for the first time. Direct examination of the aragonite lattice is usually performed only on limited areas, or on subsamples. In the present work, a micro-X-ray diffraction methodology was developed to permit the analysis of centimetric samples with micrometric resolution on reasonable time scales. This approach allowed both mineral phase identification and the determination of crystal orientation, permitting the first fullsection maps at a fine lateral resolution of the crystals which compose otoliths.

O T O L I T H S : L I F E H I S T O R Y A R C H I V E S

1.1 "Ear stones": Anatomy, function, and growth . . . . Otoliths, from ancient Greek (ωτο, "ear" + λίθος, "stone"), are accretions in the inner ear of vertebrates, including bony fishes. They are biominerals, composed primarily of inorganic mineral with a small proportion of organic templating compounds. Their growth is sequential, resembling that of a tree trunk.

1
1.1 "ear stones": anatomy, function, and growth

Otoliths are mineralised structures found in the inner ear of vertebrates (Fig. 1). In teleost1 fish, they are generally composed of (2013b).

This gure has been removed due to copyright restrictions.

It can be found in the referenced text or in the full version of this work at the Université Paris-Sud library.

(b) Dorsal view of the labyrinth systems within the head of the fish with the semicircular canals and otosacs containing the otoliths (sagittae, asteriscii, and lapilli). Adapted from [START_REF] Secor | Otolith Removal and Preparation for Microstructural Examination: A Users Manual[END_REF].

Figure 1: Location of otoliths and labyrinth systems within the fish.

aragonite (CaCO 3 ) (Carlström, 1963) and an organic matrix (Degens et al., 1969). The shape and size of otoliths is unique to each species (Nolf, 1985). Otoliths serve for audition, orientation, and motion sensing through being loosely-fixed references whose motion or vibrations relative to the surrounding membranes are detected (Nolf, 1985;[START_REF] Fermin | Otoconia biogenesis, phylogeny, compositon and functional atributes[END_REF].

The term "otoliths" refers to any one of a set of three accretions occurring in pairs on the left and right sides of the individual (Nolf, 1985). While all three otolith pairs contribute to hearing and motion sensing, it has been described that one pair in particular, depending on the species, contributes more siginificantly to audition [START_REF] Secor | Otolith Removal and Preparation for Microstructural Examination: A Users Manual[END_REF]. The proximity of the asteriscus to other structures in ostariophysi 2 suggests this is the more significant pair, while the large size of sagittae in acanthopterygii 3 for which communication is important [START_REF] Secor | Otolith Removal and Preparation for Microstructural Examination: A Users Manual[END_REF].

The three pairs of otoliths are named for their shapes: the "sagittae" (arrow), "lapilli" (pebble), and "asterisci" (star) (Carlström, 1963).

Sagittae are usually the larger (Fig. 1b), except for few ostariophysi [START_REF] Miller | [END_REF]. Note that all definitions given in footnotes are collected in the Glossary (Appendix ??). 2 A superorder of teleost fish, one characteristic of which is a "Weberian apparatus" connecting the inner ear to the swim bladder. 3 A superorder of teleost fish. (b) The structure of the saccule in transverse section as indicated in (a) highlighting the cellular structure. Hairs (black triangles) connect the otolith to the nerve. Adapted from [START_REF] Pisam | Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, Oncorhynchus mykiss and Psetta maxima[END_REF].

Figure 2: Structure of the labyrinth system with the three semicircular canals and otosacs (lagena, saccule and utricule) containing the otoliths (asteriscus, sagitta and lapillus in grey) of a typical Teleost.

The structure is seen from the angle represented by the fish schematic. The red areas indicate the macula and sulcus: where the otolith connects to nerves and the wall of the otosac.

species where lapilli or asterisci are more developed (Nolf, 1985), for example in the Ariidae family4 .

The inner ear's labyrinth system is composed of a series of semicircular canals and larger chambers (Fig. 2a). The term "otosac" can be applied to refer to any of the three otolith-containing chambers, known as the saccule (containing the sagitta), the utricule (containing the lapillus), and the lagena (containing the asteriscus). Each otosac is composed of an epithelium in four parts: the macula, the meshwork area, the intermediate area, and the patches area (Fig. 2b) [START_REF] Pisam | Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, Oncorhynchus mykiss and Psetta maxima[END_REF]. Within the otosac, the otolith is surrounded by plasmalike endolymph. Secretory cells and ionocytes found in the meshwork area and patches area supply the endolymph with organic molecules and inorganic ions [START_REF] Pisam | Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, Oncorhynchus mykiss and Psetta maxima[END_REF]. Collagen-based proteins loosely connect from the macula of the otosac to a groove on the otolith known as the sulcus. Hairs called kinocilia extend from the macula to touch the surface of the otolith. The kinocilia relay vibration or movement to the nerve cells in the macula (Nolf, 1985).

The kinocilia transmit movement or vibration of the otolith to the nerves for audition, motion, and head orientation sensing (Fig. 2b) [START_REF] Pisam | Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, Oncorhynchus mykiss and Psetta maxima[END_REF].

Otolith growth begins in the egg stage and continues until death (Campana and Neilson, 1985). Their growth continues in proportion to somatic (body) growth (Panfili et al., 2002). The core for otolith growth is the primordium, one or more small crystallised spherules that form late in the egg stage around which future layers will be deposited [START_REF] Tanaka | Effects of photoperiod and feeding on daily growth patterns in otoliths of juvenile Tilapia nilotica[END_REF]. The first increment is composed of proteinaceous matrix to form the nucleus of the otolith, which is usually spherical [START_REF] Morales-Nin | Review of the growth regulation processes of otolith daily increment formation[END_REF]. Secondary Centres of Calcification (CoCs) can occur, playing a role similar to the nucleus, with growth marks radiating from the new centre of calcification instead of the nucleus; this in turn impacts the overall form of the otolith (Panfili et al., 2002). Unlike bone or scales, once deposited otoliths are reported to become inert within the organism and are not subject to remodeling or reabsorption (Campana and Neilson, 1985).

Otoliths grow via the alternative deposition of organic-rich and organic-poor increments. The mineral component forms 90-99%

by weight with the remainder being a matrix formed of an organic mixture (Degens et al., 1969). The organic mixture divides into a water-insoluble fraction and water-soluble fraction. The water-insoluble fraction is a non-collagenous protein containing a significant amount of acidic amino acids. The water-soluble fraction is composed of a mixture of proteins, polysaccharides [START_REF] Asano | Biochemical and calcium-binding properties of water-soluble proteins isolated from otoliths of the tilapia, Orechromis niloticus[END_REF], and glycoproteins (Dauphin and Dufour, 2003).

Being supplied by the metabolism of the fish, otoliths grow according to its circadian rhythms with a relation to the individual's environment. Experiments have shown that the photoperiod determines the deposition of each layer by demonstrating that inverting the light-dark cycle also inverts the layer deposition order [START_REF] Tanaka | Effects of photoperiod and feeding on daily growth patterns in otoliths of juvenile Tilapia nilotica[END_REF]. In experiments using isotopically labelled glutamate and 45 Ca this rhythm was verified in both light and dark layer formation occurring alternately [START_REF] Mugiya | Diurnal rhythm in otolith formation in the goldfish, Carassius auratus[END_REF][START_REF] Mugiya | Phase difference between calcification and organic matrix formation in the diurnal growth of otoliths in the rainbow trout, Salmo gairdneri[END_REF]. Mineralisation was found to occur primarily during day cycles, decreasing during the night [START_REF] Wright | Otolith calcification in Atlantic salmon parr, Salmo salar and its relation to photoperiod and calcium metabolism[END_REF]. However, the relation between environmental factors and otolith growth is not direct as cyclical deposition continues during experiments under constant light [START_REF] Campana | Lunar cycles of otolith growth in the juvenile starry flounder Platichthys stellatus[END_REF] or dark [START_REF] Radtke | Increment formation in the otoliths of embryos, larvae, and juveniles of the mummichog, Fundulus heteroclitus[END_REF].

Circadian rhythms produce daily growth marks, which are bipartite structures with alternating optical and chemical characteristics (2005). (Campana and Neilson, 1985). In temperate regions, seasonal cycles produce analogous bipartite annual growth marks (Figure 3), called annuli (Campana and Neilson, 1985). Annuli are also observed in fish from tropical regions, even though seasonal climatic variation is much less than in temperate regions [START_REF] Green | Tropical Fish Otoliths: Information for Assessment, Management and Ecology[END_REF]. This observation is contrary to the belief held since the early 20 th century that tropical fishes exhibit continuous activity, without annual variation.

1.2 organisation of an otolith from atoms to a macroscopic object 1.2.1 Aragonite: the mineral in biomineral 1.2.1.1 Crystal structure of aragonite Aragonite is one of three crystal polymorphs of calcium carbonate (CaCO 3 ). The structure of aragonite was determined by [START_REF] Bragg | The structure of aragonite[END_REF] Table 1: Aragonite lattice parameters determined by various authors [START_REF] Bragg | The structure of aragonite[END_REF] De [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF] [START_REF] Bragg | The structure of aragonite[END_REF] who revealed an orthorhombic unit cell in a Pmcn space group. Other authors have since continued to refine the structure (Tab. 1). Aragonite can be considered formed of Ca 2+ and trigonal planar CO 2 - 3 ions [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF]. From this view, the ions are stacked in alternating planes containing CO 2 - 3 groups and Ca 2+ ions (Fig. 4) in a hexagonal close packing arrangement [START_REF] Bragg | The structure of aragonite[END_REF][START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF]. In each layer of CO 2 - 3 groups, the carbon atoms are superimposed and the group is rotated 180°relative to the groups above and below it. In this arrangement, each Ca 2+ is surrounded by 9 oxygen atoms from 6 CO 2 - 3 groups, sharing three edges and three corners.

More precise refinements of the aragonite structure have been published by De [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF], [START_REF] Bevan | On the structure of aragonite -lawrence bragg revisited[END_REF], and Caspi et al. (2005). The unit cell parameters determined by De [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF] closely resemble those of [START_REF] Bragg | The structure of aragonite[END_REF]. A triclinic unit cell with dimensions similar to De [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF] was proposed by [START_REF] Bevan | On the structure of aragonite -lawrence bragg revisited[END_REF], but a later study by Caspi et al. (2005) supported the orthorhombic unit cell. A summary is presented in Table 1. The unit cell parameters determined by Caspi et al. (2005) are coherent with both [START_REF] Bragg | The structure of aragonite[END_REF] and De [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF], and exhibit the highest precision. As such, for the purposes of this work the results of Caspi et al. (2005) will be used as the reference values.

Crystal twinning

A crystal is said to be twinned when it is composed of two or more domains 5 whose crystal lattices are oriented according to a symmetry rule [START_REF] Kelly | Crystallography and Crystal Defects[END_REF]. Twinning introduces some limited disorder between domains, but the crystal lattice of each remains intact. The orientation occurs on a lattice level and can be produced either through deformation or during growth. For example, a crystal of aragonite grows along the c axis (in {110} planes) in an ABCD pattern, where C and D are rotations of 180°about the c axis of A and B, respectively. As layers grow one after another, ABCDABCD, a stacking fault could produce a mirror: ABCDCBA. The crystal is then composed of two parts whose c axes are mirrored. Aragonite crystals have a strong tendency toward twinning and frequently exhibit a hexagonal habit as a result of multiple twinning along the c axis [START_REF] Bragg | The structure of aragonite[END_REF][START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF][START_REF] Bevan | On the structure of aragonite -lawrence bragg revisited[END_REF]. In fact, it is unusual to find a single crystal of aragonite without any twinning [START_REF] Bragg | The structure of aragonite[END_REF][START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF].

Incorporation of elements by solid solution

A solid solution is formed when auxiliary (ie. not part of the chemical formula) atoms or ions are incorporated into the crystal lattice [START_REF] Kelly | Crystallography and Crystal Defects[END_REF]. Incorporation can occur in one of two modes: interstitial or substitution. An interstitial solid solution occurs when the additional occupants fill what would normally be a void in the crystal structure, while substitution is the replacement of one of the majority constituents (e.g. in aragonite, replacement of a Ca 2+ ion with a Sr 2+ ion). A substituent atom which is more similar to the atom being substituted (e.g. radius, charge) forms a substitutional solid more easily.

Strontium is one of the most common impurities in natural aragonite (Caspi et al., 2005). The similarity in size and charge of the Ca 2+ and Sr 2+ ions allows Sr to substitute for Ca in aragonite with relative ease (Ruiz-Hernandez et al., 2010). Further, strontianite, the carbonate of strontium (SrCO 3 ) is isostructural with aragonite [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF]. The unit cell of strontianite is slightly enlarged compared to aragonite because the Sr 2+ ion (131 pm (Lide, 2010)) is larger than the Ca 2+ ion (118 pm); the volume of the strontianite unit cell is 14 % larger than that of aragonite (Tab. 2).

In determining their proposition of a triclinic unit cell for aragonite, [START_REF] Bevan | On the structure of aragonite -lawrence bragg revisited[END_REF] used a sample containing 1 atom% Sr. This impurity was criticised by Caspi et al. (2005) who, using a sample containing only 0.143 atom% impurity (0.058 atom% Sr) reported a structure coherent with the previous orthorhombic cell. Although the lattice Table 2: Unit cell parameters of aragonite, strontianite, calcite, and vaterite, using data from Swanson and Fuyat (1953); De [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF]; Caspi et al. (2005), and Hayakawa et al. (2008). parameters are very close (Tab. 2), this contamination appears to have distorted the crystal lattice. Sr has been experimentally shown to be soluble in aragonite up to at least 58 µmol • g -1 when synthesised from aqueous solution at equilibrium (Plummer and Busenberg, 1987), and up to 160 µmol • g -1 in solid-solid solution simulations (Ruiz-Hernandez et al., 2010;[START_REF] Kulik | Ca,Sr)CO 3 Aqueous-solid solution systems: From atomistic simulations to thermodynamic modelling[END_REF].

Because of the relatively high Sr content of otoliths and its resistance to leaching compared to other components such as Na + and Cl -, Gauldie et al. (1998) suggested that the strontium of otoliths may occur within discrete strontianite domains. Macro-scale EXAFS6 experiments have identified the presence of strontianite domains in modern and ancient samples of coral, another aragonitic biomineral (Greegor et al., 1997). Given both the solubility of Sr in aragonite, and the suggestions of strontianite domains, it seems likely that the Sr chemical environment would lie somewhere on a spectrum between pure aragonitic (CaCO 3 : Sr) and pure strontianitic (SrCO 3 ). Investigation of Sr incorporation is important to confirm the reliability of current elemental analytical practices used with otoliths. Recent macro-scale analyses of ten otoliths from modern individuals from Australia did not find evidence of strontianite or mineral phases other than aragonite (Doubleday et al., 2014). The possibility of micro-scale presence of strontianite, particularly in ancient specimens susceptible to post-mortem alteration of the original mineralogy, remains open, however.

Vaterite and calcite can be observed in otoliths

In most teleost otoliths, the calcium carbonate is almost universally in the aragonite polymorph (Degens et al., 1969;Carlström, 1963;Gauldie, 1993), but the two other crystal polymorphs of CaCO 3 , vaterite and calcite, can also be found. Vaterite is the least thermodynamically-stable polymorph, followed by aragonite, with calcite being the most stable [START_REF] Bischoff | Catalysis, inhibition, and the calcite-aragonite problem; I. The aragonite-calcite transformation[END_REF][START_REF] Bischoff | Catalysis, inhibition, and the calcite-aragonite problem; II. the vaterite-aragonite transformation[END_REF].

Vaterite is a metastable crystal whose structure has been generally accepted as hexagonal (Kamhi, 1963), although debate continues as to the nature of a minor structure which has been observed [START_REF] Bail | Microtwinning hypothesis for a more ordered vaterite model[END_REF][START_REF] Kabalah-Amitai | Vaterite crystals contain two interspersed crystal structures[END_REF]. Perfect synthetic vaterite crystals are difficult to produce, which is a hindrance to the study of its structure. Biomineral samples have therefore been employed for structure determination because of their larger size and low number of defects [START_REF] Kabalah-Amitai | Vaterite crystals contain two interspersed crystal structures[END_REF]. Calcite is a rhombohedral crystal (Swanson and Fuyat, 1953), and is the most stable polymorph of calcium carbonate at ambient conditions [START_REF] Kunzler | The aragonite-calcite transformation; a problem in the kinetics of a solid-solid reaction[END_REF][START_REF] Mucci | The solubility of calcite and aragonite in sulfate-free seawater and the seeded growth kinetics and composition of the precipitates at 25 • c[END_REF].

The formation of calcite or vaterite in pathological aberrant sagittae has been reported in a number of marine and freshwater species from different environments (Strong et al., 1986;Tomas and Geffen, 2003;Béarez et al., 2005). The presence of a second polymorph in certain aberrant aragonitic otoliths may indicate either a local change in structure of the organic template permitting the second polymorph, or the absence of the organic template in the affected area (Strong et al., 1986). Where calcite is present in aragonitic otoliths, the variation may be a result of a fast-growing area on the otolith where there is insufficient template deposited, or damage to the neighbouring macular cells (Strong et al., 1986). In many fish with aragonitic sagittae, the asteriscii are composed of vaterite (Campana, 1999); the formation of aberrant vaterite regions in a sagitta may have a genetic origin (Tomas and Geffen, 2003).

Biomineral architecture

The smallest crystal units yet evidenced in biominerals are composite organic-inorganic nanogranules. Nanogranules have been observed

in a wide range of other aragonite biominerals, including the shells of abalone, nautilus, and echinoids [START_REF] Li | Nanoscale structural and mechanical characterization of a natural nanocomposite material: The shell of red abalone[END_REF]Addadi Figure 5: The structure of an otolith from atoms to a macroscopic object. Nanogranules in a broken untreated modern Gadus morhua otolith have been evidenced through AFM (a, reproduced from Dauphin and Dufour, 2008) in which the granules, as well as the matrix layer (white arrows) are visible. Secondary electron images reveal the individual prismatic crystalline units (few µm in size) of an archaeological acid-etched section of a Micropogonias otolith (b) are aligned along the growth axis (green arrow) in bundles several µm in diameter (c). Some D-zones are indicated by white arrows. In a broken, un-etched Pliocene era Diaphus sp. otolith, the fibres formed from these bundles (d, courtesy E. Dufour, using Tescan SEM) are continuous from their origin to the edge. A view along the growth axis (pointing out of the page) reveals the discrete bundles of fibres as they appear on the outer surface of the otolith (e, courtesy E. Dufour, using Tescan SEM). A schematic of the multiscalar organisation seen in a-d. Aragonite (i) is deposited in nanogranules (ii) which are coated in organic matrix. Nanogranules accumulate to form microcrystals (iii) which are organised into bundles (iv) to form the aragonite fibres (v) that compose the otolith (vi, thin section shown).

et al., 2006;[START_REF] Decker | Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro[END_REF]. Studies of biomineralisation have begun to elucidate the complex interplay between the organic and mineral fractions that govern growth [START_REF] Cölfen | Higher-order organization by mesoscale self-assembly and transformation of hybrid nanostructures[END_REF].

While crystals can grow strictly from precipitation according to thermodynamic equilibrium, biological systems use kinetic controls to produce complex structures [START_REF] Gebauer | Prenucleation clusters and nonclassical nucleation[END_REF]. In classical nucleation, ions form unstable precritical nuclei, some of which pass the critical point where precipitation becomes more energetically favourable than dissolution and continue to grow. The classical view has been described as failing in many quantitative predictions, leading to current theories on the biomineralisation process [START_REF] Gebauer | Prenucleation clusters and nonclassical nucleation[END_REF]. Rather than unstable precritical crystal nuclei, biomineralisation is now commonly described as proceeding via stable precritical clusters of amorphous calcium carbonate (ACC). The nanocusters self-assemble into mesocrystals, wherein each nanogranule is enclosed in an organic matrix envelope, but the assembly produces a common orientation. This common orientation results in behaviour resembling a single crystal. The common orientation and single crystal-like behaviour of mesocrystals means they can be difficult to identify from single crystals. It has been suggested that mesocrystals are more common than previously thought, and will be detected more frequently in future studies [START_REF] Niederberger | Oriented attachment and mesocrystals: Non-classical crystallization mechanisms based on nanoparticle assembly[END_REF]. Ultimately, mesocrystals may undergo fusion to form a true single crystal, although the extent of this behaviour remains to be confirmed [START_REF] Wohlrab | Polymerinduced alignment of dl-alanine nanocrystals to crystalline mesostructures[END_REF][START_REF] Niederberger | Oriented attachment and mesocrystals: Non-classical crystallization mechanisms based on nanoparticle assembly[END_REF].

Fluorescence microscopy of stained Danio rerio ultrathin otolith sections has revealed a mottled appearance, strongly suggesting the presence of nanoscale structure (Söllner et al., 2003). Direct evidence of nanogranules in otoliths has been provided though Atomic Force Microscopy (AFM). Gauldie (1999) noted the presence of granules in otoliths of Macruronus novaezelandiae using transmission electron microscopy (TEM). Using AFM, granular structure was also observed on the {110} faces of the crystalline units of Pagrus auratus and Macruronus novaezelandiae otoliths. The granules observed by AFM (Gauldie, 1999, Fig. 5) have major axes of ca. 100 nm and minor axes ca. 50 nm. [START_REF] Gauldie | Atomic force microscopy of the morphology of the matrix and mineral components of the otolith of hyperoglyphe antarctica[END_REF] noted the presence of granules diameters of 40-120 nm and mean diameters of 74, 77 and 82 nm in three otoliths of Hyperoglyphe antarctica. Round nanogranules with a diameter typically below 50 nm have been documented in fractured, untreated Gadus morhua otoliths by Dauphin and Dufour (2008). A difference between the interior and exterior of the nanogranules was identified using chemically-sensitive AFM phase images, suggested to be due to the presence of the organic envelope. Even if this nanoscale organisation has only been described in sagitta, it is expected to be identical in lapilli (and asterisci) since at the higher level (prismatic crystalline units) they share similar organisation. The observation of nanogranular ultrastructure7 in otoliths of multiple species, and the identification of nanogranules of similar size and appearance in other groups (e.g. bivalves, brachiopods, echinoids;[START_REF] Jacob | Nanostructure, composition and mechanisms of bivalve shell growth[END_REF] suggests that nanogranule-based growth is at the heart of biomineralisation and that understanding of similar biominerals can be applied to otoliths.

The nanogranules join to form prismatic crystalline units a few to several micrometres in size (Fig. 5iii andb), which are aligned along the growth axis (crystalline c axis) (Dauphin and Dufour, 2008). With circadian rhythms and changing seasons, the proportion of organic matrix to mineral varies, producing variations in composition which result in growth marks. [START_REF] Cuif | Evidence of a biological control over origin, growth and end of the calcite prisms in the shells of pinctada margaritifera (pelecypod, pterioidea)[END_REF] examined the growth of calcite in Pinctada margaritifera, identifying four growth stages of the prismatic crystallite units: (i) initial crystal disk formation, (ii) layered thickening, (iii) passage to prismatic polycrystalline structure, and

(iv) degradation of the mineralisation mechanism with coverage by a different organic layer. Mineralisation of the next crystalline unit resumes on the organic layer deposited in (iv). It may be that otolith growth undergoes a similar cycle. Crystal habits indicative of epitaxial growth have been observed on fractured surfaces of otoliths [START_REF] Davies | Otolith ultrastrucrure of smooth oreo, Pseudocyttus maculatus, and black oreo, Allocyttus sp., species[END_REF].

Growth marks are composed of narrower organic-rich layers (termed D-zones because they appear darker under optical microscopy), and broader organic-poor layers (L-zones because of their lighter appearance) (Panfili et al., 2002). Daily growth marks are typically on the order of 3-5 µm [START_REF] Davies | Otolith ultrastrucrure of smooth oreo, Pseudocyttus maculatus, and black oreo, Allocyttus sp., species[END_REF]. In secondary electron images (Fig. 6), D-zones appear as narrow, dark trenches while L-zones appear as bright plateaus because sample pretreatment by etching preferentially attacks organic-rich portions to produce a topographic contrast (Campana and Neilson, 1985). D-zones are generally laid during slower growth periods as at night while L-zones are laid during the day. The first growth mark is generally formed either at hatching or upon the switch from nutrition from the yolk sac to exogenous food (Panfili et al., 2002). Many crystalline units grow in parallel with the same growth mark periodicity (Fig. 5iv andc).

Bundles of crystalline units compose the aragonite fibres several micrometres in diameter (Fig. 5v andd) and reach hundreds of micrometres in length. The fibres are uninterrupted across the growth marks and exhibit only a change in relative composition. In highmagnification views of otolith sections obtained by SEM8 , an acid etching has been performed to reveal topography, resulting in voids along the length of the fibres (Figs. 5b,5c,and 6b). The prismatic crystalline units so revealed are a result of preparation. In an unetched sample no prismatic crystalline units can be distinguished (Fractured surface in Fig. 5d, polished section in Fig. 6a). This structure of continuous fibres is the natural state, not the isolated units frequently observed. The crystallites are said to be twinned across growth marks (ie. along the c axis), which may in part be a result of the increased organic fraction in D-zones, producing a disruption in the aragonite lattice (Gauldie, 1999). A high rate of twinning as been described in otoliths of Macruronus novaezelandiae, Oncorhynchus tshawytscha, and Pagrus major [START_REF] Gauldie | Aragonite twinning and neuroprotein secretion are the cause of daily growth rings in fish otoliths[END_REF]Gauldie, 1999). The rate of twinning in otoliths can vary from the higher rate observed in crossed lamellar portions of bivalve and gastropod shells to the very low rate observed in nacre (Suzuki , 2012). Twinning may contribute to increasing the growth rate, providing control over the crystal habit, and stabilising the "preferred" aragonite polymorph [START_REF] Smith | Feldspar Minerals[END_REF]Gauldie, 1999).

The final form of the otolith is determined by multiple factors. By the simplest reasonable description, otoliths are roughly ellipsoidal, but the imprecision of this view is evident on examining a real example (Fig. 8). An otolith maintains more or less the same form over a fish's lifespan, growing with the individual to maintain a constant relation to somatic size. The overall form is species-specific, but there are some common characteristics (Nolf, 2013). The inner surface is smooth, albeit with a groove (the sulcus, Fig. 8a) where the otolith contacts the macula. The outer surface (Fig. 8c) exhibits a rougher, less orderly appearance. The form is largely determined by the surrounding otosac which supplies its component ions. [START_REF] Payan | Chemical composition of saccular endolymph and otolith in fish inner ear: Lack of spatial uniformity[END_REF] indicate the concentration of Ca 2+ is uniform throughout the endolymph, but that the protein concentration is higher on the interior, while the CO 2 concentration (inversely related to carbonate) is higher on the exterior. They suggest that the concentration gradients play a role in the otolith form. For growth to take place along the c axis on any given direction in the complex form of an otolith, the aragonite fibres must be reoriented. The orientation of the aragonite fibres is dependent on their origin: a secondary CoC provides a new origin, changing the fibre orientation (Fig. 7). Oxygen, calcium, and carbon in the form of calcium carbonate are the most abundant elements found in otoliths by orders of magnitude, both by mass and atomic measurements. A wide variety of other elements from organic elements to heavy metals are known to be present in otoliths in varying quantities (Tab. 3), with others expected in yet-undetected concentrations (Campana, 1999).

Dorsal

Strontium is generally among the most abundant minor elements, and the only divalent cation at minor concentrations9 (Sr content is 8.0-48.0 µmol • g -1 ) (Campana, 1999;[START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF]. It is the only element with Z > 20 reported to exceed 20 µmol • g -1 . The similar radius of Sr 2+ (131 pm (Lide, 2010)) to the Ca 2+ ion (118 pm (Lide, 2010)) in 9-fold coordination allows it to substitute readily into the aragonite matrix, with a 4.6% increase in the lattice parameters (Caspi et al., 2005). It has been generally accepted that the strontium ions in otoliths are isomorphously substituted for Ca 2+ [START_REF] Bath | Strontium and barium uptake in aragonitic otoliths of marine fish[END_REF] since the Ca 2+ and Sr 2+ ions exhibit similar ionic radii, and the concentration is not expected to be beyond the solubility of Sr 2+

in aragonite, approximately 160 µmol • g -1 (Plummer and Busenberg, 1987;Ruiz-Hernandez et al., 2010). Some elements are incorporated from naturally-present elements in water while others originate from anthropogenic pollution [START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF]. The incorporation of various elements into the otolith, while potentially approximated by purely physical conditions of deposition from the endolymph solution (Campana, 1999), is in reality influenced by a combination of salinity, temperature, ambient ion concentrations, and the fish's physiology (Elsdon and Gillanders, 2003). So-called vital effects can result from multiple layers of physiological filters between the ambient water and the otolith, which produce a strict regulation of some ions while others remain weakly-or unregulated (Campana, 1999). To be used as an environmental proxy10 , an element must be incorporated into the otolith in a predictable manner reflecting the physical and chemical composition of the ambient water, with a limited vital effect (ie. physiological regulation). Strontium and barium are weaklyregulated, and represent the most-studied environmentally-linked elements (Campana, 1999). While the environmental relationship is not a simple ratio as can be found in simpler organisms such as corals (Shen et al., 1996), these elements are nevertheless highly useful indicators of a fish's life history. The presence of vital effects affects the incorporation of all elements to some extent, making any calibration species-specific. Both Sr and Ba have been demonstrated

to be useful and accurate environmental proxies, in both field studies (Elsdon and Gillanders, 2005a) and laboratory experiments (Elsdon and Gillanders, 2004). Ba can be a more sensitive indicator of life history (Elsdon and Gillanders, 2005a), but Sr has been used more frequently as an environmental proxy. The greater use of Sr likely results from its easier analytical accessibility, being present at concentrations ca. 100 times that of Ba (Tab. 3).

Stable isotopic composition and controls

The abundances of a variety of different isotopes have been measured in otoliths, including those of C, O, N, S, and Sr. Currently only C and O isotopic ratios are widely used for environmental reconstruction while others are under active development [START_REF] Kennedy | Using natural strontium isotopic signatures as fish markers: methodology and application[END_REF].

Temperature-based fractionation of oxygen isotopes between solid and solution phases during deposition of inorganic aragonite has long been established [START_REF] Mccrea | On the isotopic chemistry of carbonates and a paleotemperature scale[END_REF]. This phenomenon is equally present in biogenic aragonite (Epstein et al., 1953). The isotope fractionation ratio is typically expressed as a δ value, calculated (Campana, 1999) B 0.018 -0.68 (Campana, 1999)

C No references found* N O Na
81 -170 (Campana, 1999) Strong (Campana, 1999) 117 -184 [START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF]) 3.6 -174 (Morales-Nin et al., 2012a) Mg 1.1 -1.4 (Campana, 1999) Strong (Campana, 1999) Temperature (Elsdon and Gillanders, 2003) 0.46 -3.1 (Nakai et al., 1999) 0.01 -20 (Morales-Nin et al., 2012a) Al 0.074 (Campana, 1999) < 5.9 (Nakai et al., 1999) Si 0.29 -0.54 (Campana, 1999) P 1.6 -27 (Campana, 1999) Strong (Campana, 1999) S 9 -12 (Campana, 1999) Hypoxia (Limburg et al., 2015) 6.9 -38 [START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF] Cl 6.5 (Campana, 1999) [Cl] [START_REF] Fowler | Experimental assessment of the effect of temperature and salinity on elemental composition of otoliths using solution-based ICPMS[END_REF] < 35 [START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF] K 15 -28 (Campana, 1999) 7.2 -42 [START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF] Ca 9490 -10200 (Campana, 1999) Strong (Campana, 1999) Salinity (Elsdon and Gillanders, 2003) 9510 -10240 (Nakai et al., 1999) 8800 -11100 [START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF] 8020 -12400 (Morales-Nin et al., 2012a) Mn 0.16 -0.20 (Campana, 1999) Limited (Campana, 1999) [Mn], Temperature (Elsdon and Gillanders, 2003), Hypoxia (Limburg et al., 2015) <3. 3 (Nakai et al., 1999) 0 -4.4 (Limburg et al., 2015) Fe 0.54 -11.5 (Campana, 1999) Temperature [START_REF] Gauldie | The relationship of the iron content of some fish otoliths to temperature[END_REF]) < 4.2 (Nakai et al., 1999) Co 0.002 -0.008 (Campana, 1999) Ni 0.032 -0.16 (Campana, 1999) Cu 0.016 -0.063 (Campana, 1999) Strong (Campana, 1999) Temperature (Elsdon and Gillanders, 2003) 0.03 -0.11 (Limburg et al., 2015) Zn 0.14 -0.7 (Campana, 1999) Limited (Campana, 1999) [Zn] (Campana, 1999) < 3.3 (Nakai et al., 1999) 0.03 -0.23 (Limburg et al., 2015) Continued on page 20.

Continued from page 19.

Element Range Physiological

Z Sym. (µmol • g -1 ) control Influences As 0.3 (Campana, 1999) [As] [START_REF] Fowler | Experimental assessment of the effect of temperature and salinity on elemental composition of otoliths using solution-based ICPMS[END_REF] Se 0.0019 (Campana, 1999) [Hg] [START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF] 0 -0.05 [START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF] Br 0.018 -0.025 (Campana, 1999) < 0.11 (Limburg et al., 2010a) Rb Sr 8 -24.4 (Campana, 1999) Limited (Campana, 1999) [Sr], Temperature, Salinity (Elsdon and Gillanders, 2003) 8.5 -31.0 (Nakai et al., 1999) 16 -44 [START_REF] Thresher | An Evaluation of electron-probe microanalysis of otoliths for stock delineation and identification of nursery areas in a southern temperate groundfish, Nemadactylus macropterus (Cheilodactylidae)[END_REF] 8 -51 (Albuquerque et al., 2010) 13-37 (Limburg et al., 2015) 5 -137 (Lin et al., 2015) Y Ag 0.0015 (Campana, 1999) Cd 0.0016 (Campana, 1999) I 0.003 -0.028 (Limburg et al., 2015) Hypoxia (Limburg et al., 2015) Cs 2×10 -5 (Campana, 1999) Ba 0.027 -0.080 (Campana, 1999) Limited (Campana, 1999) [Ba] [START_REF] Fowler | Experimental assessment of the effect of temperature and salinity on elemental composition of otoliths using solution-based ICPMS[END_REF], Temperature, Salinity (Elsdon and Gillanders, 2003) 0 -1.8 (Albuquerque et al., 2010) Hg 0.014 (Campana, 1999) Pb 0.0013 -0.0029 (Campana, 1999) Limited (Campana, 1999) [Pb] [START_REF] Fowler | Experimental assessment of the effect of temperature and salinity on elemental composition of otoliths using solution-based ICPMS[END_REF] Th U 0.0001 (Campana, 1999) relative to a standard reference as exemplified in Equation 1 for 18 O and 16 O.

δ 18 O(‰) = (O 18 /O 16 ) sample (O 18 /O 16 ) reference -1 × 1000 (1)
Relationships between otolith δ 18 O values and the environment in which an individual lived have been identified by multiple studies on both fish from the wild and raised in controlled conditions (Degens et al., 1969;[START_REF] Iacumin | Oxygen and carbon isotopic composition of fish otoliths[END_REF][START_REF] Patterson | Continental paleothermometry and seasonality using the isotopic composition of aragonitic otoliths of freshwater fishes[END_REF][START_REF] Thorrold | Natal homing in a marine fish metapopulation[END_REF][START_REF] Geffen | Otolith oxygen and carbon stable isotopes in wild and laboratory-reared plaice (pleuronectes platessa)[END_REF]. However, numerous equations relating water temperature to δ 18 O have been determined, indicating a strong possibility of vital effects [START_REF] Geffen | Otolith oxygen and carbon stable isotopes in wild and laboratory-reared plaice (pleuronectes platessa)[END_REF]. Contrary to the fractionation of oxygen, there is no direct temperature effect on carbon isotope fractionation but a strong metabolic effect (Kalish, 1991a;[START_REF] Solomon | Experimental determination of the sources of otolith carbon and associated isotopic fractionation[END_REF][START_REF] Dufour | Whitefish (coregonus lavaretus) respiration rate governs intra-otolith variation of delta c-13 values in lake annecy[END_REF][START_REF] Geffen | Otolith oxygen and carbon stable isotopes in wild and laboratory-reared plaice (pleuronectes platessa)[END_REF]. The relative contribution of organic metabolic sources and ambient dissolved inorganic carbon (mostly CO 2 ) vary over the individual's life and can indicate specific life events and seasonal variations in metabolism [START_REF] Dufour | Whitefish (coregonus lavaretus) respiration rate governs intra-otolith variation of delta c-13 values in lake annecy[END_REF][START_REF] Schwarcz | Stable carbon isotope variations in otoliths of atlantic cod (gadus morhua)[END_REF].

1.4 otoliths as information archives 1.4.1 Taxonomy

The shape of an otolith is species-specific and can thus be used for species identification. Nolf (1985) reports that Cuvier was the first to remark on the taxonomic utility of otolith shapes in 1836, and the first use for palaeontological identification of fish species was by Koken in 1884. Identification of fish species from otoliths in the stomach contents of animals such as birds or marine mammals can serve to determine diet [START_REF] Alonso | Stomach contents of false killer whales (Pseudorca crassidens) stranded on the coasts of the Strait of Magellan, Tierra del Fuego[END_REF].

Age and growth

Estimation of an individual's age is by far the most common application of otoliths, with hundreds of thousands of otoliths examined every year by fisheries institutes worldwide (Campana and Neilson, 1985;Panfili et al., 2002). Examining the otolith under high magnification (using either light or electron microscopy) permits distinction of D-and L-zones composing daily growth increments or annual growth increments (Pannella, 1971). Daily marks can usually be resolved in the first year of growth, but become narrower and more difficult to discern afterward (Pannella, 1971;Campana and Thorrold, 2001).

Counting the growth marks then provides an estimate for the individual's age, although the periodicity of marks must be validated to ensure accurate estimates (Campana, 1999). Life history can be inferred directly from the growth marks (Pannella, 1971), where a thicker growth mark generally indicates a prosperous year while a more narrow mark may result from various stressors, such as a lack of food or deterioration of habitat (Campana and Neilson, 1985).

Aperiodic marks can also appear in relation to significant life events such as reproduction, a change of living environment, or maturation, and can be used to reconstruct life traits (Panfili et al., 2002). The size and weight of otoliths are strongly correlated to the somatic size of the individual, with a particular size-weight relationship per species [START_REF] Béarez | Archaic fishing at quebrada de los burros, southern coast of peru. reconstuction of fish size by using otoliths[END_REF]. The establishment of these metric relations allow backcalculation of the size of fish from their otoliths (Campana, 2005).

Environment and physiology

The environmental conditions of growth, such as water temperature and salinity, can be estimated by examining the otolith chemistry.

These factors are known to influence the chemistry of otoliths in the same way as other aragonite biominerals, including corals (Finch et al., 2003). Isotopic composition can also serve as a life history tracer for fish by identifying marine residence from freshwater, relying on the relative depletion of 18 O in fresh water compared to the ocean [START_REF] Epstein | Variation of O-18 content of waters from natural sources[END_REF]Degens et al., 1969). A wide range of studies rely on elemental data as records of fish movement for questions of natal origin (Elsdon et al., 2008;Curtis et al., 2014), stock identification (Limburg et al., 2010a), migration history tracking [START_REF] Morales-Nin | Seasonal and ontogenic migrations of meagre (Argyrosomus regius) determined by otolith geochemical signatures[END_REF], or pollution monitoring [START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF]. The use of elemental fingerprinting is frequently reported to identify the natal origins of fish in a given stock by matching their otolith elemental content to known spawning grounds (e.g. [START_REF] Campana | Otolith elemental fingerprinting for stock identification of atlantic cod (Gadus morhua) using laser-ablation ICPMS[END_REF][START_REF] Thorrold | Natal homing in a marine fish metapopulation[END_REF][START_REF] Gillanders | Using elemental chemistry of fish otoliths to determine connectivity between estuarine and coastal habitats[END_REF][START_REF] Svedäng | Migratory behaviour and otolith chemistry suggest finescale sub-population structure within a genetically homogenous atlantic cod population[END_REF][START_REF] Amano | Otolith elemental and sr isotopic composition as a natal tag for Biwa salmon Oncorhynchus masou subsp. in Lake Biwa, Japan[END_REF]. For wider-scale life history tracking, measurements of the variation in elements such as Sr or Ba across the fish's life can be calibrated by environmentallycontrolled experiments to relate the elemental content of the otolith to the water conditions, such as temperature and salinity [START_REF] Edmonds | Stock discrimination by trace-element analysis of otoliths of orange roughy (Hoplostethus Atlanticus), a deep-water marine teleost[END_REF][START_REF] Secor | Identification of riverine, estuarine, and coastal contingents of hudson river striped bass based upon otolith elemental fingerprints[END_REF]. Each calibration, however, is valid only for the species in question due to strong vital effects [START_REF] Urey | Measurement of paleotemperatures and temperatures of the upper cretaceous of England, Denmark, and the southeastern United States[END_REF][START_REF] Dimaria | Temperature and growth effects on otolith elemental chemistry of larval Pacific cod, Gadus macrocephalus[END_REF]Hicks et al., 2010). Even so, it remains possible to produce approximations using environmental classifications of fresh/intermediate/salt water to monitor movements between a few known areas, particularly where diadromous movement 11 occurs (Elsdon and Gillanders, 2005a).

11 Movement between fresh and salt water, catadromy refers specifically to birth at sea and migration to freshwater while anadromy refers to birth in freshwater and migration to a marine environment.

Otoliths as archives for palaeontology and archaeology

As rich biological archives, otoliths found in excavations can bring a wealth of information. By using the otolith shape to determine the originating species, biostratigraphic analyses can be performed by examining the presence of known reference species to date a geological layer [START_REF] Reichenbacher | Biota, palaeoenvironments and biostratigraphy of continental Oligocene deposits of the South German Molasse Basin (Penzberg Syncline)[END_REF]Nolf, 2013). Such examinations also permit the palaeontological identification of the earliest appearance of a family based on the otoliths. Teleost families have been identified as early as the Jurassic period using this approach (Nolf, 2013). Taxonomic identification of fossil otoliths can also serve to reconstruct palaeoclimate 12 and palaeoenvironment 13 by placing bounds on the possible conditions by cataloguing the presence of species with known habitat ranges (Nolf, 2013). In archaeology, otoliths can be used to determine the list of species fished by ancient humans [START_REF] Béarez | Archaic fishing at quebrada de los burros, southern coast of peru. reconstuction of fish size by using otoliths[END_REF]Nolf, 2013). Such studies can help to elucidate the subsistence strategies of ancient populations in marine, riverine, or lacustrine environments [START_REF] Béarez | Archaic fishing at quebrada de los burros, southern coast of peru. reconstuction of fish size by using otoliths[END_REF]Disspain et al., 2012).

As archives of size and age, archaeological otoliths can be used to estimate the size of fish captured by ancient peoples, and thus infer the fishing technology they employed and the weight of fish flesh consumed [START_REF] Béarez | Archaic fishing at quebrada de los burros, southern coast of peru. reconstuction of fish size by using otoliths[END_REF]. The growth curve for an ancient fish population can be compared to that of modern populations, allowing identification of possible changes in maturation rate over time. For example, the impact of the fishing industry on the commercially exploited fish populations has been quantified by back-calculating size and age for medieval fish compared to modern specimens for five species in the North Sea [START_REF] Bolle | Growth changes in plaice, cod, haddock and saithe in the North Sea: a comparison of (post-)medieval and present-day growth rates based on otolith measurements[END_REF].

The elemental and isotopic composition of otoliths can serve as valuable complements to other palaeoproxies 14 such as corals.

Compared to corals, which can contain a low temporal resolution environmental record over hundreds of years in their geochemistry, otoliths can contain records over a few years to decades with a seasonal or subseasonal temporal resolution. This ability to temporally correlate data on a subseasonal time scale allows, for example, the use of otoliths to observe seasonal temperature fluctuations using δ 18 O as a temperature proxy (Patterson, 1998; The climate in the geological past.

The environment in the geological past. Proxies for environment, climate, ecology, etc. in the geological past. Béarez et al., 2011). After a long maturation period, otolith microchemistry is now a rapidly-growing field: only 6 papers were published in this domain prior to 1980 while there are now over 1000 articles published using elemental analyses and nearly 200 published on isotopic composition.

The elemental concentrations in ancient water systems cannot be known, but using a classification approach opens the possibility of application of chemical analyses to ancient specimens (Disspain et al., 2011(Disspain et al., , 2012)). Disspain et al. (2011Disspain et al. ( , 2012) ) to examine ancient ecosystems and the movements of fish within them (Disspain et al., 2011(Disspain et al., , 2012;;Long et al., 2014). Ancient human populations have been studied by inferring fishing practices based on the palaeoenvironmental and palaeoecological data recorded in the otoliths of the fish they consumed (Béarez et al., 2011;Carré and Dufour, 2012;Disspain et al., 2012;Long et al., 2014).

Isotopic analyses have so far been used in archaeological otoliths more frequently than elemental analyses. In an analysis of δ 18 O, influence of ambient water δ 18 O and temperature are examined simultaneously (Kalish, 1991a). For marine species, this does not pose a difficulty since the ambient δ 18 O in any modern ocean is very close to 0‰, and for ancient oceans the δ 18 O has been determined by long-term geological records such as ice cores. This simplifying assumption has allowed studies of the palaeoclimatic sea surface temperature on an individual's lifetime (Devereux, 1967), interannual [START_REF] Schwarcz | Stable carbon isotope variations in otoliths of atlantic cod (gadus morhua)[END_REF], or seasonal time scale [START_REF] Dufour | Stable isotopes in fish otoliths discriminate between lagoonal and oceanic residents of taiaro atoll (tuamotu archipelago, french polynesia)[END_REF][START_REF] Wurster | Advances in micromilling techniques: a new apparatus for acquiring high-resolution oxygen and carbon stable isotope values and major/minor elemental ratios from accretionary carbonate[END_REF]. Hufthammer et al. (2010) examined otoliths from a Stone Age site in Norway to determine the seasonal water temperature cycle using δ 18 O as a paleotemperature proxy. The otoliths were determined to have come from fish caught in the late winter or early spring, indicating the site was occupied in this period.

Contrary to the relative ease of temperature history reconstruction in marine species, the δ 18 O thermometer in diadromous and freshwater species is complicated by the highly variable ambient isotopic ratio in freshwater environments [START_REF] Epstein | Variation of O-18 content of waters from natural sources[END_REF].

Multiple approaches have been used to overcome this variation.

One study examined the otoliths of one ancient species living in nearly constant-temperature water to determine the ambient isotopic content [START_REF] Patterson | Continental paleothermometry and seasonality using the isotopic composition of aragonitic otoliths of freshwater fishes[END_REF]. Knowing the water chemistry allows analysis of samples belonging to a second ancient species living near the surface of the same water body to determine the seasonal temperature fluctuations [START_REF] Patterson | Continental paleothermometry and seasonality using the isotopic composition of aragonitic otoliths of freshwater fishes[END_REF].

In another case, no constant-temperature reference species were available, but approaches have nonetheless been found to determine the palaeotemperature from a single species. A constraint can be placed if it is known the selected species effectively does not grow below a certain temperature: the lowest recorded temperature on the otolith must be at least equal to the minimum growth temperature since there was no growth of the individual or its otoliths in the time the individual spent below the minimum (Patterson, 1998).

The substantial variation in δ 18 O between freshwater and the ocean can not only be overcome, but also used in its own right as a life history tracer in the same way as elemental signals [START_REF] Elsdon | Interactive effects of temperature and salinity on otolith chemistry: Challenges for determining environmental histories of fish[END_REF]. Using a time profile of isotopic composition the migration of an extinct Late Cretaceous species from South Dakota, Vorhisia vulpes, has been identified (Carpenter et al., 2003). Multiple individuals were analysed, revealing birth and initial growth in estuaries followed by migration to the ocean, contrary to the previous belief that the species was freshwater-dwelling (Carpenter et al., 2003).

As measurements of δ 18 O become increasingly common, use of In general, "neutral" resins -those which do not contain significant concentrations of elements of interest (e.g. Ca, Sr, Ba) -are selected, although there can be some metals (e.g. Fe, Cu) present in the hardener. It was suggested that a resin could also leach endogenous elements from the otolith, notably through chelation by the common preservative ethylenediaminetetraacetic acid (EDTA), which forms strong complexes with transition metal ions including Mn 2+ , Cu 2+ , and Fe 3+ (Jolivet et al., 2013;[START_REF] Miessler | Inorganic Chemistry[END_REF]. A significant contribution of embedding resin (5-30 % of the signal) has been observed in δ 13 C analyses of beluga teeth [START_REF] Stewart | Bomb radiocarbon dating calibrates beluga (Delphinapterus leucas) age estimates[END_REF].

Further complicating the impact of this contamination, the resin was unevenly distributed in the samples examined by Jolivet et al.

(2013), with the otolith core typically being more contaminated. It was suggested that further work on the impact of resin contamination is necessary.

In age estimation studies, various liquids including water, solvents, or stains are often used to improve visualisation of growth marks by altering the transparency of or contrast between D-and L-zones (Gauldie et al., 1998). For these treatments to have an effect, the water and dye molecules must be able to diffuse into the otolith (Gauldie et al., 1998). Indeed, weight increases of up to 4.18% were demonstrated after 50 hours of soaking in deionised water, assumed to be within interstitial spaces and possible microcapillaries (Gauldie et al., 1998). If the otolith is in fact a body with microchannels and pores, not a solid mass as it is usually considered, this raises questions on its stability and reliability (Gauldie et al., 1998), and opens the possibility of diffusion of ions into and out of the otolith bulk (Andrus and Crowe, 2002). In fact, freezing (Milton and Chenery, 1998), storage in chilled seawater (Proctor and Thresher, 1998), storage in ethanol (Milton and Chenery, 1998;[START_REF] Hedges | Effects of ethanol preservation on otolith microchemistry[END_REF], polyethylene or paper containers [START_REF] Swan | Effects of handling and storage methods on the concentrations of elements in deep-water fish otoliths[END_REF], regular handling (Rooker et al., 2001), forceps material used in otolith removal [START_REF] Swan | Effects of handling and storage methods on the concentrations of elements in deep-water fish otoliths[END_REF], or delaying otolith removal without special treatment (Milton and Chenery, 1998) can result in detectable differences in concentration.

The nature and intensity of the alteration are expected to be different among elements if they are considered as labile or weaklybound (e.g. Na + , Cl -), or assumed to be in substitution for Ca 2+ (e.g. Cl, but only weak signs of alteration of S, Ca, and Sr content (Proctor and Thresher, 1998). Immersion in water for several minutes was sufficient to deplete K, Cl, and S, while enhancing Na. A study based on diffusion of the cacodylate anion ((CH 3 ) 2 O 2 As -) showed that the arsenic was able to intrude into the otolith, but that the Ca, Sr, Na, and F did not appear to be altered (Gauldie et al., 1997). It was noted that strontium appears stable under most conditions, which was regarded as supporting the assumption that Sr was within the aragonite matrix (Proctor and Thresher, 1998). In a recent study involving otolith samples from modern Australian marine fishes, Doubleday et al.

(2014) supported the hypothesis of Sr randomly substituting Ca in aragonite. This likely also applies for barium, indicating that the most-used elements for environmental tracing remain largely, but not completely, unaffected by conditions or duration of storage of the fish before preparation. While this is an encouraging observation for the use of otoliths as environmental proxies, further studies are necessary to confirm its wider applicability, as other authors have suggested the presence of strontianite domains in otoliths (Gauldie et al., 1998) and corals (Greegor et al., 1997). Furthermore, if Sr in random substitution for Ca is confirmed as a general characteristic in modern otoliths, a different mode of incorporation would provide a direct indicator of post-mortem alteration. The effect on other divalent cations such as Mg, Zn, and Mn, which are also typically assumed to be strongly associated with the aragonite matrix in substitution for calcium [START_REF] Hedges | Effects of ethanol preservation on otolith microchemistry[END_REF] raises questions. Proctor and Thresher (1998) observed that although the magnitude of the elemental concentrations were altered, the overall ontogenic 15 trends were intact in full elemental profiles from the primordium to the edge for frozen and ethanol-stored otoliths. This relative stability is encouraging for the use of modern samples to observe seasonalitythe relative annual fluctuations are preserved -but poses a greater issue for reconstructions relying on the absolute concentrations.

Alteration in archaeological contexts

Archaeological otoliths having been deposited for thousands of years or longer can be readily expected to have undergone some level of post-mortem alteration. An archaeological otolith must endure conditions such as burial, exposure to ambient water, and potentially cooking between the fish's capture or death and analysis. These conditions may alter mineralogy, microstructure, and the original biogenic chemical signal. Storage of modern specimens after capture has been demonstrated to impact otolith chemistry; storage of the fish caught by ancient fishers would certainly have a similar impact. For otoliths found at coastal sites, storage duration may have been be minimal, but for those found at sites several kilometres inland, prehistoric transport and thus the effect of storage duration and conditions are expected to be non-negligible. Preparation for consumption by ancient fisher societies could present a significant

impact. An examination of the effect of five different cooking techniques on geochemical composition has been performed on otoliths from two species: Micropogonias undulatus and Bagre marinus (Andrus and Crowe, 2002). The left otolith of each fish was removed as a reference and the right remained in situ for the cooking. The five cooking techniques tested were: boiling in seawater, boiling in fresh water, roasting in a dry oven, roasting over a wood fire, and burning directly on the coals. Burning caused a discolouration, loss of structural integrity, and complete transformation to calcite, but the other cooking techniques did not produce an effect detectable by visual observation or XRD 16 (Andrus and Crowe, 2002). The δ 13 C and δ 18 O of the otoliths were also not altered except by burning. Burning produced a decrease in δ 13 C of 7.8‰ and 3.6‰ in M. undulatus and B. marinus, respectively (Andrus and Crowe, 2002). The δ 18 O decreased by 3.1 ‰ and 1.8 ‰ for the same samples. Contrary to the isotopic stability observed, all cooking treatments produced some effect on the elemental concentrations, with strontium and magnesium among the most frequently altered elements, although no element is altered in every treatment (Andrus and Crowe, 2002). In the control pair, the right (uncooked) otolith exhibited an increase in Sr relative to the previously-removed left (reference) otolith of 1.2 and 1.9 µmol • g -1 for M. undulatus and B. marinus, respectively. The cooked fish exhibited a difference (generally an increase) between the reference and cooked otoliths of up to 8 times these amounts (Andrus and Crowe, 2002).

Diagenetic alteration of otoliths over the long-term has been poorly explored, compared to other biominerals. Despite the lack of explicitly-proposed diagenetic trajectories, some hypotheses can be advanced on the basis of aragonite behaviour. Aragonite is metastable and can undergo a transformation to calcite over time (Plummer and Busenberg, 1987), suggesting that on a sufficient time scalemillions of years for a dry, ambient environment -otoliths would transform to calcite. This transformation can, however, be accelerated to laboratory timescales in the presence of water, as well as increased temperatures [START_REF] Bischoff | Catalysis, inhibition, and the calcite-aragonite problem; I. The aragonite-calcite transformation[END_REF].

Although alteration may be expected with greater severity on the outer surface in contact with the soil (Cochran et al., 2010),

it is in fact a concern for the whole otolith. Diffusion of liquids in otoliths has been demonstrated, as well as the leaching of endogenous elements intrusion of exogenous compounds. The current set of tools to detect altered specimens consists of visual examinations for morphological changes (Disspain et al., 2012), SEM examination of the microstructure (Dufour et al., 2000), and analysis of mineral phase by infrared spectroscopy, XRD (Trueman, 2013),

or cathodoluminescence (Vanhove et al., 2011). These examinations of the mineralogy and microstructure of an otolith is necessary and useful to determine the possibility of alteration, but not sufficient for geochemical analyses as alterations may occur without crystallographic alteration or visible modification (Dufour et al., 2000).

As a basic post-facto control for significant alteration, the rationality of palaeoenvironmental reconstructions should be considered within the context of the species or taxon and compared to available known modern relatives. A reconstruction suggesting a different environment than that hospitable to the species or its modern relatives should be carefully considered (Dufour et al., 2000). Table 4 summarises a compilation of recent works on ancient otoliths. These works often consider the preservation state (e.g. microstructure and/or mineralogy) of ancient otoliths, but do not always explicitly otoliths: life history archives Control for alteration is frequently performed using X-ray Approximately 96 % of all modern fish are teleosts, one-third of which are percifomes [START_REF] Nelson | Fishes of the World[END_REF].

2.2 description of selected otolith samples

Archaeological interest and geographical context

The role of the tropics in driving climate change has been recognized only since the 1970s [START_REF] Kerr | The tropics return to the climate system[END_REF]. The tropical Pacific Ocean is the center of action of the ENSO (El Niño Southern Oscillation), the strongest source of interannual climate variability on the planet.

ENSO represents one of the most important driving factors in the global climate system (Webster and Palmer, 1997;Tudhope and Collins, 2003). The costal waters of Peru are at the heart of the phenomenon (Sandweiss and Richardson III, 2008;Webster and Palmer, 1997). The modern ENSO phenomenon has a frequency of 4-7 years. Historical understanding of El Niño activity on the Peruvian coast in the past has often been limited by the proxies available (Tudhope and Collins, 2003), notably warm-water corals -absent from the cool waters of the Humboldt Current -foraminifers, and speleotherms, so new, subseasonal-resolution palaeoclimate proxies are welcomed [START_REF] Carré | Exploring errors in paleoclimate proxy reconstructions using Monte Carlo simulations: Paleotemperature from mollusk and coral geochemistry[END_REF]. Studies of mollusk assemblages as well as isotopic measurements of shells and otoliths from sites localized at different latitudes of the Peruvian and Chilean coast have brought insight into East Pacific marine conditions (Carré et al., 2012a), but interpretation of the data from such palaeoenvironmental proxies can remain controversial [START_REF] Béarez | Comment on "Otolith δ 18 O record of mid-Holocene sea surface temperatures in Peru[END_REF].

All archaeological material in this study comes from the coast of northern Peru. This region, located in the intertropical zone, is a long desert on the Pacific coast from 5-30°S (Fig. 10). The desert is bordered on the west by the cold waters of the Humboldt Current system and on the east by the Andes mountains, spanning a distance of about 20-200 km inland. The Humboldt Current is affected by strong coastal upwelling activity that brings phytoplankton nutrients to the surface enhancing biological productivity. As a result, the northern Humboldt Current ecosystem is one of the world's richest fisheries [START_REF] Chavez | The northern humboldt current system: Brief history, present status and a view towards the future[END_REF]. This region, although it is one of the most arid in the world, has been inhabited for over 13 000 years (Sandweiss and Richardson III, 2008). The desert coast is interspersed by river valleys running perpendicularly, bringing water from the Andes to the Pacific ocean. In the foothills of the mountains, verdant lomas form where moisture collects from air forced upward.

These local environments strongly influenced the location of human settlements. The reliance on abundant and reliable resources brought by the ocean and these local environments supported the long term occupation of the region (Sandweiss and Richardson III, 2008;Dillehay et al., 2012).

Archaeological otoliths

The archaeological otoliths examined in this work (Tab. 5) hold a dual potential to reveal both seasonal palaeoclimate and to support study of the humans who occupied the region (Fig. 10) over thousands of years. The otoliths in the present work originate from three sites (Fig. 11) associated with two Peruvian prehispanic periods.

Paiján

One of the first human groups of northern Peru, known as the Paiján, occupied an area near the Pacific coast approximately 11 000-8000 BP (Chauchat and Pélegrin, 1994). The Paiján were fisher-huntergatherers with a varied diet in which the primary biomass was fish (Béarez et al., 2011). These human groups were small and mobile, resulting in many small archaeological sites, each of which represents a short period of occupation [START_REF] Dillehay | Profiles in pleistocene history[END_REF]. At the time of the Paiján samples and sample preparation groups, sea levels were lower; Paiján camps currently 15 km inland would have been closer to 25 km from the shore [START_REF] Sandweiss | Terminal pleistocene through mid-holocene archaeological sites as paleoclimatic archives for the peruvian coast[END_REF].

Multiple studies suggest that in northern Peru, the ENSO was not present at the time. There is evidence based on the species identified during excavations that the coastal waters near the Cupisnique Desert were calmer, warmer, and less saline than present (Béarez et al., 2011).

This has been explained as resulting not necessarily directly from variations in coastal seawater characteristics, but rather from lagoons or estuaries fed by the mountain rivers, producing an environment amenable to species which no longer occupy this coastal region.

The otoliths recovered from archaeological explorations related to the Paiján are typical of tropical species whose modern range is incompatible with current cool saltwater conditions near the Paiján site, and only one of the 12 species identified during explorations currently inhabits the region. Barring major migrations this suggests warmer, brackish conditions resembling those at the extreme north of Peru, with additional warmer fresh water supplied and estuaries formed by the rivers descending from the Andes.

The otoliths QCD and BH17 used in this study were recovered from the surface layer during a recent exploration by Claude Chauchat (CNRS UMR 8096) and Philippe Béarez at La Pampa de los Fósiles, an inland area of the Cupinisque desert, pictured in Figure 11a; the location is marked in Figure 10.

Huaca Prieta

The Huaca Prieta (Fig. 11b) in the Chicama river valley (Fig. 10) is a monumental mound, built in layers of refuse, including ceramics, faunal remains, wood and ashes (Dillehay et al., 2012). The mound was built up over 13 700-3800 years before present (BP) to its current size of 168 × 62 × 32 m 3 (Dillehay et al., 2012;[START_REF] Grobman | Preceramic maize from Paredones and Huaca Prieta, Peru[END_REF].

It was first excavated in the 1940s by Junius Bird and was recently the site of a campaign from 2007-2011 supervised by Tom Dillehay (2012). Excavations have revealed human burials, lithic artefacts, and many floral and faunal remains. The recent campaign established a precise chronology with the use of carbon dating and close attention to stratigraphy (Dillehay et al., 2012). The present work includes three otoliths from this campaign. The otoliths originate from strata with conventional radiocarbon ages of 3588±36 BP for HP-1 (Dillehay et al., 2012), 4140±40 BP for HP-11 (E. Dufour, pers. comm.), and 6641±49 BP for HP-12 (Dillehay et al., 2012). In this time period, studies indicate the coastal waters had a cool Sea Surface Temperature (SST) (like present-day) and a low-frequency ENSO had begun to occur starting ca. 5800 cal BP [START_REF] Sandweiss | Terminal pleistocene through mid-holocene archaeological sites as paleoclimatic archives for the peruvian coast[END_REF].

Bayovar

Bayovar-01 designates a site in the Sechura Desert (Figure 11c)

in the far north of Peru (Figure 10). The site is dated to the Early Intermediate Period (1526-1255 BP) [START_REF] Goepfert | Programme archéologique desert de sechura, pérou: Rapport sur la campagne 2012 bayovar-01[END_REF]Goepfert et al., 2014, Goepfert, pers. comm.). Excavations produced 100-150 otoliths/m 2 , corresponding to species which prefer calm, littoral 1 areas [START_REF] Goepfert | Programme archéologique desert de sechura, pérou: Rapport sur la campagne 2012 bayovar-01[END_REF]. Modern coastal waters are turbulent, leading to the hypothesis that conditions have changed and there may have once been a calm lagoon with easy access for fishing. Because of the organised nature of the archaeological site, it is hypothesised that a specialised settlement was built to exploit this resource by a sedentary population that had practised agriculture [START_REF] Goepfert | Programme archéologique desert de sechura, pérou: Rapport sur la campagne 2012 bayovar-01[END_REF].The otolith Bayovar-1C included in the present work originates from a surface layer of the Bayovar-01 site.

Modern otoliths

Modern reference specimens collected by P. on its δ 18 O and δ 13 C profiles (E. Dufour, pers. comm.). A second modern M. altipinnis sample (119) was selected due to its isotopic life history profile, which indicated a progressive change in δ 18 O from -4 ‰ to 0 ‰ and δ 13 C from -9 ‰ to -2 ‰ (E. Dufour, pers. comm.).

Sample preparation

Otoliths have been examined in the laboratory for many purposes for many decades. Their preparation for examination is a process whose steps have become relatively standardised, but there remain significant variations from one research group to another in the implementation of those steps. In general -and in the present work -the otolith is embedded in a polymer block for stability, sectioned with a precision saw, and polished to reveal the growth marks.

The otoliths used in this study were prepared in such a way to maximise the exposed surface area of each isochronous growth mark to facilitate sampling by micromilling. As such, the Micropogonias sp.

otoliths have been cut in a frontal section, the G. Peruvianus in a transverse section, and Cathorops sp. in a transverse section. The three section types are shown in Figure 12. For only a visual examination to age the fish, a Micropogonias sp. otolith, for example, would typically be cut in a sagittal section to more clearly reveal the growth marks with maximum symmetry, rather than favoring surface area. (Escil, Chassieu, France). Gentle polishing did not prevent detachment of some crystals at the edge of G. peruvianus otoliths. Internal stresses during the preparation process can also induce cracks in some samples.

The section of otolith QCD could be removed from the glass slide after polishing and analysed without other support, while it was not attempted for the other sections owing to their apparent fragility.

After preparation, the otolith sections were examined for gross alteration using LM, SEM, Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD) (Yoshinaga et al., 1999(Yoshinaga et al., , 2000)). As the particle size of the standard as delivered was an order of magnitude larger than the spot size of some analyses, pellets were prepared by grinding into a fine, homogeneous powder in a clean agate mortar and pestle for 5 min, then pressing in a 13 mm die under 10 t for 20 s.

Further methodological measures were also taken to limit the impact of microscale heterogeneity of the reference eg. scan and average a Figure 14: A reference pellet of CRM22 supported by a microscope slide and mounted on the sample gonimeter, ready for analysis. The slide behind the reference pellet does not contribute to the signal, as the pellet was produced thick enough to be opaque to X-rays in the energy range used.

large area (1 mm 2 ) rather than a single point. A mounted reference pellet is shown in Fig. 14.

Chemical environment references

Three reference compounds were selected for comparison to assess the chemical environment of Sr in our samples: natural aragonite (orthorhombic CaCO 3 , Alfa Aesar, batch I15T027), natural calcite (rhombohedral CaCO 3 , Alfa Aesar, batch C08S004), and synthetic strontianite (orthorhombic SrCO 3 , 99.994% pure, Alfa Aesar, batch 23669). Each reference pellet was prepared by grinding into a fine, homogeneous powder in a clean agate mortar and pestle for 5 minutes, then pressed in a 13 mm die. The aragonite and calcite references were prepared neat. The strontianite reference was diluted to limit autoabsorption of fluoresced X-rays due to high Sr content. The required mass of strontianite for an edge jump of 1 in X-ray absorption spectra (see 3.4)was calculated using Absorbix [START_REF] Michalowicz | Max: Multiplatform applications for xafs[END_REF] spectral resolution, and the information to be gained.

Otolith sections, as life history archives, are snapshots of a few years to decades of activity. Each sample is essentially unique; even the modern counterparts cannot be re-sampled for exactly the same conditions as in years past. While even ancient otoliths are not commercially valuable, the environmental and physiological data archived within the otoliths of each individual is unique, making them scientifically precious. For rare and/or precious cultural heritage samples, nondestructive techniques are highly preferable (Bertrand et al., 2013). Nondestructive techniques as considered in this work are effectively a subset of noninvasive techniques, in that they are performed directly on the artefact without further sampling (non-invasive) and are expected to have no lasting effect such as beam damage (non-destructive) [START_REF] Janssens | Chapter 1 introduction and overview[END_REF]. In this work, analyses were performed on sections; while preparation of these is destructive to the original sample, it is also necessary for sclerochronological analysis and is thus not considered a separate sampling step.

One challenge in otolith analysis is in fact their relative purity.

Analysis of trace elements in otoliths (typically at concentrations below 0.5 %wt) can often present a challenge as the signal from ca.

40 %wt Ca can overwhelm the signal of minor and trace elements (Limburg et al., 2007). A sensitive technique with a good signal-tonoise ratio, as well as a high dynamic range, is needed to detect these weak signals among those of the major components.

In order to access the temporal information archived within each otolith, techniques must be laterally-resolved (i.e. within the plane of the sample surface) to reveal the periodic fluctuations. As growth occurs incrementally, a higher lateral resolution along the length of an aragonite fibre could allow distinction of prismatic crystalline units and potentially of the ultrastructure therein. An analytical technique providing a high lateral resolution thus provides an improved temporal resolution.

A technique capable of simultaneous analysis of many elements with good spectral resolution would be ideal to reveal potential unanticipated components (e.g. chemical elements or mineral phases).

In particular a technique allowing in-depth post-facto data treatment and analysis could help to reveal interesting trends.

Selection of analytical techniques

The analytical techniques in the present work were selected with the aforementioned needs in mind. A unified analytical methodology (Fig. 15) was planned in order to integrate multiple types of information from various techniques. The strategy considered the preparation required for each method, as well as potential sample effects from each step of the analysis. The order of analyses was planned with consideration for which analyses present the highest risk of sample effects, as well as which information may be informative for following analyses. A method of spatial referencing to align the data from multiple methods was elaborated, and the potential gain from different analyses -correlated or complementary -was considered.

A Focused Ion Beam (FIB) was used in deposition mode to place small plots of tungsten at selected points on the samples as a multitechnique coordinate marking system. Micro-X-Ray Fluorescence (µXRF) was performed using both medium-energy and tender X-rays (DiffAbs and LUCIA beamlines, respectively, at Synchrotron SOLEIL, Gif-sur-Yvette, France) to acquire laterally-resolved multielemental semi-quantitative maps. Tests of full-field XRF imaging were performed at the BAMLine (BESSY II, Berlin, Germany) as a promising alternative approach to point-by-point raster scanning for XRF mapping. Micro-Proton-Induced X-ray Emission (µPIXE) was performed at the Ion Beam Analysis Laboratory (ATOMKI, Debrecen, Hungary) in complement to medium-energy µXRF for a high lateral resolution analysis with a shallow information depth. Micro-Xray Absorption spectroscopy (µXAS) on the DiffAbs beamline was used to determine the chemical environment of Sr in otoliths at many points on several samples, as well as to perform a chemical environment mapping to identify any variations related to position or Sr concentration. Micro-X-Ray Diffraction (µXRD) was performed at the DiffAbs beamline with the dual objective of studying the microcrystalline structure of otoliths using a fast texture mapping approach and contributing to the development of the technique itself.

laterally-resolved analyses of otolith elemental geochemistry

Beginning in the 1960s, early elemental assays were performed on whole, crushed otolith samples using solution-based methodsoften Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) or Mass Spectrometry (ICP-MS) -but this approach obliterates the valuable chronological growth data (Campana, 1999).

Development of in situ beam-based approaches using ions, electrons, or photons can eliminate laborious mechanical sampling. Analyses can be performed either through a point fingerprinting strategy for studies such as natal origin [START_REF] Walther | Geochemical signatures in otoliths record natal origins of american shad[END_REF], or a transect (typically centre-to-edge or edge-to edge) for studies of life history [START_REF] Morales-Nin | Seasonal and ontogenic migrations of meagre (Argyrosomus regius) determined by otolith geochemical signatures[END_REF][START_REF] Amano | Otolith elemental and sr isotopic composition as a natal tag for Biwa salmon Oncorhynchus masou subsp. in Lake Biwa, Japan[END_REF].

A 1997 inter-laboratory experiment compared the four most common and/or promising in situ beam-based approaches for elemental quantification in otoliths at the time: Electron Microprobe Analyses (EMPA), using both Wavelength Dispersive (WD) and Energy Dispersive (ED) detectors, Laser Ablation ICP-MS (LA-ICP-MS), and Proton-Induced X-ray Emission (PIXE) (Campana et al., 1997;Campana, 1999). The techniques were evaluated on limit of detection (LOD), accuracy, precision, linearity of response, and beam or crater size. A summary of the general characteristics of these three techniques (EPMA, PIXE, and LA-ICP-MS), as well as an additional technique noted in the 1997 article (Synchrotron X-ray fluorescence), is presented in Tab. 7. ED-EPMA was found lacking in sensitivity with level of detection around 1000 ppm. WD-EPMA fared somewhat better with a LOD at 50 ppm, but ultimately LA-ICP-MS and PIXE were identified as the techniques of choice for trace analyses (Campana et al., 1997). LA-ICP-MS provided a single ppm to sub-ppm LOD, depending on element (Campana et al., 1997) and has since become the most common technique for elemental analysis (e.g. manual for the general study of otoliths, and PIXE and LA-ICP-MS are highlighted for laterally-resolved analyses with high sensitivity (Panfili et al., 2002). Several dozen elemental studies of otoliths in the 1990s and early 2000s were performed using PIXE and µPIXE. One example is the use of µPIXE life history scans to detect the exposure of individuals to runoff from mining tailings in a freshwater lake system, which revealed continuous elevated Mn, Zn, and Sr in all 69 individuals from 7 species [START_REF] Saquet | Micro-pixe analysis of trace element variation in otoliths from fish collected near acid mine tailings: Potential for monitoring contaminant dispersal[END_REF]. Development of labbased LA-ICP-MS fuelled its increasing use, and ultimately it became the most common tool: [START_REF] Thorrold | Otolith chemistry[END_REF], a reference dedicated to the study of tropical fish otoliths described only LA-ICP-MS for microchemical analyses. Lateral resolutions on the order of one growth interval, as fine as 3 µm have been obtained by LA-ICP-MS (Woodhead et al., 2005) and 5 µm by PIXE [START_REF] Sano | Ion microprobe Sr isotope analysis of carbonates with about 5 µm spatial resolution: An example from an ayu otolith[END_REF], have been demonstrated for measuring strontium in otoliths.

The first application of synchrotron µXRF for life history analysis of otoliths was published from data collected at the Photon Factory synchrotron (Japan) beamline 4A. Maps of the Sr/Ca and P/Ca ratios with a lateral resolution of 100 × 100 µm 2 (H×V) were recorded, allowing discrimination of Japanese eels which experienced the expected catadromous behaviour from those which were sea-locked (Nakai et al., 1999). XRF has been applied to otoliths for several studies related to life history (Tab. 8) but is not yet widely used.

µXRF maps of otoliths have been used to identify the movements of diadromous fish through waters of varying salinity, as well as to detect movement of fish between freshwater lakes polluted with varying heavy metal content (Fig. 16a) (Limburg et al., 2007[START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF].

The trace-level sensitivity of µXRF has been used to measure bromine content in otoliths to identify Baltic cod stocks exposed to varying levels of hypoxia (Limburg et al., 2010a). A first application of µXRF mapping on archaeological otoliths was similarly used to examine hypoxic conditions present over millennia (Fig. 16b) (Limburg et al., 2011). Further investigations of modern fish specimens suggests that primarily Mn, as well as Fe, I, and Zn, may be valuable proxies for dissolved oxygen (Limburg et al., 2015). XRF has additionally been used for physiology studies of otoliths, searching for evidence of the role of otoliths in magnetic field sensing [START_REF] Harada | Magnetic materials in otoliths of bird and fish lagena and their function[END_REF][START_REF] Harada | The relation between the migration function of birds and fishes and their lagenal function[END_REF] or for the impact of inhibiting certain enzymes in larvae [START_REF] Sakurai | The role of pigment cells in the brain of ascidian larva[END_REF]. Although µXRF for otolith elemental analyses has been used by only a few authors, it is a promising technique.

Exploration of the capabilities, optimisation of methodologies, and consideration of potential issues could aid in the wider adoption of µXRF for otolith studies.

focused ion beam preparation

Operational principle

In its more commonly-used milling mode, the FIB uses an accelerated beam of ions to progressively ablate samples at small lateral scales [START_REF] Yao | Introduction to the focused ion beam system[END_REF]. In deposition mode, a small nozzle is placed at the surface through which the desired metal is injected in a gaseous state.

The gas is typically an organometallic compound which adsorbs to the sample surface. Under irradiation of the ion beam the adsorbed organometallic dissociates. The light, organic fragments evaporate into the high vacuum while the metal atom remains adsorbed to the sample. Under properly balanced ion beam characteristics and gas flow, the metal is deposited faster than the ion beam ablates it.

analytical methods

Experimental setup and conditions

A Zeiss LEO 1530 Scanning Electron Microscope equipped with an OrsayPhysics Cobra Ga + FIB and gas injection system was used to deposit tungsten marks on the samples. Tungsten was selected as an element not present in otoliths and non-interfering with the key X-ray emission spectral lines of interest (Fig. 17), so tungsten hexacarbonyl (W(CO) 6 ) was used as the injected gas. Sample exposure to the Ga + beam was kept to a minimum to avoid potential sample alteration.

In order to position the sample in the focal plane of the ion beam, it was tilted 45°to the electron beam. Marks were laid out in a pattern of 9 lines of 20 × 0.1 µm 2 , separated by 2 µm to form a 20 × 20 µm 2 square from the perspective of the electron beam. As a result of the tilted position, the final motif was 28 × 20 µm 2 (V×H). Deposition was performed for 60 s/line with a current of 50 pA. Deposition of the marks was complicated by the insulating character of the sample;

this was partially compensated by placing strips of either carbon mounting tape or copper tape on the sample near the deposition zones, forming a bridge to the sample holder to aid in the evacuation of built-up charges. Optical and electron microscopy (Appendix C) was performed after deposition to confirm the presence of the marks and identify their precise positions on the otolith relative to the growth intervals. The deposited marks provide an absolute reference point visible under optical and electron microscopy and elemental analyses.

This gure has been removed due to copyright restrictions.

It can be found in the referenced text or in the full version of this work at the Université Paris-Sud library.

(a) Atomic cross-section of C

This gure has been removed due to copyright restrictions.

It can be found in the referenced text or in the full version of this work at the Université Paris-Sud library.

(b) Atomic cross-section of Pb (1980).

3.4 x-ray absorption spectroscopy

Physical principle

X-ray absorption spectroscopy is used to probe the charge and local environment (typically up to 6-10 Å) around the atoms of a selected chemical element in a sample.) When a photon below ca. 10 4 -10 6 eV (for C and Pb, respectively) interacts with an atom, it can either be absorbed or scattered [START_REF] Als-Nielsen | Elements of Modern X-ray Physics[END_REF]. The probability that a photon will interact with an atom of a given species, known as the atomic cross-section, depends on the photon's energy.

The nature of the interaction is also dependent on the energy. At the X-ray energies typically available on synchrotrons (10 3 -10 5 eV), absorption dominates (Fig. 18). Absorption of X-rays is a result of the photoelectric effect, in which an electron absorbs a photon, gaining kinetic energy and being ejected from the atom. X-ray absorption spectroscopy (XAS) measures the attenuation of the X-ray photon flux passing through a material.

The absorption follows Beer's Law (Equation 2), where I is transmitted intensity, I 0 is incident intensity, µ(E) is the energydependent linear absorption coefficient in units of inverse distance, and x is sample thickness.

I = I 0 e -µ(E)x (2)
In a canonical XAS experiment, I and I 0 are recorded and x is constant, permitting measurement of the energy-dependent absorption term µ(E). The absorption coefficient follows a general trend decreasing with energy but at particular energies, discontinuities known as absorption edges, occur. This series of edges occurs at the ionisation energies (each of which is associated with a particular orbital), where a photon has sufficient energy to excite a core-level

electron. An absorption edge is named for the principal quantum number n of the core level, where n=1 is the K-edge, n=2 is the L-edge, and so on. Subscripts are used to denote sublevels (e.g. L 1 , L 2 , L 3 ) corresponding to different energy levels because of orbital angular momentum ( ) and spin quantum number (s). Each element has a set of characteristic ionisation energies as a result of the interaction between the nucleus and electrons. In an experiment, one particular absorption edge (E 0 ) is typically selected (frequently the K or L 3 edge). If the photon energy is insufficient, no excitation of this edge can take place (Figure 19a). If the photon energy is equal to the difference between that of an empty orbital and the core level, the electron is excited to the first unoccupied levels (Figure 19b). This excitation produces pre-edge peaks, typically found in the high-energy end of region 1 in Figure 20a. If the photon energy (hν) is equal to or greater than the ionisation energy for the atom (Figure 19c), the electron is excited into the continuum with a kinetic energy (E K ) equal to the difference between the photon energy and E 0 (Eqn. 3).

E K = hν -E 0 (3)
As E K increases, mean free path decreases to a minimum before slowly increasing, shown in Figure 20b. This variation in E K , thus mean free path, is what marks the difference between regions 2 and 3 in Figure 20a. In the X-ray Absorption Near-Edge Spectroscopy (XANES) region (2), the electron typically has E K 50 eV, while in the Extended X-ray Absorption Spectroscopy Fine Structure (EXAFS) region E K > 50 eV [START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF]. In XANES, the photoelectron has a long wavelength and interacts with many surrounding atoms. In EXAFS, the electron has a shorter wavelength and generally interacts with a shell of atoms at a discrete distance [START_REF] Newville | Fundamentals of xafs[END_REF]. It is these interactions with surrounding atoms that produce the XANES and EXAFS oscillations seen in Figure 20a.

Experimental setup and conditions

A general schematic of a XAS experimental setup is presented in Figure 21. XAS requires a high-intensity, accurately tunable X-ray source. A high photon flux over a wide energy range, typically tens to hundreds of electron volts, is necessary to perform XAS experiments. Installations such as synchrotrons and free electron lasers are generally the only sources capable of producing an appropriate beam, although novel developments have led to compact X-ray sources capable of producing a spectrum now appropriate for XANES of light elements [START_REF] Peth | Near-edge X-ray absorption fine structure measurements using a laboratory-scale XUV source[END_REF]

(a) Regions of a XAS spectrum: the pre-edge (1), the edge and XANES region (2) and the EXAFS region [START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF].

This gure has been removed due to copyright restrictions.

It can be found in the referenced text or in the full version of this work at the Université Paris-Sud library.

(b) The mean free path of a photoelectron (λ) in matter depends on its kinetic energy, E K . Adapted from [START_REF] Newville | Fundamentals of xafs[END_REF].

Figure 20: A XAS spectrum (a) is typically considered in three parts. The XANES region (a, 2) covers from the edge to the minimum mean free path length (typically around E 0 +50 eV), and the EXAFS region covers all higher E K . The mean free path of a photoelectron (b) in the XANES region (E K <50 eV) is relatively high, with a sharp dropoff, before a more gradual rise with increasing E K in the EXAFS region. Fluorescence mode operates on the basis that fluoresced intensity of the corresponding edge is linearly proportional to the rate of excitation of that edge, that is, the absorption rate [START_REF] Newville | Fundamentals of xafs[END_REF].

In this work, the strontium K-shell was probed, so the fluorescence two Si(111) crystals and provides a spectral resolution E/ΔE of ca.

10 4 . Using a DCM with fixed exit geometry maintains the vertical output beam position constant at all energies. The second crystal is curved to provide primary horizontal focusing. The beam exits the monochromator and passes through a slit to reject the undesired energies before being vertically focussed using a second mirror.

This produces a spot size of 250 × 250 µm2 with an on-sample flux of 6 × 10 12 photon/s (ca. 1 × 10 13 photon/s/mm 2 ) designated "widebeam mode".

For "micro-beam mode" experiments, a Kirkpatrick-Baez mirror setup was used to focus the beam to 6 × 11 µm 2 (V×H) Full

Width at Half Maximum (FWHM) 2 at the Sr K-edge to attain micrometric resolution with an on-sample flux of 1 × 10 9 photon/s (ca. 2 × 10 13 photon/s/mm 2 ). Note that the areal flux of photons is not significantly increased in the micro-beam relative to the wide beam, contrary to what may be expected. The DiffAbs beamline was not designed with the micro-beam in mind; the capability was added by the use of a removable Kirckpatrick-Baez (KB) mirror system in order to provide a "cleaner" micrometric spot than simply using the preexisting slits to reduce the size. The KB mirror uses a pair of bent crystals, one for each of the X and Z axes (Fig. 23), to focus the beam to a small spot size independent of wavelength with a low loss of flux [START_REF] Mocuta | Fast pole figure acquisition using area detectors at the DiffAbs beamline -Synchrotron SOLEIL[END_REF]. Three different Vortex SII silicon drift diode fluorescence detectors (Quad-element Vortex ME-4, 170 mm 2 active area; monoelement Vortex-60EX, 100 mm 2 active area; monoelement Vortex-EM, 50 mm 2 ; Hitachi High-Technologies Science America Inc., Northridge, CA, USA) were used during different experimental sessions. The detector was placed in the same horizontal plane at 90°from the beam with the sample mounted in-air on a goniometer at 45°to both the incident beam and detector. This resulted in an effective spot size on the sample of 6 × 16 µm 2 (V×H). Samples were mounted with the growth axis parallel to the short axis of the beam to maximise the spatial resolution across isochronous layers.

The monochromator energy was calibrated using a reference sample of known composition and Sr speciation (strontianite), and determining the shift between the measured edge energy (determined using the edge jump approach described in Sec. 3.4)

and the literature value. For both µXANES and µEXAFS, the signal was recorded by fluorescence yield using the same configuration as in Figure 23. For micro-beam measurements, the µXAS signal was calculated by numerically integrating a window 600 eV wide centered on the Sr Kα fluorescence peak in order to ensure that the entire peak was integrated, including the tails at high concentrations.

Only krypton and thallium have characteristic energies in the 13 865-14 465 eV range. Both were expected to be present in trace quantities at most, and the lines that are present all have relative intensities below 15 [START_REF] Thompson | X-ray Data Booklet[END_REF]. The integrated signal was divided by the recorded I 0 from the thin film diode for normalisation.

µXANES spectra of otolith samples were recorded with a count time of 4 s/energy point over the 16 023-16 083 eV energy range in 2 eV steps, 16 084-16 134 eV in 1 eV steps, and 16 135-16 373 eV in 2 eV steps. Some XANES spectra were stopped after the first major oscillation to sample more positions on the sample more rapidly (16 223 eV).

Reference XANES spectra of Sr-bearing aragonite and diluted strontianite were recorded in transmission mode using a wide (250 × 300 µm 2 V×H) X-ray beam to limit the impact of microscale

Data treatment

The spectra were processed using the iXAFS suite version 2.2

including ATHENA for data extraction and ARTEMIS for fitting of the spectra, alongside IFEFFIT for calculation of theoretical spectra (Ravel and Newville, 2005). EXAFS fitting was performed on the interval 1-6 Å in R-space.

Extrapolation
Occupancy of each shell was fixed at theoretical values.

multi-energy µxanes mapping µXANES mapping was performed using a reduced energy dataset on the section of QCD, detailed as follows. Five diagnostic energies were selected to provide significant discrimination between aragonite and strontianite based on the µXANES spectra of reference inorganic aragonite and strontianite (as identified in Fig. 48). A SR-µXRF map was recorded at each of these five energies. The data were processed using procedures written under the 'R' statistical environment [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF]

I Exp (E) = β[a × I Arag (E) + b × I Stro (E)] + × E + δ (4)
Background removal and normalisation of both EXAFS reference spectra was performed in ATHENA. The linear system was solved using four of the energy values (16 149, 16 170, 16 removal of an electron from an atom's inner levels to form a hole, and the following relaxation of higher-level electrons to fill the hole (Fig. 24).

In the present work, XRF designates an analysis using photoionisation by X-rays to produce a hole, although other excitation sources are possible, such as gamma rays from decay of a radioactive isotope. In photoionisation (Fig. 24a), a photon with sufficient energy strikes an electron and the electron is ejected and a hole is created. An electron from a higher energy level transitions into the hole, emitting a photon of characteristic light. The photons emitted are at characteristic energies for each level of each element. The number and energy of photons can be detected using an energy-sensitive3 detector.

The flux of characteristic photons can be used to quantify the elemental concentration. The theoretical detected photon flux can be derived using a fundamental parameters approach, as in [START_REF] Mantler | Quantitative analysis[END_REF]. A hypothetical experimental setup is used for the derivation (Fig. 25). The detected flux of characteristic photons (I F, Det ) with energy E F , characteristic of an element z is dependent on a set of parameters relating to the sample, the elements being examined, and the experimental setup: I 0 , the incident photon flux of energy E 0 ; S K z , the absorption edge jump ratio (in this case for the K-shell) of element z; τ z (E 0 ), the total photoabsorption coefficient for the element z at an infinitesimal thickness (dx) within the sample (distance x). The incident X-ray beam (I 0 ) intersects the flat sample surface at an angle of (ψ ). The resulting characteristic photons (I F ) exit at an angle (ψ ) toward the detector with a given surface area (Ω). Adapted from [START_REF] Mantler | Quantitative analysis[END_REF].

I F, Det = I 0 S K z -1 S K z τ z (E 0 ) C z p z ω z 1 sinψ Ω 4π γ(E F ) 1 µ * (5) 1 µ * = 1 -exp -µ 0 (E 0 ) sinψ + µ F (E F ) sinψ T µ 0 (E 0 ) sinψ + µ F (E F ) sinψ (
Furthermore, it is possible to simplify the expression if a sample can be considered infinitely thick ("bulk") or infinitely thin ("thin film"). In order to be considered a bulk sample, the exponential term must be much less than 1, such that the denominator in Equation 6dominates. Using the attenuation length in CaCO 3 (ρ=2.93 g • cm -3 ) of 16.2 keV incident X-rays and 14.1 keV characteristic photons (for Sr), the minimum thickness for ±1 % accuracy using the bulk approximation can be placed at 440 µm. In the samples of the present work, thicknesses ranged over 130-710 µm; the bulk approximation is not sufficiently accurate for approximately half the samples.

At the lateral resolutions employed in the present work, inhomogeneities within standards may produce erroneous resultsusing powder standard with a particle size of 100 µm, a micro-beam of 6 µm could detect variations in concentration with in the particles.

Standards fulfilling these requirements are relatively easily found for macro-scale XRF experiments, but they are less available at the micro-scale. An ideal standard would have homogeneous elemental concentrations throughout in a matrix identical to that of the sample under investigation. Even in a homogeneous standard with large particles, surface effects are possible due to varying escape depths, so a particle size substantially smaller than the beam is preferable. Solid glass standards are frequently used in LA-ICP-MS elemental analyses of otoliths and exhibit a homogeneous elemental distribution as well as eliminating concerns over particle size, but the vastly different matrix (SiO 2 ) precludes their use as XRF references for otoliths.

Data treatment

The software "PyMca" (Solé et al., 2007 and Appendix E) allows quantification of XRF data based on a spectrum deconvolution approach (Solé et al., 2007). The characteristic fluorescence spectrum of a given element with a given excitation energy (or energies) is represented by a single sum curve of all contributing lines, including the characteristic peaks, escape peaks, and pile-up peaks. The approximate matrix composition is specified beforehand (aragonite in the present work), as well as sample thickness (or multiple sample layers), experimental geometry, the detector material and thickness, and any absorbers in the X-ray path (including beam window, atmosphere, and detector window, for example). This well-specified analytical environment allows many of the terms from Equations 5 and 6 to be collected into a single representation for the entire energy spectrum for each element.

In Equation 7, the peak area (A) for an element in a spectrum depends on the mass fraction (C), the incident flux (I 0 ), the detector efficiency (γ), the detector solid angle (Ω), and the summation of an element's detected spectrum over all energies j with a given excitation energy ( j R j ). I 0 and Ω are given as experimental parameters. j R j is the set of characteristic peaks for an element, incorporating the characteristic energies as functions (Gaussian, Voigt 4 , or Hypermet 5 ) in proportion to their theoretical ratios as well as escape peaks 6 , with accounting for noise broadening and matrix attenuation. Thus, the only variable remaining to fit is the mass concentration.

A = I 0 • C • γ • Ω 4π j R j (7)
The peak area of each contributing element is adjusted to minimise the overall quadratic error. Once the optimal fit has been determined, the mass fraction of each element can be estimated. The detector solid angle is calculated by PyMca from the detector chip area and distance from the sample, both required parameters from the experimental setup. The incident flux was determined by manually adjusting the value on a reference (CRM22) until the appropriate strontium concentration was obtained.

However, when attempting to fit other measurements, attention must be paid to the incident flux as it can change from one to another.

The most significant changes occur when readjusting the beamline setup, such as the monochromator pitch and roll 7 , to compensate for small variations (e.g. crystal thermal expansion, electron source position) which affect the X-ray beam position and angular spread.

Such a change in I 0 invalidates any quantification results using the standard without correction. Because the concentration is linearly dependent on the flux, a linear scaling factor derived from the beam A linear combination of a Gaussian and a Lorentzian function allowing more accurate representation of both the instrumental and core hole lifetime broadening.

A peak shape function consisting of a Gaussian function combined with a lowenergy tail which decays exponentially [START_REF] Phillips | Automatic analysis of gamma-ray spectra from germanium detectors[END_REF].

A peak below the energy of the characteristic line, where a photon has been only partially absorbed by the detector. The energy difference between the characteristic line and the escape peak is characteristic of the detector material (e.g. 1.74 keV for a silicon detector).

Orthogonal angular axes of the monochromator used to align the beam for downstream optics.

monitors can be used to correct for variations both between maps and within a single map. This correction was applied on a per-pixel basis in order to normalise the variation within a map, expected to be <1 % due to beam decay and reinjections, but sometimes observed to be more significant, possibly due to beam drift over the course of an acquisition. The results were recorded total counts for a given channel, so must be corrected for the detector livetime, which varies between samples and within a sample as the elemental composition varies.

Equation ). The second factor corrects variation between the sample mean I 0 and the reference mean. The third factor corrects variations in the livetime (LT ) on the i th pixel to standardise at 1 s. beamline is a wavelength shifter producing an on-sample photon flux of 10 12 photon/s in the hard X-ray range. In this experiment, an 18 keV beam in a spot size measuring up to 1.5 × 1.5 cm 2 (photon flux density ca. 4 × 10 9 photon/s/mm 2 ) was used in conjunction with a so-called colour X-ray camera [START_REF] Ordavo | A new pnCCD-based color X-ray camera for fast spatial and energy-resolved measurements[END_REF][START_REF] Scharf | Compact pnCCD-based X-ray camera with high spatial and energy resolution: A Color Xray camera[END_REF].

C Real = C Calc • I Sample 0 I i 0 • I Reference 0 I Sample 0 • 1 s LT i (s) (8
This system allows recording an hyperspectral image of the XRF signal in an area of 12.5 × 12.5 mm 2 using a CCD (264 × 264 pixels, 48 × 48 µm 2 pixel size, 152 eV energy resolution; PNSensor GmbH, München, Germany). A polycapillary lens directs the photons emitted by the sample to a pixel of the camera, rejecting those with too large an incident angle while maintaining a high transmission efficiency. This permits the recording of a spatially-resolved image in one shot without scanning the sample. The projected pixel size (pps) is function of the characteristics of the polycapillary lens used. A 1:1 and a 1:6 polycapillary were employed, providing effective pps of 48 µm and 8 µm, respectively. Total count times ranged from 1 to 8 h per hyperspectral image. Peak areas of the calcium and strontium Kα peaks were calculated by integration on a region of interest (ROI) 300

and 600 eV wide, respectively.

proton-induced x-ray emission

Physical principle

Proton-induced X-ray emission (PIXE) is a technique used for measurements of elemental composition. PIXE employs characteristic X-rays emitted from the elements composing the sample, as in XRF, but instead of X-ray photons, protons with kinetic energy in the MeV range are used to excite electrons from the atomic core levels and produce a stimulated emission. Compared to XRF using X-rays of a few tens of keV, the ionisation cross-section -the probability of excitation -of lighter elements (Z<40) by protons is high, which results in both a high signal per incident flux and a shallow information depth [START_REF] Malmqvist | Comparison between PIXE and XRF for applications in art and archaeology[END_REF]. As a result of this high absorption cross-section, PIXE experiment are generally carried out under vacuum to minimise both the attenuation and spreading of the incident beam [START_REF] Arai | An attempt to analyze fish otoliths by in-air PIXE[END_REF]. PIXE has been shown to be a powerful technique for archaeological studies, as for many samples it requires negligible preparation beforehand and is often described as nondestructive [START_REF] Malmqvist | Comparison between PIXE and XRF for applications in art and archaeology[END_REF][START_REF] Araújo | Comparison of EDXRF and PIXE in the analysis of ancient gold coins[END_REF][START_REF] Carmona | Advantages and disadvantages of PIXE/PIGE, XRF and EDX spectrometries applied to archaeometric characterisation of glasses[END_REF], but not in all cases (Huszank et al., 2009).

Experimental setup and conditions

Experiments were performed at the ATOMKI ion beam analysis laboratory in Debrecen, Hungary [START_REF] Rajta | The new atomki scanning proton microprobe[END_REF][START_REF] Uzonyi | Realization of the simultaneous micro-pixe analysis of heavy and light elements at a nuclear microprobe[END_REF]Kertesz et al., 2005). This facility is based upon a Van der Graaf generator with a tunable voltage of 0.8-5 MeV. The protons are produced from hydrogen gas excited by an inductively-coupled radiofrequency source before being accelerated using the electrostatic charge built up in the Van der Graaf generator. After acceleration, a magnetic field is used to isolate only those protons of the desired energy before being passed into the scanning microprobe system.

The microprobe focusses the ions into a micrometric spot using electrostatic lenses. Under computer control, this spot is then raster scanned across the analysis area.

A proton beam energy of 2.5 MeV was selected to provide a good excitation of elements with Z 38 while maintaining a relatively shallow estimated information depth of ca. 30 µm for Sr Kα fluorescence based on calculations in SRIM software [START_REF] Ziegler | The stopping and range of ions in matter[END_REF] and Appendix E). The experimental setup is shown in Fig. 27.

Experiments were carried out under primary vacuum. A stage is used to position the polished thin section (ca. 100-400 µm) sample in the desired position to a precision of a few tens of micrometres with its surface normal to the incident beam, after which the proton beam is raster scanned across the sample. The raster area is continuously adjustable using analogue electrostatic lenses. Scans are carried out within the raster area in a grid of 256 × 256 pixels defined by the OMDAQ acquisition and control software. The lateral resolution attainable is therefore dependent on the raster area. As a compromise between lateral resolution and throughput, scans were performed on a square of 2 × 2 mm 2 , yielding an 8 µm step size. Higher lateral resolutions were obtained by scanning reduced areas of 1000, 500 and 250 µm, yielding step sizes of 4, 2 and 1 µm, respectively. For all but one sample, the 2 mm scan size was insufficient for a full centre-toedge transect, so two or more scans were made and stitched into one. Characteristic photons were measured using a pair of Si (Li) detectors, one with a two-layer 25 µm Be and 125 µm polyimide (Kapton) window (d,Fig. 27), and one with a 0.2 µm polyimide window (e, Fig. 27). The first detector records photons up to 30 keV while the second provides an increased energy resolution below 9 keV and sensitivity down to the C K signal. PIXEKLM was used for quantification of the µPIXE data [START_REF] Uzonyi | Calibration of an UTW Si(Li) detector in the 0.28-22.1 keV energy range at a nuclear microprobe[END_REF][START_REF] Uzonyi | PIXEKLM-TPI -a software package for quantitative elemental imaging with nuclear microprobe[END_REF]. Because of limited statistics, PIXEKLM was first used to determine the average concentrations of the analysed area, from which the mass fraction required per photon detected can be calculated and used to calculate the elemental concentrations in each pixel.

One semi-quantitative map was collected on a 413 × 183 µm 2 zone of QCD. During recording there was a slow position derivative, which is likely a result of drift of the sample on the support, despite care taken to tightly fix the sample in place. This derivative was corrected by segmenting the recorded data into 20 data 'slabs', each representing 24 min of measurement time. The position of the tungsten mark was determined in each slab, and the slabs were reassembled with appropriate translation to produce a realigned map, largely free of derivative.

x-ray diffraction

Physical principle

X-ray diffraction (XRD) is a widely-used technique for the characterisation of crystalline materials. It is exclusive to long-range ordered materials, as it relies on repeated infinite planes to produce constructive interference of each reflected photon (Fig. 28). This condition is described by Bragg's law (Eqn. 9), where n is the reflection order (almost always 1), λ is the X-ray wavelength, d is the spacing between the repeated atomic planes, and θ is the angle between the atomic planes and incoming X-rays.

n • λ = 2d • sinθ (9)
In a perfect single crystal, X-rays are diffracted as a series of repeating beams. Projected on an image plane, the beams would produce an array of points representing each diffracting family of planes [START_REF] Jenkins | Introduction to X-ray Powder Diffractometry[END_REF]. When analysing a polycrystalline material -such as an otolith -there are many such crystals diffracting. Each of the thousands or millions of crystals composing the polycrystalline sample can be differently oriented and thus produce an array of points with a particular rotation corresponding to the orientation of the crystal. For example, in an otolith composed

F 1 2 3 1' 2' 3' A B C D E d d θ θ (hkl) (hkl)
(hkl)

Figure 28: Bragg's law illustrated by analogy to optics. Parallel, in-phase X-rays (1-3) intersect a family of parallel atomic planes (hkl) at an angle θ and are reflected (1'-3'). Ray 2 travels a distance AB+BC farther than ray 1 and ray 3 travels DE+EF farther. For a given interplanar spacing d, there is an angle θ where AB+BC=λ, resulting in a phase delay of 1 wavelength. For further (hkl) planes and trajectories (ray 3) DE+EF=2λ, with a phase delay of 2 wavelengths, and so on for many additional (hkl) planes. With integer phase delay, constructive interference occurs between rays 1', 2', 3', and so on to produce a diffracted beam. Adapted from [START_REF] Jenkins | Introduction to X-ray Powder Diffractometry[END_REF], and [START_REF] Cullity | Elements of X-ray Diffraction[END_REF].

of 50 nm diameter spherical nanogranules, a 6 × 11 µm 2 beam passing through a 350 nm section intersects ca. 10 8 nanogranules.

A given reflection from each crystal produces a beam with the same 2θ but at a different rotation about the incident beam (ψ).

Summing together the beams from many randomly-oriented crystals produces a diffraction cone with a homogeneous dispersion of spots;

projecting this onto an image plane then produces a solid Debye ring (Fig. 29a) [START_REF] Jenkins | Introduction to X-ray Powder Diffractometry[END_REF]. The physical size of a Debye results in variations of intensity at positions related to the orientations of the crystals.

Experimental setup and conditions

Experiments were performed on the DiffAbs beamline using a 17.2 keV incident beam. The configuration is shown in Fig. 30. A Kirckpatrick-Baez mirror (Kirkpatrick and Baez, 1948) was used to focus the incident beam to an on-sample FWHM spot size of 6 × 11 µm 2 (V×H) with a flux of ca. 2.5 × 10 9 photon/s. Beam divergence after the focal point is 0.1°in the vertical and 0.04°in the horizontal. The sample sections were mounted vertically and quasi-normal to the incident beam (Fig. 30). X-ray diffraction (XRD) images were collected using an XPAD [START_REF] Delpierre | XPAD: A photons counting pixel detector for material sciences and small-animal imaging[END_REF]. The area detector was mounted downstream from the sample for transmission detection, out of the transmitted beam axis and so as to maximise 2θ and ψ coverage (ca. 7° 2θ 27°at 17.2 keV, equaivalent to 15° 2θ 61°a

t Cu Kα, see Fig. 31). For better comparability, all 2θ values in the was mounted in the horizontal plane of the beam and normal to the beam path 39 mm from the beam-sample intersection point to minimise Compton scattering.

Raster scanning was performed in steps of 100 µm in both the horizontal and vertical axes with a real exposure time of 1 s per pixel in order to complete the mapping in a reasonable collection time of several hours with simultaneous recording of XRF and XRD signals.

Data treatment

Initial integration of the images from the XPAD detector was performed following the protocol in [START_REF] Mocuta | Fast pole figure acquisition using area detectors at the DiffAbs beamline -Synchrotron SOLEIL[END_REF] using ImageJ software [START_REF] Schneider | Nih image to ImageJ: 25 years of image analysis[END_REF]. For each pixel in the raster map, the corresponding raw image (Fig. 31a) was adjusted to match the physical layout of the detector chips (Fig. 31b). The resulting image was then transformed into Cartesian (2θ,ψ) coordinates for direct integration while allowing a visual feedback on the quality of the coordinate transformation (Fig. 31c). Integration was performed

analytical capacity using selected techniques

The objectives outlined in Section 3.1 were achieved using the techniques described in this chapter. Focused ion beam marking provided a means to locate analysis points in multiple techniques.

Chemical speciation imaging and point analyses were carried out using a combination of X-ray absorption techniques to acquire in-depth information across large sample areas with micrometric lateral resolution. Elemental mapping using both X-ray fluorescence and proton-induced X-ray emission was performed on millimetric length scales with micrometric lateral resolution, compatible with single growth increments in otoliths. Studies of the crystal lattice at the micrometric level were carried out using a state-of-the-art X-ray diffraction mapping technique which permitted rapid acquisition over large areas.

4

E L E M E N TA L M A P P I N G B Y H I G H -D E F I N I T I O N S Y N C H R O T R O N µ X R F 4.

chapter introduction

Elemental analysis of fish otoliths is a well-established and expanding field of fish and fisheries research (Campana and Thorrold, 2001). Elemental fingerprinting, often coupled to sclerochronological analysis, has been used for questions of natal origin and population movement determination, stock identification, migration history tracking of diadromous species, or pollution monitoring (Elsdon et al., 2008;Limburg et al., 2010a,b;[START_REF] Morales-Nin | Seasonal and ontogenic migrations of meagre (Argyrosomus regius) determined by otolith geochemical signatures[END_REF]Kerr and Campana, 2014). To be used as an environmental proxy, an element must be incorporated into the otolith in a predictable manner reflecting the physical and chemical composition of the ambient water. At least 32 elements have been observed in otoliths, mostly in trace quantities, with others expected in yet-undetected concentrations (Campana, 1999). Some elements are naturally present at significant levels in ambient water (Kalish, 1989). Content in others is due to, or largely overprinted by, anthropogenic pollution [START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF]. In terms of absolute and relative concentrations, the incorporation of elements other than calcium, carbon and oxygen into the otolith, while potentially approximated by purely physical conditions of deposition from the endolymph solution (Campana, 1999), is in reality influenced by a combination of temperature, elemental mapping by high-definition synchrotron µxrf total salinity, and ion concentrations of the water inhabited by the individual, and the individual's physiology (Elsdon and Gillanders, 2003). Multiple "physiological filters" between the ambient water and the otolith produce a strict biological regulation of some ions while others remain weakly or unregulated (Campana, 1999). The alkaline earths strontium and barium fall into the weakly regulated group and are therefore the most studied environmentally-related elements. The variation in concentration of these elements has been demonstrated

to be useful and accurate environmental proxies, both in field studies (Elsdon and Gillanders, 2005a) and laboratory experiments (Elsdon and Gillanders, 2004), even though the incorporation is more physiologically regulated than in simpler organisms such as corals (Shen et al., 1996). Conversely, in mollusk shells, strong physiological regulation has been shown to produce a departure from thermodynamic equilibrium in the incorporation of trace elements during biomineralisation (Schöne et al., 2010). Random substitution of Sr for Ca within the aragonite matrix has been proposed in modern otoliths (Doubleday et al., 2014), supporting the reliability of Sr analyses. There is however some discussion about the formation of SrCO 3 domains in other calcium carbonate palaeoproxies (Greegor et al., 1997;Finch et al., 2003).

Otoliths can be found in abundance in some geological and archaeological contexts (Nolf, 1985). Taxonomic identification of fish assemblages from otoliths whose morphology is preserved over geologic time scales has been applied to biostratigraphy, palaeoecology and palaeoenvironment reconstructions (Nolf, 1985(Nolf, , 2013)). The potential of ancient otoliths as archives for past biology and environments has been extended by studying their composition.

Stable isotopic analyses have been used for instance for the study of fish migration or metabolism (Carpenter et al., 2003;Wurster and Patterson, 2003), of palaeoenvironmental and palaeoclimatic conditions from the Jurassic to the Holocene (Patterson, 1998;Aubert et al., 2012;Vanhove et al., 2012) or fishing practices of past human populations (Hufthammer et al., 2010;Carré and Dufour, 2012). A limited number of works have recently applied elemental analyses in the same way for fish palaeoecology (Limburg et al., 2011), palaeoenvironment (Disspain et al., 2011), and past human subsistence strategies (Disspain et al., 2012). Diagenesis might hamper such reconstructions from ancient samples, and the level of induced alteration in the otoliths has to be estimated (Dufour et al., 2000;Disspain et al., 2011). When performed, the identification of altered from unaltered specimens is sometimes limited to visual examination for morphological changes of the otoliths overall. Other criteria are occasionally used based on microstructural features: presence of daily and annual increments, crystal habit, and identification of CaCO 3 polymorphs (Dufour et al., 2000;Vanhove et al., 2011;Kern et al., 2012). Preservation of the microstructure and mineralogy is often taken to indicate the geochemistry therein is pristine. Absence of morphological and mineralogical change does not guarantee a pristine geochemical signal (Dufour et al., 2000). Furthermore, when alteration occurs, the extent and nature of alteration is not necessarily homogeneous across the specimen and indeed can be very limited in area. The outer increments are expected to be more vulnerable to alteration during burial (Cochran et al., 2010).

Unified coordinate marking

A series of tungsten marks were deposited on the samples using a FIB in selected locations near the intended analysis areas (Fig. 32a). A rectangular motif of parallel lines constitute a compromise between a practical deposition time and a sufficiently visible mark; producing a solid rectangle on the order of 560 µm 2 would take several hours, where the motif selected, totaling approximately 25 µm 2 required only 10 minutes. Under LM, the deposits are highly reflective (Fig. 32b), particularly with grazing incident light, and opaque under transmitted light.

The W deposits were easily detected by SR-µXRF even though only approximately 90 pg/pixel were deposited; W Lα fluorescence yield is 0.255 and it is not naturally present in otoliths, producing a high SNR and a highly contrasted image (Fig. 32c). The W marks, presenting both a chemical and physical change in the sample surface, are detectable by multiple probes (Fig. 32d). The motif, with features smaller than 50% of the spot size, provides a homogeneous appearance under XRF with micrometric lateral resolution. Integrating the W Lα peak over its FWHM at the points marked shows an average 435 counts/pixel within the mark compared to a background of 10 counts/pixel outside of the mark (Fig. 32e). They were used to quickly calculate the relative position of any point on the otolith and later align SR-µXRF results with microstructure. In addition, they were valuable landmarks for positioning and alignment during the SR-µXRF experiment itself. Ca ×10 Over all samples, 13 intrinsic elements were detected. Ca, Fe, Zn, Rb, and Sr were detected in all 6 samples, either in the otolith or in the surrounding resin. Cu and Pb were detected in 5 samples.

Ba was detected in four samples, Br in three, and K and Mn in two. Hg was detected in one sample. Y was detected in all three samples analysed using appropriate incident beam energy to excite

it. An additional two elements, Ga and W were detected on the 2 otoliths which had been marked by FIB, exclusively on the marks themselves, as shown in Fig. 32 The core is not visible, but the symmetry axis of the otolith is indicated by the line of . K, Mn, Br, Ba, and Hg were not detected. See text for explanation of marks.

particular Sr, is expected to be reliable and absolute quantification of Sr accurate.

A combination of periodic and unique features can be seen in all elemental maps, which will be exemplified for the Micropogonias sp. sample QCD (Fig. 33). In QCD, Ca, K, and Sr, in order of decreasing concentration, are present over 10 µmol • g -1 . K, Ca, Rb, Rb exhibits very weak fluctuations which are correlated to some extent with Sr, but not entirely (Fig. 39). In Bayovar-1C, the Sr near the core, in the range of 15-30 µmol • g -1 , and one toward the edge at 20-42 µmol • g -1 . [Sr] fluctuations in 424 are within a more restricted range of 14-22 µmol • g -1 . In both samples, there is no overall difference in the edge position between Ca and Sr, but 424 exhibits an indentation in the Ca map (E) which is smaller in the Sr map. An increased concentration of Ca is visible at the edge of both samples. A crack is visible near the core of Bayovar-1C (C2).

In the archaeological sample HP-12 (Fig. 36), taken as an example of G. peruvianus samples, 10 elements were detected. Ca, K, and Sr in order of decreasing concentration, were detected over 10 µmol • g -1 .

K, Ca, Br, Rb, Sr first attempts in full-field elemental imaging of otoliths Elemental maps were collected using an energy-and laterally-resolved X-ray camera [START_REF] Ordavo | A new pnCCD-based color X-ray camera for fast spatial and energy-resolved measurements[END_REF].

Information volume of medium-energy SR-µXRF in otolith thin section analysis

As otoliths grow incrementally from the core, they form a three dimensional structure which can be compared to a "stack of dinner plates". Taking a thin section of an otolith, these plates are oriented approximately orthogonally to the surface. The on-sample spot at

DiffAbs is an ellipse with FWHM of 6 × 16 µm 2 (V×H), thus allowing a higher lateral resolution in the vertical axis. As for any material studied by XRF, the precise lateral resolution attainable in this experiment is not a simple function of spot size, but is also dependent on the sample matrix (Janssens et al., 2000;Kanngießer et al., 2005).

The effect on attainable lateral resolution can be largely decoupled on the horizontal and vertical axes. In the present work, the growth increments visible on the thin sections were oriented horizontally (corresponding to the X axis in It can be clearly seen in Fig. 41 that the volume containing 50% of the information is significantly smaller than that containing 75%.

The lateral resolution is dependent on the total information volume.

In effect, the best attainable lateral resolution for Ca is smaller than that for Sr because the signal is recovered from a much more limited depth. The relative homogeneity of otoliths should produce a largely constant information volume across the sample (Janssens et al., 2000;Kanngießer et al., 2005). After the difference in information volumes was observed, it may be inaccurate to present [Sr]:[Ca] ratios, so absolute concentrations are presented herein. This information volume disparity is one particularity of XRF, and is exacerbated by the 10 keV difference in characteristic energies between the two elements.

Other element pairs with more similar Z and characteristic energies (B) In the vertical, the sample plane is positioned perpendicular to the incident beam, so there is no additional impact on lateral resolution. While this orientation relative to the X axis can be controlled, the internal slant of the increments (corresponding to the XZ plane) cannot be easily evaluated and could produce temporal averaging by measuring not only the visible surface, but also the subsequent growth underneath. A sharp peak in Sr content, for example, could be detected in several pixels at varying depth, increasing its apparent width.

To ensure the majority of the data in a single measurement originates from one isochronous volume, the angle to the XZ plane must be within a certain margin. For example, in sample QCD (Fig. 32) under light microscopy some subannual growth increments are homogeneous at lengths of approximately 40 µm. With an incident beam FWHM of 6 µm in the vertical axis, penetrating 161 µm, the angle between the increment and the "ideal" XZ plane must be less than 12°for the information volume. To perform two consecutive measurements within this increment, the angle must be less than 10°. Because the analysed thin sections have been taken including the core of the otolith, the slant of the growth increments relative to the surface should be minimised and this condition should be met in increments outside of the core. At or close to the core, the volume of aragonite representing one isochronous increment decreases and the curvature of each increment increases. It is possible to obtain a nonaveraged measurement only if the information volume can fit within this increment. From the previous calculations for Sr, assuming a spherical core, this would not be possible for a radius at least 161 µm around the core, and most likely more depending on experimental geometry and thickness of these initial growth marks.

At the otolith edge, the information depth also produces effects. In some samples the apparent edge position depends on the element being examined. In QCD (Fig. 33), the apparent edge is farther from the core in the Sr map than the Ca map; in Bayovar-1C (Fig. 34), there is an indentation at the edge in the Ca map which is shallower in the Sr map. As previously noted, the information depth of the analysis can vary significantly between elements such as Ca or Sr. With a higher energy, the Sr Kα signal can escape from deeper in the sample than the Ca Kα (see Tab. 9), leading to such edge displacements where a subsurface structure exists within the resin. The Ca signal is reabsorbed by the intervening material, while the Sr signal is capable of escaping and evidencing this subsurface structure. In other cases, such as HP-12, there is an inversion of this geometry, where the otolith curves inward. It is visible on the surface by LM and in the Ca map, but not in the Sr map, because the smaller Ca information volume is filled with the otolith, but the larger Sr information volume is only partially occupied. The discontinuity in matrix between the otolith and the surrounding resin also has an influence on the chemical signal. More characteristic photons passing through the resin can reach the detector than the aragonite bulk (Tab. 9). Where the edge between otolith and resin is nearly perpendicular to the sample surface, the density change between aragonite and resin can produce signal enhancement, as seen in Fig. 34. The core and the edge of the otolith are areas of particular interest for environmental or palaeoenvironmental applications, including determination of natal origin or location of capture. These areas might be particularly affected by a decreased temporal resolution or decreased analytical precision. The question of information depth and its interaction with internal morphology presents a point of reflection for all analytical techniques applied to otoliths. An appreciable information volume is not unique to SR-µXRF and can be observed in other techniques as well. Ablation is commonly observed in both LA-ICP-MS (Jones and Chen, 2003) and EPMA. The total integration volume in LA-ICP-MS has been shown to significantly impact the measured concentrations in a life history scan (Hoover and Jones, 2013). In addition, this ablation makes these techniques ultimately destructive, whereas SR-µXRF exhibits very limited beam damage. Good alignment along the horizontal axis of the majority of the growth increments was achieved for all samples except Bayovar-1C and GP-292 (Figs. 34 and 37). An equally sensitive approach permitting analysis of a more restricted sample volume, such as reducing the incident X-ray energy, or using PIXE may be of value.

Lateral resolution and data alignment in sclerochronological archives

The most common techniques applied to the elemental analysis of otoliths are LA-ICP-MS, EPMA and to a lesser extent PIXE. While all three techniques can be used for mapping (Hinz and Kohn, 2010;Huszank et al., 2009;Shirai et al., 2014), on otoliths they are generally used for transect analyses. Maps of otoliths from wild specimens by PIXE (Huszank et al., 2009;Limburg et al., 2003) and Sr-tagged specimens raised in tanks by SEM-EDX (Morales-Nin et al., 2012a) have been previously shown. SR-µXRF scanning on a macro scale is a recent and actively developing technique in the study of both modern and ancient materials (Bertrand et al., 2012;Lintern et al., 2009;Anné et al., 2014). Application of milli-to microbeam synchrotron XRF mapping to otoliths is in its early stages, with some environmental and ecological studies on modern samples (Nakai et al., 1999;Limburg et al., 2007;Lochet et al., 2010;Limburg et al., 2010a,b;Nakazawa et al., 2012), and two seeking to determine the variation in hypoxic conditions using Mn content as a proxy (Limburg et al., 2011(Limburg et al., , 2015)). The high incident flux and sensitive detectors found in state-of-the-art SR-µXRF setups permit collection of maps of whole otoliths or significant portions in reasonable time periods. In the present study, maps were collected with a step size as small as 4 × 10 µm 2 (V×H) across significant portions of otoliths to obtain a per-sample analysis time of a few hours, rather than possibly longer than a day (Limburg et al., 2007).

In sclerochronological archives such as otoliths and mollusk shells a high lateral resolution yields a high temporal resolution. Otolith analyses using the most common techniques (LA-ICP-MS, EPMA, and PIXE) are rarely carried out with a lateral resolution better than around 10 µm; higher lateral resolution is attainable, although sometimes requiring specially-designed apparatus (Shirai et al., 2014;Woodhead et al., 2005). Even if one measurement per season (warm/cold, dry/wet season) is sufficient to access the seasonality, more points permit better discrimination of the annual cycles and more accurate identification of the true extremes (Shirai et al., 2014).

Maps of several years' growth of bivalve shells (ca. 100 µm per year) by EPMA have been previously presented, but only for a portion of a shell of the long living Arctica islandica, representing 15 years of the individual's life (Shirai et al., 2014). In our Micropogonias sp. otoliths annual growth increments are observed with widths of 70-140 µm. A 4 µm lateral resolution permits a monthly to biweekly period, while a lateral resolution of 10 µm typically permits a temporal resolution of 1-2 months (depending on growth rate that varies both ontogenically and seasonally). The resolution attained in our SR-µXRF analysis reveals subseasonal variations, observable, for example, in the highfrequency fluctuations in QCD just above the Sr minimum (Fig. 33, S1) or in HP-12 between the core and the Sr maximum (Fig. 36,S3).

Sample features, including impacts of previous experiments, aid in sample alignment. Precision of location by surface features, particularly in the Ca maps, can vary depending on the dimensions of the feature, with smooth features such as LA-ICP-MS craters presenting significantly less precision than steep ones such as cracks. Due to the absence of any visible marks left by SR-µXRF characterisation, relocating the points analysed has previously been noted as more difficult than with techniques which leave a trace (Zumholz et al., 2007). Using the W marks permits alignment of even sub-zones of samples which have limited features which could be used for alignment. We developed in the present study multi-technique on-sample coordinate marking, described here for the first time on this type of samples. These marks decrease the uncertainty in positioning to one pixel with minimal intervention on the sample surface. They enhance the ease and reliability of comparisons to sclerochronological data and to results obtained from other geochemical analyses. The marks proved particularly helpful in initial positioning during a single synchrotron experiment. They were also efficient during later experiments, both for repositioning and accurate intercomparison of maps.

Quantification process

Semi-quantitative XRF analysis relies upon accurate determination of the area of peaks at characteristic energies (Janssens et al., 2000). This work applied curve deconvolution using PyMca software to identify and quantify the constituent elements. However, the superposition of very strong peaks and weak peaks can produce difficulties, as seen for elements whose characteristic peaks fall close to the Ca K (Kα=3690 eV) or Sr K (Kα=14 165 eV) peaks.

Sample matrix effects are typically countered by calibration from known references of an identical or similar material to the sample, which was performed in this study using pellets of CRM22 certified reference material (Yoshinaga et al., 1999(Yoshinaga et al., , 2000) ) composed of ground otoliths. The standard and samples used in this work should be very similar and variations matrix effects should therefore be negligible.

Detected Sr content of all samples fell in the range of 14-101 µmol • g -1 . Quantification of other elements, however, is more problematic. The absolute Ca quantities measured in our otoliths were 6000-19 000 µmol • g -1 , while pure aragonite contains 9990 µmol • g -1 (40%wt). We believe that the spectral resolution of the detector varying over the 10 000 eV range from Ca Kα to Sr Kα, and calibration on the single minor element (Sr) are responsible for this discrepancy. A linear correction factor was applied to the elements whose signals occur in the portion of the spectrum below 9650 eV (K, Ca, Mn, Fe, Cu, Zn, Ba), calculated to bring the median Ca concentration in line with that of the CRM22 reference (38.8%wt, or 9690 µmol • g -1 ). The absolute quantification of other elements may also be inaccurate as a result of poor SNR, due to their low concentrations and the short count time accepted as a compromise for faster mapping of Sr. Using longer count times and/or higher incident photon flux, quantitative maps of trace elements in otoliths by SR-µXRF, including Br, Ba, and Se have been reported in the literature (Limburg et al., 2007(Limburg et al., , 2010a,b;,b;Lochet et al., 2010;Limburg et al., 2011). SR-µXRF point analyses with 1000 s count times have successfully quantified Sr, Ba, and detected Y, Zr, and Nb in cuttlefish statoliths (Zumholz et al., 2007).

Insights into the reliability of the Sr signal

Our SR-µXRF maps reveal for all specimens very regular major fluctuations with a thickness on the order of 100 µm, as expected for individuals several years of age [START_REF] Kalish | Determinants of otolith chemistry -seasonal variation in the composition of blood-plasma, endolymph and otoliths of bearded rock cod Pseudophycis barbatus[END_REF]. More rapid fluctuations on the order of 1-2 pixels, or ca. 20 µm can be observed, for example shortly before the minimum in Sr content in QCD (S1, the distribution of values. Kernel density plots1 are shown for two archaeological specimens and one modern specimen (424) in Fig. 42. Kernel density plots offer a similar view of the data to histograms, but reduce the impact of bin width (analogous to kernel width) and eliminate the need to select an origin, both of which can alter the distribution's appearance (Silverman, 1998).

The kernel density plots reveal an additional dimension beyond the total spread of the data, and the maps in (Campana, 1999;Nakai et al., 1999;[START_REF] Morales-Nin | Seasonal and ontogenic migrations of meagre (Argyrosomus regius) determined by otolith geochemical signatures[END_REF]Albuquerque et al., 2010;Lin et al., 2015). The distributions for the other two samples are narrower, extending over 14-22 µmol • g -1 in 424, and 27-51 µmol • g -1 in QCD. Sample 424 is bimodal, with a primary mode centred at ca. 16 µmol • g -1 and a secondary at ca. 20 µmol • g -1 .

QCD exhibits a multimodal distribution, with a primary mode ca.

33 µmol • g -1 , and excursions to both higher (ca. Taking a mean of 5 points around the highest value in the SR-µXRF profile yields a more consistent average value of 80 µmol • g -1 .

The importance of sufficient lateral resolution to distinguish the true extremes has been previously demonstrated for the example of isotopic variations in mollusk shell by simulating a varying sampling width and comparing the extreme values identified to those of the simulated curve (Shirai et al., 2014). Using the pixels as a set of data permits examination of data reliability by revealing variations within isochrones, can reveal additional trends or extreme values, given sufficient lateral resolution, and therefore enhances confidence in measurements by providing multiple analogous data points.

Biogenic versus post-mortem signal

A full examination of fish life history and biological characteristics is beyond the scope of the present effort, but the capabilities of the technique used in this work, and the data treatments applied have implications for the examination of the quality of the otolith elemental signals. Different criteria have been used to assess the level of diagenetic alteration of ancient fish otoliths (Dufour et al., 2000;Disspain et al., 2011). The elemental signal itself can be considered a criterion to identify post-mortem alteration, assuming that elemental concentrations found in archaeological fish must be coherent with those found in modern specimens (Dufour et al., 2000;Disspain et al., 2012). In order to define a "fingerprint" of alteration, beyond Sr concentration, one can consider other elements which are either (a) not expected to be present in the otolith in significant quantities, or (b) regulated by the organism and expected to be present at certain levels.

Other than Sr, elements with Z > 20 are not typically present in otoliths at significant concentrations. Some heavier trace elements, which can be relatively common in the environment, can be present in otoliths (Campana, 1999). Y has been previously used as an indicator of diagenesis, as have rare earth elements (Brand and Veizer, 1980;Pucéat et al., 2004;[START_REF] Reynard | Trace elements and their isotopes in bones and teeth: Diet, environments, diagenesis, and dating of archaeological and paleontological samples. Palaeogeography[END_REF]; Fe, Cu, and Zn could be expected to serve similar roles as markers of the arrival of exogenous elements if the otolith examined exhibits relatively low mean levels coherent with those reported in literature. In this work, the concentrations of Fe, Cu, Zn, and Y were always higher in the resin than in the otolith within a given thin section, and decreased toward the inner otolith. Mn, Fe, Cu, and Zn were detected on the outer edges of archaeological samples QCD and Bayovar-1C, as well as modern sample 424 (Figs 33,34,and 35). Where present, these elements show a maximum at the otolith-resin interface with a generally smooth decrease to zero or near-zero over ca. 500 µm from the edge toward the core of the otolith. A similar presence on the edge of the modern otolith as the archaeological samples indicates that diagenetic alteration is not involved in the appearance of these elements at the interface between the samples and the resin.

In three cases (QCD, Bayovar-1C, HP-12), cracks in the samples also showed localised concentrations of at least one of Fe, Cu, and Zn.

It therefore appears that Fe, Cu, Zn, and Y originate primarily from the resin, and either infiltrated during embedding, or were spread across the otolith surface during polishing, as described by Jolivet et al. (2013) for otoliths of Merluccius merluccius. The resin used for embedding the otoliths was selected to have limited concentrations in trace and minor elements which may interfere with analysis.

This was indeed the case for K, Ca, and Sr, but not for Fe, Cu, Zn, or Y, at least one of which was detected in the resin of every sample . Resin contamination has been shown to impact analysis of the organic fraction of otoliths, and the potential impact of elemental impurities in the resin was noted (Jolivet et al., 2013).

Although contamination by the resin therefore made the use of Fe, Cu, Zn, or Y as indicators of diagenesis-related exogenous elements impossible in our samples, the contamination is generally limited to a few hundred micrometres. The sensitivity of SR-µXRF to the minute contaminations does bode well for detection of potential exogenous elements. As general advice, care must be taken in the selection of materials and preparation of thin sections to reduce alteration or contamination during preparation as much as possible for palaeoenvironmental proxies.

Na, S, Cl, and K are the only minor elements typically expected in otoliths at significant concentration other than Sr (Campana, 1999).

They are, however, poorly accessible by in-air XRF because of the low energies of their characteristic fluorescence lines, and only K was detected in any samples in this work. Interestingly, where detected Figs 33 and 36), we did not observe depletion in K at the sample periphery. It is generally expected that K is one of the more easily soluble elements in otoliths because it is presumed associated with the organic matrix rather than in the aragonite lattice (Gauldie et al., 1998;Proctor and Thresher, 1998). This is an indication of limited post-mortem alteration of these samples.

The Sr concentrations in archaeological Micropogonias sp. and Cathorops sp. otoliths measured in this study are mostly within, although some points are slightly above, the compiled literature biogenic values (<51 µmol • g -1 , Albuquerque et al., 2010). In contrast, some of the Sr concentrations measured in HP-12 and GP-292 exceed the range observed in literature up to present, with the archaeological HP-12 exceeding in some portions the maximum concentration of its modern counterpart. The data analysis performed in this work (Figs 42 and 43) supports that the obtained values are not analytical errors or the result of surface defects or contamination. The highest Sr concentrations measured in HP-12 and GP-292 exceed the 58 µmol • g -1 equilibrium solubility of Sr in bulk aragonite reported from crystallisation experiments (Plummer and Busenberg, 1987).

Theoretical studies, however, indicate significantly higher values (up to 1100 µmol • g -1 ) (Ruiz-Hernandez et al., 2010), well above the highest Sr concentrations measured in this work. To the authors' knowledge no elemental data have been previously published for G. peruvianus. The Sr concentrations observed herein for a modern specimen suggest a need to expand the expected biogenic range of [Sr] values for otoliths of marine fish. Further measurements are necessary to more accurately determine an expected Sr concentration range for the species of the coast of northern Peru. Until such a corpus of data is available, it is difficult to interpret the highest Sr levels in HP-12 as biogenic or resulting from post-mortem alteration based on the concentration itself. The various excursions or modes in Sr concentration observed in the archaeological specimens of this study (highlighted for HP-12 and QCD in Figs 42 and43) are within the defined biogenic range and are located at different positions along the growth axis within each otolith and not, as may be logically expected, only on the outer edge which is most susceptible to post-mortem alterations (Trueman, 2013). The enrichment is only truly at the edge in Bayovar-1C. In QCD the enrichment reaches its maximum some 100 µm from the actual edge and has a MAD of 0.4 µmol • g -1 , which indicates high intra-band homogeneity. Neither of the archaeological lapilli (HP-12 and BH17) exhibit such near the edge, so this is not a constant among all the archaeological samples in this work, while the modern sagitta 424 and the modern lapillus GP-292 both exhibit similar increases in Sr concentration. Given these observations, the modes and excursions in Sr observed in the archaeological otoliths might not be evidence of post-mortem alteration but might rather reflect variations in Sr content of habitats frequented by the fish over their lives. As such, the 28 µmol • g -1 mode measured in QCD (Figs 42 and43b) with an MAD of 0.2 µmol • g -1 may indicate an episode of life in freshwater as previously suggested by other proxies (Béarez et al., 2011;Aubert et al., 2012). We are not able to define conclusively if the Sr concentration increase near the edge of QCD evidences postmortem alteration. However, the lack of Sr enrichment beyond a few hundreds of micrometers deep seems to confirm the viability of the Sr signal as a palaeo-environmental indicator in our archaeological samples.

intermediate conclusion

Synchrotron micro-X-ray fluorescence multi-elemental mapping at micrometric lateral resolution is a fast-developing technique. This work sought to assess its the potential and limits for application to archaeological otoliths and bring insight to examination of the elemental compositions to better distinguish biogenic signal from post-mortem and post-depositional sources of alteration. SR-µXRF using medium X-ray energies provided accurate maps of Sr concentration with semi-quantitative calibration. This mapping approach allows for the investigation of intra-and inter-incremental variations with a subseasonal temporal resolution, which was coupled to a coordinate marking strategy for increased precision in positioning. Analysis of zones, complementing that of transects, Ca in aragonite of otoliths was recently demonstrated in modern otoliths (Doubleday et al., 2014). The stability of this substitution over long time scales, if proven, would be another strong indicator of the preservation of the corresponding environmental signal.

No alteration of the elemental signal was detected deeper than a few hundred micrometres into the otoliths, which supports their reliability for use in palaeo-environmental reconstructions.

Sr concentrations detected in some otoliths extend the range of typical reported values from literature, indicating a need to expand the knowledge in Sr concentration range for distinct species and environments. SR-µXRF presents some limitations in attainable lateral resolution as a result of the integrated information volume, comparable to that presented by LA-ICP-MS. Examining the Sr L fluorescence lines would significantly decrease the information volume and permit a further increase in lateral resolution, at the expense of a greater sensitivity to surface artefacts and heterogeneities. In this work a few core to edge maps were produced per day, which is a throughput comparable to common techniques such as LA-ICP-MS and EPMA. This rate can be significantly increased using high-throughput methods coupled to faster detection and recording while retaining a high sensitivity analysis (Medjoubi et al., 2013;Barberie et al., 2014). SR-µXRF mapping could therefore open new avenues to the analysis of stratified biocarbonate palaeoproxies thanks to its high sensitivity, speed, and data reliability.

chapter introduction

During accretional growth the otolith aragonite and the organic matter can incorporate essential and nonessential elements. Among the at least 27 minor and trace elements that have been reported in otoliths, Na, K, P, and Sr are typically found in the highest concentrations (Campana, 1999). Sr concentration profiles (normalised to Ca content) is the most widely used environmental tracer in fish and fisheries sciences.

In precipitation from solutions, Sr incorporation in aragonite follows thermodynamic fractionation relations (Plummer and Busenberg, 1987). In fishes, incorporation of Sr into otoliths is dependent primarily on water chemistry, and on the ambient temperature (Elsdon andGillanders, 2004, 2005b). Physiological effects and a possible impact of diet can be observed (Elsdon and Gillanders, 2003).

The elemental and isotopic signals from ancient otoliths are being applied more frequently for various types of studies (Campana, 1999(Campana, , 2005)): biological and environmental archives for the study of past fish migration or metabolism (Carpenter et al., 2003;Wurster and Patterson, 2003), palaeoenvironmental and palaeoclimatic conditions from the Jurassic to the Holocene (Patterson, 1998;Aubert et al., 2012;Vanhove et al., 2012;Disspain et al., 2011;Long et al., 2014), and fishing practices of past human populations (Hufthammer et al., 2010;Disspain et al., 2012;Carré and Dufour, 2012;Long et al., 2014).

The species-specific shapes of otoliths and quality of morphology preservation over geologic timescales enable taxonomic identification of fish assemblages from archaeological excavations (Nolf, 2013).

Identifying the species in such assemblages and comparing them to modern species with known behaviour and ecology enable biostratigraphic studies and reconstructions of palaeoecology and palaeoenvironment (Nolf, 2013(Nolf, , 1985)). Nevertheless, the reliability and permanence of the geochemical information encoded within ancient otoliths is critical to their use as information archives.

Diagenesis might hamper such reconstructions from ancient samples, and the level of this alteration in the otoliths has to be estimated (Dufour et al., 2000;Disspain et al., 2011).

Examination of the integrity of the internal and external morphology and mineralogy of ancient otoliths is a prerequisite to use of their geochemical signals. As a standard practice, altered specimens are distinguished from unaltered ones by detection of microstructural alteration and/or change in the identity of calcium carbonate polymorph. An intact microstructure is defined by the presence of growth marks typical of otoliths (daily and yearly increments), long needle-like (prismatic) aragonite crystals, and the absence of recrystallised or neoformed minerals (Hufthammer et al., 2010;Kern et al., 2012;Dufour et al., 2000;Vanhove et al., 2011).

Aragonite is metastable and can undergo a transformation to calcite over time (Plummer and Busenberg, 1987). Transformation to a crystal polymorph can be detected by X-ray diffraction (Béarez et al., 2005;Patterson, 1998;Dufour et al., 2000;Wurster and Patterson, 2001;Vanhove et al., 2011;[START_REF] Geffen | Highlatitude climate variability and its effect on fisheries resources as revealed by fossil cod otoliths[END_REF], vibrational spectroscopy (Kern et al., 2012), or cathodoluminescence (Kern et al., 2012;[START_REF] Geffen | Highlatitude climate variability and its effect on fisheries resources as revealed by fossil cod otoliths[END_REF]Béarez et al., 2005), as well as from the absence of apparent vein-like incursions in between the crystals at the millimetre and micrometre scales as viewed under light miscroscopy (LM) and scanning electron microscopy (SEM). The absence of calcite and vaterite is considered an indicator of geochemical preservation. The quality of preservation of the pristine geochemical signal measured in ancient otoliths can also be assessed through comparison to the otoliths of modern individuals of the same species or of a taxonomically close species which are expected to exhibit similar geochemical signatures (Dufour et al., 2000). Otoliths of taxa with different ecologies can be compared in order to reveal expected contrasts in different geochemical signatures (Vanhove et al., 2011).

However, these three parameters (mineralogy, microstructure, geochemistry) may not allow concluding on the low impact of diagenesis on the geochemical signal. For instance, diagenetic alteration may lead to an artifactual increase in elemental concentrations yet still within a realistic range for living fishes.

Central questions are whether: (a) samples with no evident alteration can be considered as intact at all length scales below a few micrometres, (b) atypical sample areas with significant elemental or isotopic excursions could show a specific behaviour, (c) outer surfaces of samples could be particularly affected. Working at the microscale is necessary as alteration could occur not only in the whole otolith, but also in localised zones (Dufour et al., 2000;Disspain et al., 2011).

However, to our best knowledge, no previous work controls in detail for speciation or potential post-mortem microscale alteration of ancient fish otoliths. The outer zones of the otolith may be expected to be more susceptible to alteration as a result of their direct contact with the surrounding environment (Trueman, 2013), including fluids such as those from the decaying animal. Heterogeneous post-mortem alteration is more likely to occur on the outer surface in contact with the burial environment (Cochran et al., 2010), but is not restricted to this zone (Dufour et al., 2000).

Regarding Sr used as a palaeoenvironmental proxy, its local mode of incorporation and stability within the organic fraction of otoliths and/or in the aragonite biomineral are likely to influence crucially its long-term behaviour, and therefore its reliability as a palaeoenvironmental proxy. Elements bound within the aragonite lattice may be less susceptible to exchange than those adsorbed on the surface or incorporated in the organic material (Gauldie et al., 1998). Over the past years, synchrotron-based spectroscopy and imaging have appeared as particularly adapted to the examination of the Sr chemical environment in modern and ancient biominerals (Doubleday et al., 2014;Finch et al., 2003;Allison et al., 2001) Furthermore A synchrotron micro-beam permits analysis of a small selected sample volume.

The lateral resolution afforded was used to select limited X-ray illumination areas with different Sr concentrations for analysis, such as the outer edge or a significant excursion. Millimetric area on an archaeological sample were examined using a fast speciation mapping approach. The Sr chemical environment detected was universally Sr in aragonite with no evidence for the presence of other polymorphs on the scale of these analyses. We discuss the palaeoenvironmental and diagenetic implications of these results.

samples examined in this chapter

Archaeological otoliths from excavations at Pampa de los fósiles, Huaca Prieta, and Bayovar-1 (northern Peru), were chosen because of their potential for palaeoclimatic information (Béarez et al., 2011;Dillehay et al., 2012;Goepfert et al., 2014). They form part of a larger study of palaeoenvironmental conditions and past human occupation in a region that lacks palaeoclimatic proxies. Otolith specimens of five archaeological and three modern fishes from the tropical Pacific coast of South America have been examined (Tab. 10).

The otolith sections were examined for gross alteration using LM, SEM, Fourier transform infrared (FT-IR) spectroscopy, and Xray diffraction (XRD). FT-IR spectra were collected in reflection mode with a spot size of 350 µm using a Bruker Fourier Equinox 55 FT-IR spectrometer coupled to a Bruker A-590 microscope (PALEOPROXUS, IRD, Bondy). XRD data (not shown here) were collected from subsamples of otoliths with Cu Kα radiation by the Gandolfi method using a custom-built diffractometer where an Sr K-edge macro-XANES spectra on the archaeological otolith sample QCD, and the inorganic strontianite and aragonite references were collected to provide reference spectra with a high signal-to-noise ratio while averaging possible microscopic fluctuations (Fig. 48).

The spectrum of the inorganic aragonite reference exhibits the The spectrum of QCD also exhibits some similarity to that of the strontianite reference. However, characteristic differences are observed: the low-intensity feature at +20.8 eV (iv) and the shoulder with those reported in previous works on carbonate biominerals and strontianite chemistry (Doubleday et al., 2014;Finch et al., 2003;Singer et al., 2008;O'Day et al., 2000). Fits of the EXAFS spectra resulted in good agreement to the experimental data for the first O, C, and Ca shells up to 4.0 Å in aragonite and QCD, and to 4.5 Å in strontianite.

Inclusion of additional O shells and multiple scattering paths was not attempted, as not requested for this work. This explains the absence of good fit of the contribution in the 4-5 Å range, also observed and not fitted by other authors (Doubleday et al., 2014).

Between one and 8 Sr K-edge µXANES spectra per sample were collected for all samples and references (Tab. 10) to permit analysis of a wide variety of zones and check for intra-sample homogeneity.

Representative µXANES spectra of the samples are compared to that of the aragonite reference in Fig. 50. The characteristic features, including the small oscillation at +20.8 eV, oscillation and shoulder at +43.4 and +65 eV, and the oscillation at +94.1 eV are consistent for all otoliths (Fig. 50). Between 5 and 8 µXANES spectra were recorded on each of QCD, HP-11, and HP-12 at the locations depicted in Fig. 47, with an overall Sr concentration range of 27-95 µmol • g -1 . These spectra all show superimposable features (Fig. 51). No variation is observed in the position of E 0 nor any of the characteristic oscillations.

The relative intensity and sharpness of the white line does vary slightly in some areas, as mentioned previously.

µXANES mapping of chemical environment

To assess the representativeness of point X-ray absorption measurements over large 'spatial dynamics' (Bertrand et al., 2013), µXANES mapping was carried out using five energies selected to correspond to characteristic features observed in the Sr K-edge XANES spectra of the aragonite and strontianite standards (marked by the dashed lines in Fig. 48). SR-µXRF maps were collected at these energies across areas up to 570 × 500 µm 2 on archaeological sample QCD (Fig. 53).

A transmission LM image (Fig. 53b) reveals variations in the luminosity, corresponding to approximately 1.5 years' growth. A crack in the otolith was observed in the Ca map and using LM, which was excluded from the analysis zone, leaving a final map width of 450 µm.

The map of aragonite fraction a derived from Eqn. 4 is shown in (Devereux, 1967) and is now fostering significant development in archaeology. In particular, otoliths have the capacity to elucidate fishing activities, interaction with aquatic ecosystems and seasonality of climates. Stable isotope analysis has suggested more humid conditions at the beginning of the Holocene for the arid northern coast of Peru (Béarez et al., 2011) and the winter settlement at different coastal Stone Age sites of Norway (Hufthammer et al., 2010). Oxygen isotope and Sr content analysis showed that Australian aboriginal settlement on the shores of Lake Mungo (Australia) correlates to the lake water levels at the time of the Last Glacial Maximum (Long et al., 2014). The type of habitat exploited by ancient Mid-to Late-Holocene fishermen were documented in two South Australian waterways by Sr and Ba content analysis (Disspain et al., 2011(Disspain et al., , 2012)). Elemental analyses also revealed the significant impact of human activities on hydrologic conditions of these waterways and provide a baseline reference dating before the arrival of Europeans in support of conservation and restoration efforts (Disspain et al., 2011(Disspain et al., , 2012)). Interpreting the geochemistry of ancient biominerals is, however, challenging, requiring an understanding of biological processes of incorporation (element partitioning or isotopic fractionation) and interpretation of post-mortem changes linked to pre-and post-burial environment. One of the major issues is that biominerals undergo post-mortem alteration that could impact the reliability of the biogenic geochemical information encoded. In this respect, otoliths have far less been studied than other biominerals such as bones (Nelson et al., 1986;Hedges, 2002;Collins et al., 2002;Trueman, 2013), enamels (Hoppe et al., 2003;Hinz and Kohn, 2010), shells (Chappell and Polach, 1972;Buchardt and Weiner, 1981;Cherns et al., 2011), or corals (James, 1974;Webb et al., 2009). No comprehensive study of otolith diagenesis has been conducted so far.

The present work constitutes a first effort to understand the long term behaviour of Sr in archaeological otoliths by examining the chemical environment of this element.

Incorporation of secondary elements into carbonate biominerals can occur in one of the five main modes illustrated in Fig. 64 that can be broadly categorised into four mechanisms: (1) incorporation into the organic material, (2) adsorption onto the surface of micro-or nano-crystals of aragonite, intra-crystalline incorporation, comprising [START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] substitution for Ca and (4) incorporation into interstitial spaces, and (5) as discrete strontianite phases. Sr (or any other element) adsorbed on the surface or incorporated into the organic material is expected to be less stable than in the crystal lattice (Gauldie et al., 1998). In aragonite, Ca 2+ is coordinated to 9 oxygen ions (while coordination is 6 in calcite). Sr 2+ is assumed to substitute isomorphously for Ca 2+ owing to the similar charge and ionic radius of the two ions: 118 pm for Ca 2+ and 131 pm for Sr 2+ in 9-fold coordination (Tab. 2) (Speer, 1983;Shannon, 1976;[START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF].

Sr is not significantly leached out during handling or preparation (cleaning, sectioning, polishing, staining, and etching) of otoliths (Gauldie et al., 1998;Milton and Chenery, 1998;Andrus and Crowe, 2002;Proctor and Thresher, 1998). This has generally been interpreted as Sr incorporation into the aragonite lattice (Milton and Chenery, 1998;Andrus and Crowe, 2002) but has alternatively be interpreted as the presence of stable strontianite domains (Gauldie et al., 1998). for Ca is confirmed as a general characteristic in modern otoliths, a different mode of incorporation in ancient otoliths would provide a direct identification of post-mortem alteration.

Micrometric point µXAS analyses ubiquitously indicates Sr in aragonite

In (Kalish, 1989).

Our work. We can therefore rule out the presence of calcite or vaterite as significant environment for Sr in any otolith.

Distinction of aragonite from strontianite is a more difficult task (Greegor et al., 1997;Finch et al., 2003). µXANES spectra of both carbonate compounds are relatively similar owing to their isostructurality (Tab. 2). However, XANES spectra did allow the identification of discriminating features (Fig. 48), attributed to the change in geometry and crystal lattice parameters, as well as Sr-Sr paths. Detectable differences are observed in the EXAFS Fourier transform (FT) (Fig. 52). In the strontianite reference, the Sr metal signal and peak occurs at 3.95 Å (Fig. 52). Inorganic aragonite exhibits a Sr-metal peak at ca. 3.90 Å, which is shared by all otolith samples.

The positions of these peaks are coherent with those previously presented for Sr in aragonite (Finch et al., 2003;Doubleday et al., 2014). The imaginary part of the FT of the otolith µEXAFS signal distinctly differs from that of strontianite while it is in very good agreement with the inorganic aragonite standard (Fig. 52,bottom). The majority of published works using XAS have observed no sign of calcite or strontianite formation in biomineral aragonites. The random substitution of Sr for Ca has been demonstrated in different species of corals, both modern (Finch et al., 2003;Finch and Allison, 2003;Allison et al., 2005Allison et al., , 2001;;Doubleday et al., 2014) and ancient (Allison et al., 2001;Finch and Allison, 2003), the shell of one modern bivalve species (Foster et al., 2009), and statoliths or cuttlebones of two modern cephalopod species (Doubleday et al., 2014). However, one work identified strontianite domains within modern and ancient aragonitic corals (Greegor et al., 1997) by fitting the FT magnitude of wide-beam EXAFS analyses with linear combinations of pure references (strontianite and Sr in aragonite).

Indeed
A macroscopic XAS analysis, as in Doubleday et al. (2014), typically produces an analysis volume at least 6.3 × 10 7 µm 3 , assuming a 250 × 250 µm 2 spot size in transmission and a 100 µm thick sample.

Our work employed a Gaussian-shaped X-ray spot with FWHM dimensions of 6 × 11 µm 2 (V×H) on sample. At the energy range and geometry employed, this produces a stretched information volume along the beam axis, penetrating ca. 260 µm deep into the sample and across the horizontal. The corresponding information volume totals 3.7 × 10 4 µm 3 (for the volume producing 75 % of the signal). Taken as an example, the DiffAbs beamline provides 10 12 photon/s in widebeam mode, and ca. 3 × 10 9 photon/s in micro-beam mode at the Sr K-edge. A greater photon flux in wide-beam mode results in an improved (lower) detection limit than in micro-beam mode. However, the smaller spot size in micro-beam mode results in a cleanly-shaped beam of a greater photon flux density (ca. 45 photon/s/nm 2 ) than in wide-beam mode (ca. 13 photon/s/nm 2 ). In a homogeneous system, the higher signal-to-noise ratio (SNR) of macro-mode provides a higher sensitivity, but in our biomineral system which could present microstructural heterogeneities, increased lateral resolution yields an increased sensitivity to local alterations. Even assuming an order of magnitude higher detection limit for the microscale compared to macro-scale (e.g. 10 000 ppm vs. 1000 ppm, respectively), a microscale analysis has the potential to reveal heterogeneous alteration 1-2 orders of magnitude smaller. The combination of µEXAFS and µXANES in the present study has permitted analysis of otoliths on the microscale with an increased sensitivity to small-scale alterations.

No alternate phases were detected on the micrometric scale.

Examination at a further increased lateral resolution, approaching that of the nanometric crystallites, would require increasingly complex and prohibitive sample preparation (Stangl et al., 2013). closely resembles the Sr map (Fig. 53d). This is a good indication that the model is accurate and that there are no tertiary phases.

Indeed, normalising the β factor by the Sr concentration at each pixel produces a homogeneous narrow distribution (mean=1.8 × 10 -3 , SD=8.3 × 10 -5 ).

The estimated aragonite fraction a is largely homogeneous across the map (mean=91 %, SD=7.2 %) while the contribution from strontianite-type domains is estimated to be ca. 9 %. However this figure is similar to the observed noise, attributed to the limited Sr XRF counts per pixel, and we do not see any outlier hotspot of strontianite.

Without completely ruling out the possible presence of strontianite domains as a minor component of the probed information volumes, a purely aragonite-type system where Sr would randomly substitute for Ca suffices to explain the observed data. The presence of Sr-Sr neighbours resulting from random substitution has been suggested as a possible source for strontianite-type contributions to XAS spectra in aragonite regions of higher Sr concentration (Doubleday et al., 2014). However, it appears relatively unlikely in our case. The expected probability to find Sr atoms with more than another Sr neighbour is very low -1.6 × 10 -5 for the highest Sr concentration observed in the mapped zone. Contributions from random Sr-Sr pairs would therefore be negligible in our absorption spectra. Although no constraints were applied to the model, the majority of points fell within the expected aragonite fraction range of a = (0, 1). Some outlier points (ca. 1.4 SD from the mean) occurred above 1.0, to a maximum of a=1.18. This further supports the model's accuracy.

In all otoliths analysed, a great attention was taken to orient the growth axis vertically to maximise the lateral resolution along this growth axis (see Fig. 53a). Annual growth increments in fish otoliths are perpendicularly oriented relative to the growth section and were observed to be 70-140 µm in width in QCD. As a result, the QCD maps crossed two sequential isochronous increments, and allowed measurements at high resolution within a single increment. The information volume for this analysis is identical to that previously described for point µXAS and is on the same order of size as the needle-like microcrystals composing individual increments which are visible under SEM (Fig. 64; Fig. 2 in Dauphin and Dufour (2008)).

Fast µXANES mapping at high resolution over a large area offers for the first time the ability to study Sr speciation over 'large spatial dynamics' (Bertrand et al., 2013) covering 2 orders of magnitude from ca. 10 µm to the millimetre range, in order to identify any heterogeneity. Ancient biominerals can exhibit heterogeneous alteration at submillimetric scales (e.g. Guzmán et al., 2009;Webb et al., 2009). Mapping on the edge of QCD was chosen because, during burial, the outer surface of the otolith is expected to be the most vulnerable to alteration by being in direct contact with the decaying fish, as well as fluids and sediments from the environment (Trueman, 2013). µXRF showed that this zone exhibits significant variation in Sr content, including the largest variation for the whole specimen, that could alternatively be interpreted as resulting from post-mortem alteration or an event during the life of the specimen. However, the mapping demonstrates that no heterogeneity in Sr speciation is present within the limits of detection.

At the length scale of the increment thickness and at the limit in terms of attainable lateral resolution, an intriguing and yet unexplained behavior was observed. The Ca content of the otoliths was observed to vary up to 3.5 % of the median in the core to edge profiles produced by SR-µXRF (Fig. 65). This fluctuation appears mostly anticorrelated to the Sr concentration. This is particularly noticeable for the most extreme Sr concentration peaks observed in the archaeological specimens QCD and HP-12.

It is unlikely that this anticorrelation results from improper XRF matrix correction as the attenuation length of the Ca signal is altered by no more than 0.25 % across the range of Sr concentrations found in otoliths (0.05-0.5 %at). The variation in Ca is too large to be accounted for purely through increased substitution by Sr. Ca-depleted regions are likely to correspond to zones richer in organic matter. However, in our samples, even in those zones, Sr appears to substitute randomly for Ca in an aragonitic chemical environment, with no evidence of Sr in another chemical environment. This anticorrelation may come unnoticed in measurements using Laser ablation-inductively coupled plasma-mass spectrometry or SR-XRF as Sr concentration is usually normalised to Ca concentration (e.g. Limburg et al., 2011), and deserves further investigation.

We collected multi-energy µXANES maps to obtain spatiallyresolved speciation information. XAS imaging is undergoing a significant development for ancient materials studies (Bertrand et al., 2012). For such measurements, two general options are offered: using a full-field illumination or a scanning microbeam. Full-field approaches have been implemented in transmission geometry at several synchrotron beamlines (Fayard et al., 2013;Meirer et al., 2013).

At an identical SNR (i.e. count per pixel), full-field imaging allows one to decrease the flux per pixel by several orders of magnitude, and suppresses dead times due to sample movement. However, the attenuation length of Sr K-edge photons is ca. 330 µm. Collecting a signal in transmission would require thinning down samples to this typical thickness. Samples of very small areas could be selfsupported, but larger samples would have to be fixed on a low X-ray absorbing support with attached risks of contamination. In both cases, this would contravene strongly to our aim to work as much as possible non-invasively on historical samples that were studied with complementary characterisation techniques. Use of fluorescence detection in a reflection geometry in a full-field mode is emerging but remains limited due to the low solid angle of detection of 2D XRF detectors in full-field mode (Tack et al., 2014).

Using a scanning microbeam, data collection is also possible using fluorescence detection in a reflection geometry. Recording a full XAS spectrum at each pixel with such an approach requires both a very high flux at each beam impact position and a very long total acquisition time. High fluxes may increase radiation-induced side effects (Bertrand et al., 2015). Even a relatively fast acquisition of 5 min per XAS spectrum, would take over an unrealistic 19 days.

The five-energy µXANES mapping approach employed in this work (570 × 500 µm 2 map with a step size of 5 × 10 µm 2 ) resulted in a map obtained in 10 h on a bending magnet beamline, while comparatively vastly reducing sample exposure to radiation. In our experiments, two scan strategies were possible: (i) positioning the sample and collecting successive XRF spectra, or (ii) setting successively each energy and collecting a full XRF map at each energy. This last option is usually preferred as it limits movements of the monochromator that may impose significant additional dead times. However, in this second option, the precision of the mechanical setup should be high enough to allow repositioning each energy map at the pixel level, while the stability of the synchrotron beam position is also essential.

We have discussed the complexity of interpreting X-ray absorption data collected at a limited number of excitation energies, however, it provided a unique insight into the large-scale homogeneity of the Sr 2+

substitution in otolith aragonite in realistic experimental conditions applicable to schlerochronological and ecological studies.

Post-mortem alteration of Peruvian archaeological otoliths

According to the common criteria which are used to assess the lack of diagenetic alteration in ancient otoliths and other biocarbonates (Dufour et al., 2000;Guzmán et al., 2009), the five Holocene Peruvian specimens of this work can be considered as intact. As aragonite is not the most thermodynamically stable of the three pure CaCO 3 polymorphs under terrestrial surface conditions, aragonite (biogenic or abiogenic) tends to transform to calcite over time (Cherns et al., 2011). There was no calcite present in our samples from the macroscale (seen with XRD and FTIR) to the microscale of the Sr chemical environment (demonstrated with µXAS). Textural observations showed no evidence of recrystallization at the level of the needle-shaped microcrystals (ca. 10-100 µm long and 1 µm in diameter), as observed under .

Otolith texture presents further levels of organization that were not explored in the present work, but that may play a significant role during diagenesis. Microcrystals are polycrystalline, composed of crystallites on the order of several hundreds of nanometers embedded completely in an organic matrix (Falini et al., 2005;Dauphin and Dufour, 2008;Söllner et al., 2003). The crystallites themselves are composed of granules a few tens of nanometers in diameter and wrapped in organic envelopes (Gauldie, 1999;Söllner et al., 2003;Dauphin and Dufour, 2008). Even if this level of organization has only been described in sagitta, it is expected to be identical in lapilli (and asterisci) since at the higher level (microcrystal units) they share similar organization (Dauphin and Dufour, 2008). Alteration of the microstructures within the prismatic calcitic and crossed lamellar aragonitic layers in the shell of Concholepas concholepas has been recognized even if these layers appear well preserved (Guzmán et al., 2009).

A third criterion to assess diagenetic alteration is the examination of the geochemical signals themselves (Dufour et al., 2000;Disspain et al., 2011Disspain et al., , 2012)). Elements that are known to be endogenous such as Na, P, K, and Sr can be studied to detect possible alteration on the basis of significant deviation from usual content. Exogenous elements diffusing into the otolith directly mark alteration. µXRF maps revealed that the Peruvian otoliths exhibit large variations in Sr concentrations (Fig. 47). While [Sr] A porous mineral organization that permits the circulation of fluids promotes mineralogical change and elemental exchange in abiogenic minerals and biominerals. The complex multiscalar organisation down to tens of nanometers increases the surface of contact with fluids. The organic matrix is present at all scales of crystalline organisation in otoliths (Falini et al., 2005;Dauphin and Dufour, 2008). Water diffusion in otoliths has been directly evidenced by mass increases up to 4.18 %wt after soaking otoliths in water for 48 h (Gauldie et al., 1998). This water is not bound to the matrix, but is mostly molecular water according to the characteristic IR peak, and is therefore available for ionic transport (Gauldie et al., 1998). Diffusion of ions and molecules into and out of the otoliths is also indirectly evidenced by the increased contrast of internal features when otoliths are soaked in water, oil, solvents, or dyed by large molecules to facilitate the reading of growth marks under light transmission (Gauldie and Coote, 1997). As a consequence, changes in elemental contents were noticed when using several analytical preparation and storage methods (Rooker et al., 2001;Milton and Chenery, 1998;Proctor and Thresher, 1998). This was reported for "labile elements" such as Na, K, S or Cl (Proctor and Thresher, 1998). On the contrary, the Ca and Sr contents of modern specimens, immersed in solutions, were noted as relatively stable. This observation was made in the limited timeframe of laboratory experiments -a few hours to several days, and may not correspond to the long-term behaviour of archaeological samples over thousands of years. After the fish death, decay of the organic matrix could further increase exchange of elements with the environment. According to Guzmán et al. (2009), the dissimilarity in the diagenetic changes between the calcitic and aragonitic layers within shells of C. concholepas are related to the difference in quality and quantity of the preserved organic matrix.

The Peruvian otoliths studied here are likely to have experienced limited exposure to fluids after their initial taphonomy, due to the exceptional climatic conditions that prevail on the coast of northern Peru. Coastal fogs that form during the austral winter are the main source of humidity in the very arid environment while intense precipitation is mostly limited to El Niño events.

Moreover, the otoliths in the present work originate from two sandy surface sites (Bayovar-1 and Pampa de los fósiles) deprived of pore water and from one stratified mound (Huaca Prieta) not susceptible to significant ground water exposure. Prevailing climatic conditions on the Peruvian and Chilean coasts have favored the conservation of Holocene bivalve or gasteropod shells (Carré and Dufour, 2012;Guzmán et al., 2009), limiting the diagenetic alteration, or on the contrary led to their rapid diagenesis, thereby precluding palaeoenvironmental reconstructions based on geochemical analyses (Labonne and Hillaire-Marcel, 2000). In the latter case, the shells originated from emerged marine terraces. Macroscopic examination showed that the aragonitic layer was frequently pulverulent. Early diagenesis during marine immersion has been evidenced by a marine U/Th signal (Labonne and Hillaire-Marcel, 2000). Later contamination by sea spray could be present as an agent of alteration, but the effects are expected to be very limited compared to immersion.

Samples from marine sites may undergo significant alteration as a result of the circulation of pore water in the surrounding sediment, so future examination of otoliths from varying deposition environments is necessary.

The diagenetic trajectory followed by our samples is more favorable for preservation as post-mortem contact with environmental fluids is minimal in a continental arid environment. The otoliths studied in this work have not been in direct contact with seawater or sediments with circulation of pore water, as the fish was caught and prepared, and the otoliths later discarded with the carcass in an arid continental context. They could however have been affected by cooking operations. Cooking and burning were shown to alter the mineralogy and elemental concentrations in otoliths (Andrus and Crowe, 2002). Even though cooking operations may not always leave recognisable evidence, we intentionally chose otoliths with no sign of burning or macroscopic alteration. The samples examined in the work appeared well-preserved by the common microstructural and mineralogical criteria; further work examining samples which exhibit visible alteration maybe valuable to expand knowledge of otolith taphonomy and reliability.

A D VA N C E D C R Y S TA L T E X T U R E R E C O N S T R U C T I O N O F O T O L I T

chapter introduction

Texture, the preferential orientation of crystallites in a polycrystalline material, has a significant impact on its properties, such as resistance to strength, toughness, fracture behaviour, and optical response [START_REF] Engler | Introduction to Texture Analysis[END_REF]. Determination of the distribution of crystal orientation on macroscopic samples has been performed by following variations of intensity within Debye-Scherrer rings collected in Xray powder diffraction for nearly a century [START_REF] Debye | Interference on inordinate orientated particles in x-ray light. iii[END_REF][START_REF] Engler | Introduction to Texture Analysis[END_REF]. Texture is often displayed in pole figures, which are 2D representations of crystallite orientations in 3D space [START_REF] Engler | Introduction to Texture Analysis[END_REF]. To produce a pole figure, typically with neutron or X-ray macrobeams, a series of diffractograms must be recorded with varying sample angular orientation throughout a large number of angles. Here we will refer sensu stricto to 'texture' determination when all crystal orientations are known, while 'crystal orientation' determination will be used to describe incomplete texture information, such as result from projection of one or more axes onto a single plane, known to be insufficient to describe the full texture.

In addition to the determination of crystal orientation at macroscopic scales, that of spatial fluctuations within samples is particularly relevant to a variety of fields related to such studies, for example materials science and biomineralisation research. Crystal orientation in a given localised zone can be determined at the microscopic scale by direct observation of crystallite habit using optical microscopy, SEM, or atomic force microscopy [START_REF] Gauldie | Atomic force microscopy of the morphology of the matrix and mineral components of the otolith of hyperoglyphe antarctica[END_REF][START_REF] Lausch | Determination of crystal grain orientations by optical microscopy at textured surfaces[END_REF], although this approach is rather labour-intensive, can require special sample preparation, may be impractical for larger sample areas, and may not be applicable for all samples. The development of electron backscatter diffraction (EBSD) in the early 1990s made mapping of crystal orientation a realistic endeavour for many applications, with lateral resolution at the microscale [START_REF] Sr | Orientation imaging: The emergence of a new microscopy[END_REF][START_REF] Engler | Introduction to Texture Analysis[END_REF]. Current EBSD measurements allow proper determination of crystallite orientation through Kikuchi patterns, yielding local (inverse) pole figures and determination of misorientation statistics in 10-1000 ms/pixel with nanometric spatial resolution [START_REF] Humphreys | Characterisation of fine-scale microstructures by electron backscatter diffraction (ebsd)[END_REF][START_REF] Mainprice | A personal and practical guide to the history, installation and future of the Electron Back-Scattered Diffraction (EBSD) system[END_REF].

There are however limitations to EBSD. The lateral dimensions of the analysis zone is often limited (Schulz-Mirbach et al., 2013a).

Additionally, EBSD probes a few tens of nanometres at the sample surface. This depth resolution is a clear advantage for many experiments but also places significant constraints on sample characteristics, preparation, radiation damage and further limits the dimensions of areas that can be studied [START_REF] Nowell | Ebsd sample preparation: Techniques, tips, and tricks[END_REF].

EBSD may also not be well adapted to stratified samples that are mostly '2D', where integrating on the depth dimension may be desirable to attain better count statistics while preserving most of the spatial information. While texture measurements have most commonly been performed using electron beams in transmission or reflection [START_REF] Engler | Introduction to Texture Analysis[END_REF], neutron or X-ray beams have been employed in many works. Development and implementation of appropriate apparatus to produce intense, focused X-ray beams has, for example, permitted micro-scale texture measurement of large cellulose molecules in wood via scattering experiments [START_REF] Keckes | Cell-wall recovery after irreversible deformation of wood[END_REF]. Thin films of nickel and cerium oxide have been examined for microtexture and other crystallographic properties to suggest that in heteroepitaxial systems increased lattice mismatch can enhance growth, contrary to the expectation of enhancement from decreased mismatch [START_REF] Budai | X-ray microdiffraction study of growth modes and crystallographic tilts in oxide films on metal substrates[END_REF]. Full texture information is a valuable source of understanding for crystalline systems. It is, however, time consuming, and compromises using partial informationdetermination of crystal orientation rather than full textureare often accepted. Diffraction patterns of carbon fibres collected using a 2D detector and an X-ray nanobeam have been used to identify the buckling of bent carbon fibres under compression and compare the strain and compression behaviour of fibres from different manufacturing processes [START_REF] Loidl | Direct observation of nanocrystallite buckling in carbon fibers under bending load[END_REF]. Orientation of macromolecules determined by wide-angle X-ray scattering has been used to demonstrate that increased exposure of spiders to carbon dioxide alters the amino acid makeup of the extruded silk, consequently altering its material properties [START_REF] Riekel | Influence of CO 2 on the micro-structural properties of spider dragline silk: X-ray microdiffraction results[END_REF].

Crystal orientation measurements have revealed preferred orientation in tooth enamel which varies relative to the biting surface and to the position on the tooth, as well as variations in lattice parameters bewteen the outer enamel and the dentin junction, hypothesised to be a result of variations in chemical composition [START_REF] Al-Jawad | 2d mapping of texture and lattice parameters of dental enamel[END_REF]. An approach permitting significantly faster X-raybased characterisation of polycrystalline samples, both phases and orientations, over a large area would prove useful in many areas of materials science.

The development of micro and nano X-ray focussing, and that of Xray detectors, is considerable, and has allowed a fast development of a range of X-ray imaging modalities. XRD raster-scanning experiments can be performed with lateral spot sizes down to tens of nanometers (Stangl et al., 2013;[START_REF] Tamura | X-Ray microdiffraction of biominerals[END_REF]. Such experiments require a high photon flux, both due to the fact that focusing may result in a decrease of typically 1-3 orders of magnitude of the integrated flux in the spot, and because the total collection time will vary linearly with the number of pixels. Synchrotron radiation is therefore a source of choice for microfocus experiments. By coupling a pixel area detector to a synchrotron source, the equivalent of thousands or millions of angular positions with a point detector can be recorded in a single exposure. With sufficient X-ray flux, collection of diffraction images in sub-second exposure times can be achieved, which reduces the time to record hundreds or thousands of images to the range of hours, making micrometric resolution mapping of crystal orientation on millimetric or centimetric scales a realistic possibility [START_REF] Barna | Development of a fast pixel array detector for use in microsecond time-resolved x-ray diffraction[END_REF]Medjoubi et al., 2013;[START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF]. While an efficient collection scheme using a point detector may require 30 min per sample to record the thousands of angular positions required for a pole figure, using a two-dimensional detector can reduce this to less than one minute per pole figure [START_REF] Mocuta | Fast pole figure acquisition using area detectors at the DiffAbs beamline -Synchrotron SOLEIL[END_REF]. Further reducing counting time is critically important as recording using a fast texture determination of 1 min/pixel still requires unrealistic exposure times for laterally-resolved texture maps that would comprise hundreds or thousands of pixels. Such developments are particularly important in the case of materials where the properties can be engineered on the basis of local crystal orientation in materials science, and for those which need to be studied as is, such as from biology, Earth and environmental sciences. Heritage and ancient materials also fall in the latter category (Bertrand et al., 2013). There is therefore a great interest in increasing efficiency and sensitivity through exploration of enhanced methodologies using state-of-the-art instrumentation in order to allow large-scale determination of crystal orientation in samples with a microbeam.

Here we present and discuss developments in synchrotron micro-X-ray crystal orientation mapping of whole real polycrystalline biocarbonate samples. Analyses of centimetre-scale zones measured at micrometric resolutions using a large-area pixel detector are demonstrated. Employing a hard X-ray beam permits examination of samples up to hundreds of micrometres thick depending on the material, and an on-sample spot size of several micrometres permits a high lateral resolution over centimetric distances. The use of a large-area X-ray pixel detector permits efficient recording of diffraction images with texture sensitivity in a single-shot approach.

The setup used is a step towards further implementation of the continuous scanning rapid acquisition system (flyscan; Medjoubi et al., 2013;[START_REF] Barrea | Fast-scanning high-flux microprobe for biological X-ray fluorescence microscopy and microXAS[END_REF]. This approach is demonstrated in this work though application to one modern and one archaeological fish otolith. To our knowledge the present work represents the first laterally-resolved investigation of otolith crystal orientation on the full scale of the sample using a probe of the crystal lattice parameters. This investigation supports existing descriptions of crystal orientation [START_REF] Ralston | Analyzing the width of daily otolith increments to age the hawaiian snapper, Pristipomoides filamentosus[END_REF][START_REF] Davies | Otolith ultrastrucrure of smooth oreo, Pseudocyttus maculatus, and black oreo, Allocyttus sp., species[END_REF][START_REF] Gauldie | Atomic force microscopy of the morphology of the matrix and mineral components of the otolith of hyperoglyphe antarctica[END_REF]Dauphin and Dufour, 2008 Figure 66: Diffraction pattern integrated over the entire section of otolith QCD with aragonite reflections labelled. Some non-aragonite reflections are visible in the "Maximum" curve, although they produce a minimal effect on the median plot, suggesting very few pixels with non-aragonite signals.

simultaneous detection of X-ray fluorescence was performed to attain semi-quantitative elemental mapping. Coupling of elemental content determination with texture are expected to become standard approaches on full real samples when local fluctuations in texture could be correlated with composition in trace or minor elements.

development of fast crystal orientation mapping of aragonitic otoliths

Otoliths are polycrystalline biomaterials, and the sections employed in the present work are expected to be approximately two-dimensional, representing a thin section through the threedimensional structure. The approximation to two dimensions allows a simpler mapping approach to derive the crystallite orientations, rather than needing a full three-dimensional analysis.

Sections of one archaeological (QCD) and one modern ( 424) otolith (Tab. 5) were mapped using X-ray diffraction. In both samples the mineral phase aragonite CaCO 3 was ubiquitously identified across both entire otolith sections (Fig. 66).

Using images collected with the area XRD detector, maps of crystal orientation were constructed in the form of intensity variation about the angle of elevation (ψ). The twofold symmetry of aragonite would require a ψ range of 180°to fully explore the orientations (i.e. half of the 360°in a full diffraction ring projected; see Sec. 3.7, Fig. 29). Under the conditions described in Section 3.7, the images collected give access to somewhat over half this range, 100°in ψ (although the range varies with 2θ), allowing measurement of the crystal orientation for at least two axes in most cases.

The nonisotropic distribution of reflections on ψ (Fig. 67) illustrates the presence of preferential orientations within the crystalline units of the samples. To better observe this, maps for three selected reflections (021, 012, and 112) are represented after integration of the diffraction rings on successive segments of 10°in ψ in Fig. 68.

These reflections are well resolved and exhibit among the strongest reflections across the otoliths overall. The nonisotropic distribution can also be observed using other reflections, notably those purely related to the main axes of the aragonite crystals. In an orthorhombic system, the odd-numbered reflections orthogonal to the unit vectors are forbidden, so the even reflections 200, 020, and 002 can be used instead (Fig. 69), although they are also relatively weak in aragonite (Fig. 66). The second column from the right is labelled These values are too close to attempt to directly discriminate one from another on this basis, particularly in such a highly-oriented sample.

In both samples, maps of reflection intensities binned by ψ reveal a "sweeping" pattern of radial sectors centred on the otolith centre with varying ψ. As oriented in the present work the bottom half of the otolith sections (the interior face in the organism) exhibits a more orderly growth, even based on visual microscopic examination . This portion of the otolith is therefore most commonly used in studies, and will be the focus of this work, although the upper (exterior) portion shows interesting behaviour nonetheless. For most of the reflection planes examined, the reflections are more homogeneous in intensity in otolith 424 than the archaeological sample. The 002 reflection, however, is less homogeneous. In section 424 it exhibits a high intensity in only one ψ bin (60° ψ 70°). This may be related to the position of the section: QCD was cut from the centre of the otolith, including the nucleus, while 424 is a nucleusadjacent section, so it may be that the c axis is not well within the plane of the section of 424. Section 424 exhibits a similar but more curved horizontal discontinuity along the horizontal centre line in the maps. In otolith QCD, a second discontinuity, indicated by an arrow in the LM image (Figs. 68 and 69), is visible in the "sweep" about the nucleus. This discontinuity is also visible under LM. It does not follow the gradual transition of orientation about the nucelus, but rather is an abrupt interface between two distinctly oriented domains.

Elemental mapping

Simultaneous with µXRD mapping, the µXRF signal was recorded to produce elemental maps. Simultaneous recording offers useful advantages by enabling direct correlation studies as well as confirming sample positioning. For instance, in Fig. 70 including water chemistry and temperature, at the time of deposition (Campana, 1999). This incorporation produces a permanent biological archive of an individual's life history (Campana and Neilson, 1985).

Geochemical analyses of modern otoliths are often used for tracking of natal origin, stock identification, and individual movements (Panfili et al., 2002). Geochemical analyses of archaeological otoliths have been used with increasing frequency to access the life history archives therein to, for example, identify migrations between fresh and salt water of a now-extinct species from the Late Cretaceous (Carpenter et al., 2003) and demonstrate a correlation of Australian aboriginal settlements on the shores of Lake Mungo (Australia) and the lake water levels at the time of the Last Glacial Maximum (Long et al., 2014). However, the crystal polymorph present has been shown to impact the incorporation of trace and minor elements in otoliths [START_REF] Brown | Elemental distribution within polymorphic inconnu (stenodus leucichthys) otoliths is affected by crystal structure[END_REF]. For example, strontianite has a much lower solubility in calcite than aragonite; if an environmental reconstruction were performed using otolith which is part calcite, a lower salinity might be estimated due to lower Sr content.

This variation of Sr content is particularly marked in the map of 424 (Fig. 70), where regular fluctuations -possibly annuli -are distinguishable in the µXRF map. Some variation is also apparent in the XRD reflection intensity between the various regions (Fig. 70, regions 1-4). In region 3, a high-medium heterogeneous reflection intensity is correlated to a low Sr content, and in region 4 a lower intensity is correlated to a high Sr content. This variation in intensity cannot be due to other phases as none were detected. It may be expected that the Sr increases the X-ray absorption due to the smaller energy separation between its K-edge (16.1 keV) and the beam energy (17.2 keV). However, within the Sr concentration range observed, the impact is limited: the attenuation length in aragonite (ρ=2.93 g • cm -3 ) is 394 µm for 0.1 % atomic Sr, and 391 µm for 0.2 % atomic Sr [START_REF] Thompson | X-ray Data Booklet[END_REF]. As Sr fluctuations are also seasonal, the absorption variation may also result from an overall change in the otolith density due to variations in organic content, which is difficult to model accurately due to its commingled incorporation in varying fractions. With fixed 0.2 % atomic Sr and 10 %wt organic content, density decreases to ρ=2.78 g • cm -3 , the attenuation length rises to 412 µm. Furthermore, the anticorrelation between Sr content and reflection intensity is not valid in region 1, which exhibits higher Sr content and higher reflection intensity. It is difficult to draw conclusions on the relation of optically-distinct annuli with reflection intensity as the annuli occur at a similar length scale to the pixel size, but further work at higher lateral resolution may reveal a relation between growth marks and diffraction properties which may assist in sclerochronological interpretations.

post-mortem

Archaeological otoliths, after hundreds or thousands of years, may have undergone alteration of the mineral phase, ie. partial conversion to more thermodynamically stable calcite (Dufour et al., 2000;[START_REF] Cook | Evaluating post-mortem alteration in biocarbonate palaeoenvironmental proxies: Coupled synchrotron X-ray absorption and fluorescence spectroscopy[END_REF]. Environmental reconstruction from archaeological otoliths should therefore be carried out with some concern to potential alterations, either from pathological growth or taphonomy. Some studies employing archaeological samples use powder XRD of mechanically-sampled portions or of ground whole otoliths to verify they are mineralogically unaltered (Patterson, 1998;Vanhove et al., 2011). For such applications, detection of minor or trace calcite or other mineral phases in the presence of the majority aragonite is a useful capability, particularly when coupled to the ability to locate those phases and estimate their potential impact.

In this work, several non-aragonitic reflections were detected that did not contribute significantly to the mean diffractogram. On QCD, two pixels exhibit reflections characteristic of calcite CaCO 3 (Fig. 71a and b) which occur inside the sample. There is a similar particle on the interior of section 424 exhibiting weak calcite reflections. On those internal pixels with a calcite signal, the aragonite signal remains stronger and of a similar magnitude to pixels with only aragonite signals. This therefore suggests single pixels of the section with surface contamination by calcite during preparation/handling, and not intra-otolith conversion of aragonite to calcite. Some trace phases on both samples did not present sufficient reflections for a positive identification (eg. Fig. 71c andd) but these were only detected at the edge of the otolith. The location and limited number of these pixels suggest that they are likely local contamination of the outer edge of the otolith from storage, sample handling or preparation, or in the case of the archaeological section from the natural environment.

Crystalline orientation

In this work, the ultrastructure measured was averaged within the information volume of the X-ray beam. The beam size results in a probing of a volume that includes many of these microcrystals. As a result of the volume examined, the measurement is not truly for one microcrystal, but for the average orientation of the volume.

Total diffracted intensity

In interpreting the variations in diffraction intensity, it is assumed that any two pixels have a similar number of nanogranules, and possibly crystalline units. It is likely that nanogranules are always approximately the same size, so the first assumption likely holds. On the contrary, the number of crystalline units in a given volume likely varies along the growth axis -closer to the otolith core where daily increments are wider and more easily distinguished, crystalline units are likely to be smaller. Outside of the first few hundred micrometres, the size of crystalline units should not be as significant a factor since the growth rate decreases and crystal size increases. The rate of orientation of the nanogranules and crystalline units -the fraction of domains aligned to within a given variance -may vary as a result of the presence of organic matter, post-mortem alteration, or local chemistry. A variance in the rate of orientation would thus alter the total observed diffraction intensity.

It is also the case that some reflections would have been missed, as the entire reciprocal space was not explored. However, the relatively homogeneous appearance in most of the mean maps (Figs. 68 and 69, bottom row) suggests a mostly equal and sufficient rate of coverage across the whole sample, and it may therefore be possible under favourable circumstances (e.g. high symmetry, well-known system) to perform a complete reconstruction from a single image.

Preferential orientation

The maps binned by ψ (Figs. 68 and69) provide a view on the coverage of reciprocal space and which reflections are stronger overall. The maximum value achieved ("Max. Value" row) highlights the presence or lack of a given reflection overall across the section, while the mean over all ψ ("Mean" row) indicates regions with relatively stronger or weaker reflections.

Changes in the reflection intensity from a given area with varying ψ is an indicator of a macroscopic crystal texture. Variations in the rate of orientation around ψ (Fig. 68) may be related to deviation from an ideal powder-like collection of nanogranules in the beam information volume. Rather than appearing as smooth variations with ψ and 2θ, there is significant "spottiness" in the maps. If the nanogranules and crystalline units were oriented along the growth axis with smooth variation between any two parts of the otolith, it would be expected that the map would reflect this with a smooth transition from strong reflection to extinction in the ψ bins. The heterogeneous spottiness indicates that some measured volumes have fewer nanogranules that have the same ψ 0 , i.e. there is some level of misorientation.

Estimation of crystallite size

Using the Scherrer equation (Eqn. 10), an upper bound can in principle be placed on the size of crystallites 1 composing a polycrystalline material [START_REF] Jenkins | Introduction to X-ray Powder Diffractometry[END_REF].

τ = Kλ β cosθ ; β = β Exp -β Ins ( 10
)
The mean crystallite dimension (τ) depends linearly on the shape factor 2 (K) and the X-ray wavelength (λ), and inversely on the peak width (β) and the cosine of the Bragg angle (θ). The peak width is the experimental Full Width at Half Maximum (β Exp ) with instrumental broadening (β Ins ) subtracted. In this experiment the instrumental broadening is conservatively estimated at 0.1°.

On average, we estimate the maximum size of the nanogranules in otoliths to be below 200 nm based on the FWHM on 2θ and the range of intensity variation in the Debye rings. However, the experiment was not optimised for resolution in 2θ, nor was the instrumental broadening in this configuration quantified. That the estimate of <200 nm is of a similar order and coherent with the previouslyobserved 50 nm granules is, however, encouraging.

1 The microscopic crystals which compose a polycrystalline material. 2 S The spotty appearance (Fig. 72) reveals that at different points, although the major orientation remains the same, the fraction of nanogranules/crystalline units exhibiting this orientation is not constant.

Heterogeneity of otolith crystal orientations

Feasibility of mapping of twinning rate of the aragonite

A method to determine twinning rate along the c axis in aragonite samples has been previously proposed [START_REF] Suzuki | Quantitative xrd analysis of {110} twin density in biotic aragonites[END_REF] Overall, the observed twinning rate in this work appear greater than those discussed by [START_REF] Suzuki | Quantitative xrd analysis of {110} twin density in biotic aragonites[END_REF]. In their work, twinning rates in otoliths were measured to be ca. 2.5 % or less. In the present work, samples were analysed as largely intact section, while those Twinning rate varies both along the growth axis and within isochronous increments. A lower twinning rate near the nucleus is counter to what may be expected, as this region exhibits faster growth and more narrow growth increments, which would be expected to imply more frequent twinning.

of the previous study were ground in before analysis. While sample preparation may have played a role in apparent twinning density, it may be that both measurements are accurate and that the otoliths of the present work exhibit a higher twinning rate than those examined previously as a result of species-related characteristics -the otoliths are from fish in different families -or ontogenic vital effects. The points were selected in a continuous line across 1.1 mm of the map (arrows in Fig. 74). The series of points show a smooth progression in ψ 0 in Fig. 74, except for the leftmost two pixels (X=7.3 and 7.4 mm, both of which deviate abruptly. The smooth progression of ψ 0 is evident in the above curves, including a shoulder near ψ=70°in the X-7.4 mm curve. However, the fitting procedure detected the spike near ψ=-35°, causing a sudden shift in measured orientation. This illustrates one of the challenges in automatic treatment of large bodies of data and the need for care in interpretation.

Toward full texture measurement

To more precisely observe the orientation of the crystalline units in each volume the 002, 020, and 200 planes were examined. These planes give direct access to the unit vectors of the lattice. At each pixel, the intensity curve along ψ was fitted with a Gaussian for each reflection (002, 020, and 200). The orientation was plotted using sticks (Fig. 74) whose direction indicates the projection of the unit vector on the sample plane with vertical being ψ=0°. The orientation of the sticks for all three reflections (200, 020, and 002) reveals the projection of the mean domain orientation. There are some pixels whose orientation is not coherent with their neighbours, for example to the right of the X=7.3 mm position in the 002 map (Fig. 74). Examining the curves in ψ, the source of the "misorientation" is clear: a narrow peak at ψ=-30°which was fitted, rather than the tail visible at high ψ. While the spike does not resemble the smoother features present in the other curves, it indicates a collection of tightly-oriented fibres or crystalline units. The origin of such a formation is not apparent and has not been previously reported to our knowledge. Given the abrupt change in position of ψ 0 , it may be associated with a change in ultrastructure -potentially on or just after a growth increment.

As it is often noted that the aragonite fibres in otoliths are twinned across growth marks, a high lateral resolution texture analysis has now been demonstrated to be capable of distinguishing this change.

The ability to examine aragonite fibres at the crystal lattice level may yield valuable insight to the biomineralisation process, as well as providing insight into local abnormal behaviour that may be a new source of information in biomineralogical and archaeological studies.

intermediate conclusion

With materials engineered at ever-smaller scales and growing interest in the ultrastructure in natural samples, the ability to measure crystal orientation or texture is of increasing interest. The ability to measure "micro-bulk" information volumes of a sample provides an additional tool which complements current methods, and the measurement of centimetric areas with micrometric lateral resolution in hours or less is a valuable capability. In this work, the rapid measurement of orientation was demonstrated using an XPAD pixel area detector, which provides a view over a wide range in both 2θ and ψ (ca. 50°and 100°, respectively). Use of this method to examine sections of fish otoliths highlights their highly-oriented nature and provides the first full-section crystallographic maps. Neither the modern nor the archaeological otolith examined showed evidence of altered mineral phase. Both showed limited levels of post-mortem contamination, and can be considered reliable environmental proxies.

The otoliths exhibit the expected high level of crystal orientation. The archaeological otolith QCD exhibits a discontinuity in the orientation, which appears to be a result of growth. Twinning in the two otolith sections was estimated using the procedure of Suzuki et al. Investigations of the elemental distributions have been carried out, providing detection of both endogenous and exogenous elements with a lateral resolution on the order of single crystalline units -a few micrometres. The incorporation of strontium into otolith aragonite was investigated at the microscale, as well as its stability over archaeological time. The mineral phase and crystal texture were investigated using a mapping approach to provide the first fullsection views. concentration range of strontium Some isochronous increments of some otolith sections exhibit concentrations of strontium beyond those commonly found in the literature, although the majority of detected concentrations fall within the typicallydescribed ranges. A series of increments near the outer edge of the section of QCD (Micropogonias sp.) reaches 51 µmol • g -1 , at the edge of commonly-observed ranges (Tab. 3;Campana, 1999;Albuquerque et al., 2010;Lin et al., 2015). The G. peruvianus specimens fall within the typical Sr concentration range for the majority of the maps, but all exhibit localised increments with concentrations often significantly higher. The modern section (GP-292) reaches 55 µmol • g -1 while the ancient specimens HP-11 and HP-12 reach 180 and 101 µmol • g -1 , respectively. The localised concentration maxima in both ancient specimens reach a range in which the solid solution of strontium in aragonite may be metastable (over 58 µmol • g -1 , Plummer and Busenberg, 1987). These high concentrations may result from different effects: exposure to exceptionally high Sr concentrations in the water, an effect of species physiology on the incorporation of Sr at a particular stage of life, or diagenetic alteration leading to concentration of the Sr content. Such high Sr concentrations are described in otoliths, albeit rarely (Munro et al., 2005;Lin et al., 2015). Coupled to the homogeneity of the isochronous increments, that the concentrations observed in the present work are likely physiological, not diagenetic. There do not appear to have been any previous elemental composition studies for G. peruvianus otoliths.

These findings suggest a need for further examination of otolith elemental composition across additional individuals and species from northern Peru.

distribution of other endogenous elements Other elements, including Na, K, and Cl, are typically expected in easilydetectable concentrations in otoliths, although they are mostly lighter, with Z<20. As such, their characteristic photons experience a high attenuation in air and are difficult to detect under the experimental conditions of the present work. In the two archaeological samples where K was detected, its concentration was homogeneous across the examined zone, whether near the edge or the centre. As K is regulated by the individual's physiology, its concentration is expected to be relatively constant. K has been shown to be one of the more labile elements through experiments immersing otoliths in water (Gauldie et al., 1998). The pristine concentration in K is a positive indicator for the samples where it was detected, as it still retains the expected behaviour and its concentration would be expected to be one if the elements most easily depleted during alteration. In experiments performed under primary vacuum using 2-7 keV X-rays on LUCIA beamline (not shown in the present work), lighter elements including K, Na, Mg, and P were detectable. Further experiments carried out under vacuum may permit the use of more labile light elements to detect potential depletion of endogenous elements.

exogenous elements

Appreciable concentrations of elements, particularly transition metals, were observed on the outer surfaces of the otoliths examined. These outer surface and near-edge concentrations are likely to result from exposure to soil during deposition for ancient samples. There may also be a contribution from handling before and/or during preparation, as similar concentrations of Fe, Cu, and Zn were observed on the outer surface of modern specimens. In the event that surface contamination penetrated the otolith (which Gauldie and Coote (1997) randomly substituted for Ca 2+ in the aragonite lattice, independent of location on the otolith, Sr concentration, or archaeological age of the otolith. This mode of substitution in ancient specimens shows no change compared to that reported in both modern counterparts studied here and the mode identified by a previous macro-scale analysis of modern otoliths from teleost fishes (Doubleday et al., 2014).

In addition, it is identical to most other bioaragonitic systems (e.g.

deep sea corals). This work demonstrates the stability of Sr in otoliths and reinforces the validity of Sr concentration in fish otoliths as an invaluable biological and palaeoenvironmental archive.

mineral phase

The fast XRD mapping approach demonstrated in the present work has permitted full-section analyses of one modern and one archaeological sample. Previously, controls for changes in the mineral phase of ancient otoliths examined whole otoliths as bulk samples, in small subsamples, or at selected points with low lateral resolution. This method permits an examination for alteration with the ability to localise any changes on a micrometric scale. In both samples examined, isolated points of calcite were identified, along with some unidentifiable phases. On the otolith surface, these points were concluded to be not alteration, but contamination from the post-mortem environment. Calcite-containing pixels were also present within the otoliths, but the calcite signature was very weak, with an aragonite signature on par with "clean" areas, so these points were concluded to be contamination by dust on the surface of the section.

observations on otolith biomineralisation

This work presented the first laterally-resolved examination of otolith crystal orientations over a large area using a direct probe showing nanogranules in the 50-100 nm range presented by other authors (Söllner et al., 2003;[START_REF] Gauldie | Atomic force microscopy of the morphology of the matrix and mineral components of the otolith of hyperoglyphe antarctica[END_REF]Gauldie, 1999;Dauphin and Dufour, 2008). The limit is, however, not homogeneous throughout a section, and further examination, potentially with improved resolution in 2θ, may yield new understanding of otolith structure, from nanometre-scale ultrastructure to centimetre-scale macrostructure.

methodological contributions

The optimisation of synchrotron-based methodology to maximise the quality of data collected on otoliths and improve its interpretation was central to the objectives of this work. Consideration was given to the order of analyses to minimise impact of earlier analyses on those which were performed later, as well as to apply and refine nondestructive techniques. In the present work, imaging and mapping were central aspects. In order to manage the large amounts of data resulting from these approaches, existing programs and packages were extended (Appendix E) and substantial new routines were written in R (Appendix F) to treat and interpret the data.

on-sample coordinate marking

In order to correlate results from optical microscopy, µXRF, µXAS, and other possible analyses, performed of various apparatus with different conditions at different times, a coordinate marking technique was elaborated. Tungsten marks deposited with a Focused Ion Beam (FIB) provide fixed points of reference on a micrometric scale, detectable under all techniques employed in the present work (optical and electron microscopy, elemental analyses), are a fast and accurate way to temporally correlate datasets. The FIB marks allow repositioning of datasets within a few minutes each, with an uncertainty of one pixel (for pixels in the micrometre range), which greatly facilitates intercomparisons.

gains using a mapping approach for elemental analysis Mapping approaches have been previously applied to otoliths, but development has been relatively limited. Mapping provides a substantially greater body of data than a centre to edge transect analysis. Elemental maps of otoliths have previously been used to calculate mean concentrations in particular regions (Limburg et al., 2010a) or to extract a profile of the points on a given trajectory (Limburg et al., 2015). In the present work, a novel approach integrating a group of 11 or more pixels within each isochronous increment at many positions along the growth axis was developed to produce classic centre to edge elemental profiles. The isochronous increments in elemental maps can be integrated in this way to produce a profile with increased reliability of the data deriving from a certainty that any point contamination can be excluded and that the integrated value is an averaged measurement along a growth increment, reducing the potential impact of "hotspots" while maintaining high temporal resolution along the growth axis. With This methodology provides laterally-resolved analysis of crystal phases and orientations, and is expected to be a valuable tool.

The possibility of such approaches becoming routine now exists, providing a complement to techniques like EBSD to enable not only the analysis of centimetre-scale areas with micrometric resolution, but also the analysis of the bulk of a sample rather than a small depth at the surface, which reduces the dependency on surface characteristics and may help avoid effects from handling and preparation. Figure 76: First attempts to compare [Sr] with δ 18 O and δ 13 C profiles in otolith HP-11. Transmission light microscopy image (a) of the transverse section of otolith HP-11 used to examine growth marks to temporally align the elemental and stable isotopic profiles. Aligned ontogenic profiles of [Sr], δ 18 O, and δ 13 C (b) show correlated fluctuations. The image above the plot is identical to that of (a), scaled to match the plot and with enhanced contrast to better reveal growth marks. Alignment was made along the yellow dotted line, which is in the same position in both images. This may yield a better understanding and clearer interpretation of life histories, although the effort needed for precise alignment is time consuming. For example, an interesting correlation between the high-Sr increment position and the δ 18 O profile is indicated by a red arrow.

perspectives comparison of elemental and stable isotopic compositions

The elemental composition of otoliths is impacted by the individual's environment, as is the stable isotopic composition in O and C (δ 18 O and δ 13 C). The mechanism influencing each compositional parameter and its relation to the environment can be different and complementary, so the comparison of elemental and stable isotope data may help to improve the interpretation of an individual's life history and provide a complementary record of past environment.

For an accurate comparison, the different datasets (maps or profiles) must be precisely aligned, which can be laborious. In the example of HP-11 shown (Fig. 76) the isotopic data was collected first, the section re-polished, and the elemental mapping performed. Re-polishing re-

sults in what can be effectively considered a different (albeit slightly) section and therefore a different examination of growth marks to form the ontogenic history. Alignment of the data therefore requires precise examination of the sclerochronology (Fig. 76a) to produce an accurate alignment of the data. Integration of isotopic data to a unified analytical methodology (Fig. 77) would suggest nondestructive elemental analysis first, in order to measure the isotopic composition on the same sample volumes. The tungsten marks proposed should not affect the isotopic measurement as its atomic mass is far above that of the ions measured in classic isotopic ratio mass spectrometry for δ 18 O and δ 13 C analysis.

improved lateral and volumetric resolution An even finer lateral resolution for elemental analysis may provide a higher temporal resolution for analysis of ontogenesis and life history by providing access to ever-smaller growth increments, approaching daily growth (Fig. 79). Compared to medium-energy X-rays, the use of tender X-rays as available on the LUCIA beamline improves the volumetric resolution and reduces the integrated information volume (Fig. 79). A reduced integration depth would also reduces the effect observed at the sample edge resulting from different information volumes for different elements. 4 µm. With a highly restricted information volume, analysis of just one or a few aragonite fibres is possible (Fig. 78). Moving to techniques such as Hard X-ray Photoelectron Spectroscopy or Scanning Transmission X-ray Microscopy with a consequent decrease in information volume, provided they can attain the necessary nanometric lateral resolution. Although such measurements could be highly sensitive to preparation, they may be able to analyse individual nanogranules with a higher attainable temporal resolution for life history reconstructions, provide further understanding of biomineralisation processes, and yield insight into diagenesis on a submicrometre scale.

impact of on-the-fly measurement techniques

With the introduction of rapid on-the-fly measurement approaches such as flyscan, new types of experiments become viable. Fly-scan is a novel approach to measure samples while moving instead of stopping at each point currently under develoment at Synchrotron SOLEIL (Medjoubi et al., 2013). The implementation of such fast XRD and XRF mapping in the present work mapped an entire otolith section (ca. 120 µm 2 ) with a 150 × 150 µm 2 resolution in under an hour, where a step-by-step approach would take 4-5 h for the same analysis.

Refinements to the recording system have further reduced this measurement time to less than 5 min for a comparable map. With such a fast acquisition, recording approximately 20 angular positions of a sample to acquire a compete pole figure map is realistic in under an hour. With such high throughput, measurements can be performed with a lateral resolution over previously unreasonable areas. Elemental transects by EPMA or LA-ICP-MS often require one to two hours. Although restrictions may apply with respect to signalto-noise ratio for some elements on short measurement times, in the same few hours a full section could be mapped with µXRF, at a higher lateral resolution.

continuing investigation of otoliths and diagenesis

While a good lateral resolution has been obtained in the present work, the overall spatial resolution is less ideal. Using mediumenergy X-rays produces an integration volume ca. 37 000 µm 3 due to penetration into the sample. This volume averages many individual aragonitic fibres and, depending on internal geometry, may intersect multiple isochronous increments.

This work is one of very few examining the potential post-mortem alteration of ancient otoliths. While some efforts have been made, further investigations would support the development of otolith geochemistry as a palaeoenvironmental proxy. The understanding obtained from examinations of otoliths has proven valuable, but with state-of-the-art techniques an even more detailed understanding may be possible. Andrus and Crowe (2002) examined the impact of cooking, and identified alterations in both elemental an isotopic composition. In their study, the otoliths were examined as whole, ground samples, which cannot answer critical questions: is the alteration limited to the outer increments? How homogeneous is the alteration within isochronous increments? Is alteration concentrated in particular regions, such as organic-rich or organic-poor zones of growth marks? µXRF elemental mapping is well-suited to answer these questions, and would begin to place limits on the extent of modification likely in ancient otoliths. Similar works examining the effects of storage and handling (e.g. Milton and Chenery, 1998;Proctor and Thresher, 1998;Gauldie et al., 1998) could be similarly extended. The work of Gauldie et al. (1998) in particular could be augmented by tender X-ray µXRF, allowing a high lateral resolution map of the intrusion of a tracer element which may help to elucidate the existence and nature of the pore network suggested by Gauldie and Coote (1997). In the study of otolith alteration, the impact of the pore network is assumed to be significant, and a better understanding could improve the reliability of ancient samples, as well as potentially reveal information about the biomineralisation process itself. Phasecontrast microtomography may be useful to directly image internal structure to reveal internal pores [START_REF] Deyhle | Comparison of propagation-based phasecontrast tomography approaches for the evaluation of dentin microstructure[END_REF]. The pore network may be associated with the organic fraction, which could be examined directly by elemental mapping techniques (e.g. Dauphin et al., 2003), or techniques for organics analysis such as ultravioletvisible luminescence [START_REF] Jamme | Synchrotron uv fluorescence microscopy uncovers new probes in cells and tissues[END_REF] or nano-FTIR [START_REF] Huth | Nano-FTIR absorption spectroscopy of molecular fingerprints at 20 nm spatial resolution[END_REF].

Archaeological otoliths have been and will doubtless continue to be used as life history archives. Continued study to better understand their post-mortem alteration in various environments over longer geological time scales will extend their known reliability and support further research employing ancient specimens.

liths with micro-X-ray fluorescence and absorption spectroscopies"

•

Introduction

Otoliths (oto, "ear" + lithos, "stone") are accretionary structures that form a part of the auditory, orientation, and motion sensing system of teleost fish (Nolf, 1985;Panfili et al., 2002). The term 'otolith' can refer to any one of three pairs of accretions, occurring in otosacs filled with otolymph on the left and right sides of the individual (Pan- * Corresponding authors Email address: loic.bertrand@synchrotron-soleil.fr (Loïc Bertrand) URL: http://ipanema.cnrs.fr (Phil K. Cook) 1 Current address: Centre de recherche sur la conservation (CRC, USR 3224), Sorbonne Universités, Muséum national d'Histoire naturelle, Ministère de la Culture et de la Communication, CNRS; CP21, 36 rue Geoffroy-Saint-Hilaire, 75005 Paris, France fili et al., 2002). The largest otoliths usually have a speciesspecific shape which is used for taxonomic determination. Growth occurs from before hatching until death via the deposition of mineral calcium carbonate (CaCO 3 ) (Campana and Neilson, 1985), templated by an organic mixture of proteins, glycosaminoglycans and lipids (Degens et al., 1969;Dauphin and Dufour, 2003) from ions and organic compounds dissolved in the endolymph [START_REF] Borelli | Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima)[END_REF][START_REF] Jolivet | Understanding otolith biomineralization processes: New insights into microscale spatial distribution of organic and mineral fractions from Raman microspectrometry[END_REF]. In most teleost species, CaCO 3 is in the aragonite polymorph (Carlström, 1963;[START_REF] Jolivet | Understanding otolith biomineralization processes: New insights into microscale spatial distribution of organic and mineral fractions from Raman microspectrometry[END_REF]. The accretional growth of otoliths results in alternating translucent and opaque increments. Pairs of opaque and translucent increments form annual growth marks which can be counted to estimate a fish's individual age (Pannella, 1971;Panfili et al., 2002). Unlike bone, otoliths are metabolically inert and are not remod-eled or reabsorbed as a source of calcium (Campana and Neilson, 1985;Panfili et al., 2002). They therefore constitute permanent elementally-zoned archives that have been widely used to reconstruct fish ages and life traits (Campana and Neilson, 1985;Elsdon and Gillanders, 2003).

Elemental analysis of fish otoliths is a well-established and expanding field of fish and fisheries research (Campana and Thorrold, 2001). Elemental fingerprinting, often coupled to sclerochronological analysis, has been used for questions of natal origin and population movement determination, stock identification, migration history tracking of diadromous species, or pollution monitoring (Elsdon et al., 2008;Limburg et al., 2010a;[START_REF] Morales-Nin | Seasonal and ontogenic migrations of meagre (Argyrosomus regius) determined by otolith geochemical signatures[END_REF][START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF]Kerr and Campana, 2014). At least 32 elements have been observed in otoliths, mostly in trace quantities, with others expected in yet-undetected concentrations (Campana, 1999). Some elements are naturally present at significant levels in ambient water (Kalish, 1989). Content in others is due to, or largely overprinted by, anthropogenic pollution [START_REF] Limburg | Selenium detected in fish otoliths: A novel tracer for a polluted lake?[END_REF]. In terms of absolute and relative concentrations, the incorporation of elements other than calcium, carbon and oxygen into the otolith, while potentially approximated by purely physical conditions of deposition from the endolymph solution (Campana, 1999), is in reality influenced by a combination of temperature, total salinity, and ion concentrations of the water inhabited by the individual, and the individual's physiology (Elsdon and Gillanders, 2003). Multiple physiological filters between the ambient water and the otolith produce a strict biological regulation of some ions while others remain weakly or unregulated (Campana, 1999). To be used as an environmental proxy, an element must be incorporated into the otolith in a predictable manner reflecting the physical and chemical composition of the ambient water, meaning it must fall into the weaklyregulated group. The alkaline earths strontium and barium fall into this group and are therefore the most studied environmentally-related elements. The variations in concentration of these elements have been demonstrated to be efficient environmental proxies, both in field studies (Elsdon and Gillanders, 2005) and laboratory experiments (Elsdon and Gillanders, 2004), even though the incorporation is more physiologically regulated than in simpler organisms such as corals (Shen et al., 1996). Conversely, in mollusk shells, strong physiological regulation has been shown to produce a departure from thermodynamic equilibrium in the incorporation of trace elements

The following abbreviations are used in the manuscript: EDX: Energy Dispersive X-ray spectroscopy; EPMA: Electron Probe Micro-Analysis; FIB: Focused Ion Beam; FT: Fourier Transform; FWHM: Full-Width at Half Maximum; LA-ICP-MS: Laser Ablation Inductively Coupled Plasma Mass Spectrometry; LM: Light Microscopy; MAD: Median Absolute Deviation MCA: Multi-Channel Analyser; PIXE: Proton Induced X-ray Emission; RISE: Radiation-Induced Side Effects; ROI: Region of Interest; SEM: Scanning Electron Microscopy; SNR: Signal-to-Noise Ratio; SR: Synchrotron Radiation; XRF: X-ray Fluorescence; µXRF: Micro-X-ray Fluorescence.

during biomineralisation (Schöne et al., 2010). Random substitution of Sr for Ca within the aragonite matrix has been proposed in modern otoliths (Doubleday et al., 2014), supporting the reliability of Sr analyses. There is however some discussion about the formation of SrCO 3 domains in other calcium carbonate palaeoproxies (Greegor et al., 1997;Finch et al., 2003).

Otoliths can be found in abundance in some geological and archaeological contexts (Nolf, 1985). Taxonomic identification of fish assemblages from otoliths whose morphology is preserved over geologic time scales has been applied to biostratigraphy, palaeoecology and palaeoenvironment reconstructions (Nolf, 1985(Nolf, , 2013)). The potential of ancient otoliths as archives for past biology and environments has been extended by studying their composition. Stable isotopic analyses have been used for instance for the study of fish migration or metabolism (Carpenter et al., 2003;Wurster and Patterson, 2003), of palaeoenvironmental and palaeoclimatic conditions from the Jurassic to the Holocene (Patterson, 1998;Aubert et al., 2012;Vanhove et al., 2012) or fishing practices of past human populations (Hufthammer et al., 2010;Carré and Dufour, 2012). A limited number of works have recently applied elemental analyses in the same way for fish palaeoecology (Limburg et al., 2011), palaeoenvironment (Disspain et al., 2011), and past human subsistence strategies (Disspain et al., 2012). Diagenesis might hamper such reconstructions from ancient samples, and the level of induced alteration in the otoliths has to be estimated (Dufour et al., 2000;Disspain et al., 2011). When alteration occurs, the extent and nature of alteration is not necessarily homogeneous across the specimen and indeed can be very limited in area. The outer increments are expected to be more vulnerable to alteration during burial (Cochran et al., 2010).

Local alterations are sometimes difficult to detect. They could significantly impact elemental concentrations measured through point or transect geochemical analysis, and thereby bias palaeoenvironmental reconstructions.

Current common techniques for elemental analyses of otoliths include Laser Ablation Inductively-Coupled Plasma Mass Spectrometry (LA-ICP-MS), Electron Probe Microanalysis (EPMA), and Proton-Induced X-ray Emission (PIXE). LA-ICP-MS and EPMA are both destructive and/or damaging to the sample, the former as a fact of its operation, and the latter as a result of the operating conditions to measure concentrations of minor elements [START_REF] Gunn | Electronprobe microanalysis of fish otoliths -Evaluation of techniques for studying age and stock discrimination[END_REF]. PIXE has also been observed to produce discolouration (Huszank et al., 2009). These techniques are typically employed with a resolution in the plane of the sample surface ("lateral resolution") on the order of 10 µm. Although some works demonstrate the potential for better lateral resolution, they are not in common use (Woodhead et al., 2005;Kertesz et al., 2005;[START_REF] Kovács | Installation and performance of the budapest-hamburg proton microprobe[END_REF]. Furthermore, the measurement time for a typical 2 mm core to edge transect is typically one hour or more (Campana et al., 1997; see also Supplementary Information SI.1). While LA-ICP-MS, EPMA, and PIXE can be used for mapping, there are relatively few examples of this approach on otoliths, likely related to longer acquisition times [START_REF] Durholtz | Nuclear microprobe mapping of statoliths of chokka squid loligo vulgaris reynaudii d'orbigny[END_REF]Limburg et al., 2003;Kertesz et al., 2005;Morales-Nin et al., 2012a). The implementation of elemental mapping by SR-µXRF in the present work opens the possibility of observing unexpected behaviours in elemental distribution (Limburg et al., 2010a) as well as providing a larger, more reliable dataset than transect measurements. SR-µXRF is a technique undergoing active development, with new approaches appearing which promise an order of magnitude faster recording while retaining a high sensitivity analysis (Medjoubi et al., 2013;Barberie et al., 2014).

The utility of otoliths as geochemical environmental information archives is well-known in modern specimens. Extension to archaeological samples requires the ability to decouple the biogenic signal from post-mortem and post depositional alterations. In the present work, archaeological otoliths from the Pacific coast of South America are compared to modern specimens. Quantitative synchrotron µXRF maps up to 3.2 × 1.0 mm 2 showing the concentration of Sr and other elements are presented herein at a lateral resolution as high as 4 µm. In preparation for SR-µXRF mapping, a coordinate marking strategy has been elaborated that allows rapid and precise alignments of large high-definition maps. Two-dimensional mapping provides a significant increase in the quality of data available. Mapping approaches reveal trends invisible to transect approaches and permit an easy identification of heterogeneities that might occur in biocarbonates. The multielemental technique improves analysis by revealing unexpected elements, typically absent from living tissue, and expected elements outside of their physiological concentration ranges. Synchrotron µXRF was implemented in the present work with a discussion of its potential and present limitations in order to further validate its use in this field and facilitate further study. The methodology and data treatment are discussed in detail to provide a base for future work and move toward a wider adoption of µXRF for analyses of otoliths and other geochemical palaeoenvironmental proxies. An analysis of the overall dataset is carried out to examine the distribution of Sr concentration values and evaluate potential post-mortem or post-depositional alteration.

Materials and methods

Archaeological and modern fish otoliths

Four archaeological otoliths and two modern counterparts from the tropical Pacific coast of South America (details and sample references in Tab. 1) were selected for their archaeological interest. The studied set of otoliths is composed of three sagittae from Micropogonias sp. (Sciaenidae) and three lapilli from Cathorops sp. and Galeichthys peruvianus (Ariidae). All species examined are coastal marine species. G. peruvianus only inhabits marine environments, while Micropogonias sp. can inhabit bays, lagoons, and estuaries, and Cathorops sp. is primarily marine but can frequent coastal rivers. The archaeological otoliths come from three sites in northern Peru. This region, although one of the most arid in the world, has been inhabited for over 13 000 years (Sandweiss and Richardson III, 2008). The oldest site and the most recent site (Pampa de los fósiles and Bayovar-1, respectively) were specialised nonpermanent fishing settlements (Béarez et al., 2011;Goepfert et al., 2014), while the Huaca Prieta is a monumental mound resulting from thousands of years of occupation (Dillehay et al., 2012). The modern otoliths were selected as species and environmental counterparts to the archaeological specimens. A Micropogonias manni was collected in Chile and a Galeichthys peruvianus northern Peru as species and environmental counterparts to the archaeological samples. Huaca Prieta otoliths were provided by T. Dillehay (Vanderbilt University, Nashville, USA), all others by P. Béarez (UMR 7209,Paris,France).

Sections were prepared from otoliths embedded in resin to provide support and prevent breakage during handling and preparation. Polyester resin (Buehler) was used for HP-12, 292, and 424;epoxy resin (ref. K2020, Brot Technologies) was used for QCD, Bayovar-1C, and BH17. Embedded samples were then cut into frontal (sagittae) or transverse (lapilli) sections using a diamond disk or wire saw. Sections were then mounted to microscope slides using Crystalbond 509 (Structure Probe Inc., Paris, France) for QCD, Bayovar 1-C, and BH17, or Brot D200 glue (Brot Technologies) for HP-12, GP-292, and 424. QCD was later removed from the slide. Sections were carefully polished using progressively finer grit, first by hand using 13 µm then 3 µm silicon carbide suspensions (Brot Technologies), finishing by polishing machine with a 0.1 µm alumina suspension (Brot Technologies) on a velvet disk (Escil, Chassieu, France). Gentle polishing did not prevent detachment of some crystals at the edge of G. peruvianus otoliths. Internal stresses during the preparation process can also induce cracks in some samples. Micropogonias sp., G. peruvianus, and Cathorops sp. otoliths are typically large (several millimetres) and exhibit growth increments on the order of up to a few hundred micrometres (Béarez et al., 2005(Béarez et al., , 2011)). Otolith preparation resulted in thin sections revealing increments along the growth axis from the core to the edge, as seen under transmitted light in Fig. 4a for archaeological sagitta QCD. These sections are 130-710 µm thick (Tab. 1) and relatively large, with core to edge distances ca. 3 mm. The sample set in this work has undergone previous analyses from which some surface effects remain visible. EPMA analyses were performed using a CAMECA SX-50 system (CAMPARIS platform, Université Pierre et Marie Curie, Paris, France) on QCD, HP-12, 424, and GP-292, producing some surface ablation on these samples. Craters from LA-ICP-MS using a NewWave 213 nm laser ablation system remain on QCD.

3 (Chauchat and Pélegrin, 1994) 260 µm

2.2. Marking of sample using focused ion beam tungsten deposition The Zeiss 1540XB CrossBeam with an OrsayPhysics Cobra Ga + Focused Ion Beam (FIB) and gas injection system at the Centre de Sciences Nucléaires et de Sciences de la Matière (Orsay, France) was used in deposition mode to place tungsten marks on samples QCD and Bayovar-1C. Deposition of the marks was complicated by the insulating character of the sample; this was partially compensated by placing strips of either carbon mounting tape or copper tape on the sample near the deposition zones, connected to the sample holder, to aid in the evacuation of built-up charges. Tungsten was selected as an element not present in otoliths (Campana, 1999) with emission lines non-interfering with the key spectral lines of interest in XRF spectra. Being a metal, it was expected to display a high contrast when observed using the SEM or LM. Sample exposure to the Ga + beam was kept to a minimum to avoid potential RISE, notably ablation of the sample which could alter the surface geometry. The marks were laid out in a pattern of 9 lines of 20 × 0.14 µm 2 , separated by 2.8 µm to form a rectangle. Deposition was performed for 60 s/line with a current of 50 pA. A rectangular motif of parallel lines constitute a compromise between a practical deposition time and a sufficiently visible mark; producing a solid rectangle on the order of 560 µm 2 would take several hours, where the motif selected, totalling approximately 25 µm 2 required only 10 min. Optical microscopy was performed after deposition to confirm the presence of the marks and identify their precise positions on the otolith section.

Synchrotron micro-X-ray fluorescence measurement (SR-µXRF)

Multielemental SR-µXRF mapping was performed on all 6 samples using the DiffAbs beamline (SOLEIL Synchrotron, Gif-sur-Yvette, France). The beamline's X-ray source is a bending magnet. The beam was focussed to a The incoming monochromatic X-ray beam (intensity I 0 ) was focused onto the sample surface. The sample was mounted on a goniometer in air at an angle φ=45°to the incident beam, producing an effective spot size of 6.0 × 16 µm 2 (y ×x) projected on the sample surface. Samples were mounted with the growth axis parallel to the y-axis to maximise the lateral resolution across isochronous increments. A Vortex SII monoelement silicon drift fluorescence detector (Hitachi) was placed in the same y plane at 90°to the incident beam to minimise X-ray scattering contribution. When using the linearly horizontal plane polarized X-ray beam, Compton scattering is eliminated, in theory, at this angular position. The sample-detector distance was optimised for each sample to maximise the detected signal but remain within the detector's range of linear response.

spot size of 6.0 × 11 µm 2 FWHM (V×H) with on-sample flux of ca. 5 × 10 9 photons/s. The experiment setup is described in Fig. 1. The maps collected at 17 200 eV excite K-lines for elements with Z ≤ 39 and L-lines for Z ≤ 89. For all SR-µXRF maps, count time was 1 s/pixel. A 25 µm filter of 99.999 % pure Al (Lot 322521, Goodfellow, Huntingdon, United Kingdom) was placed over the detector window on indicated samples, as suggested in (Limburg et al., 2007). Mapping was performed for sample sections measuring up to 3.2 × 1.0 mm 2 (V×H) including both the core and the edge of the otolith (scan steps: 10 × 15 µm 2 (V×H), count time: 1 s/pixel for good signalto-noise ratio (SNR) and fast acquisition). Areas of interest up to 420 × 400 µm 2 , identified by a change in the Sr fluorescence signal were then examined with oversampled step sizes of 4 × 10 µm 2 to look for variations in the 4 elemental signals on a smaller spatial scale. Additional single-point XRF spectra were collected on the QCD archaeological sample for 10 s/pixel on locations of interest at an improved SNR. Pellets from reference materials were analysed. 10 × 10 pixel SR-µXRF maps were collected near the centre of each reference sample, on a 1 × 1 mm 2 zone (1 s/pixel).

Identification of the emission lines in the SR-µXRF spectra was carried out from the map sum spectra using in-house algorithms developed on 'R' and the SpectralImage package (S. Cohen, IPANEMA). Elemental quantification was performed by deconvolution of the XRF spectra using the PyMca software (Solé et al., 2007). Further details on the fitting process and final result are detailed in Supplementary Information Section SI.2. The fundamental parameters were experimentally determined from processing the mean spectrum collected as a 1 × 1 mm 2 grid of 10 × 10 points on a pellet of a fish otolith Certified Reference Material no. 22 (CRM22, National Institute for Environmental Studies, Tokyo, Japan) reference pellet (Yoshinaga et al., 1999(Yoshinaga et al., , 2000)).

Elemental quantification was calibrated on certified Sr and Ca contents of CRM22. After determining the basic fitting parameters, the photon flux was estimated manually beginning with an approximation of 1×10 9 ph/s, then adjusted manually using the reference's certified Sr mass fraction of 0.236% (±5%) (Yoshinaga et al., 1999(Yoshinaga et al., , 2000) ) to obtain the correct value of the total photon flux. The matrix was set appropriately for aragonite, with sample thicknesses as shown in Tab. 1. The elements to fit were set individually for each sample based on the peak identification carried out from the average spectrum. When a batch fitting was completed, the χ 2 map, representing the quadratic sum fitting error at each pixel was observed for any trends or notable deviations, potentially indicating an area of poor fit that may result from the presence of an unexpected element. An increased error in the content estimation of elements with low characteristic photon energies such as Ca appeared in the fitting process. A correction factor was applied to the elements whose signals occur below 9650 eV (K, Ca, Mn, Fe, Cu, Zn, Ba), assuming that the median Ca concentration in the samples is equivalent to that of the CRM22 reference.

The results for each fit were imported using further custom scripts in 'R' for detailed processing including the obtention of profiles and mean measurements for zones of interest as well as correction for variations in excitation beam intensity (within ±1%). To plot the maps, the Cacontaining (thus in-otolith) pixels were isolated, and the values of the 0.5 th percentile and the 99.5 th percentiles of the intensity distribution of the isolated pixels were used as endpoints for a color scale. The colour scale provides a wide range of contrast and ensures that local "hot spots" will not inappropriately bias the image. Concentration distributions were visualised using kernel density plots, in which a distribution function is calculated by integrating over all pixels the convolution of a Gaussian curve with the value at each pixel.

Results

Micrometric fluctuation in elemental composition revealed by SR-µXRF mapping

Figures 2 and 3 present the quantified SR-µXRF maps, recorded with incident energy of 16 200-17 200 eV, count time 1 s/pixel, and effective on-sample FWHM spot size of 6.0 × 16 µm 2 (V×H) for the 6 analysed otoliths. In the maps, the otolith core is always oriented to the top with the outer edge and embedding resin visible at the bottom. In the Cathorops sp. and some G. peruvianus samples, resin can be seen at the top of the map as well. The size of the maps varied with the otolith, from (3000-4250)×(400-1000) µm 2 (V×H), with total acquisition time varying consequently. The largest map required 8 h.

Over all samples, 13 intrinsic elements were detected. Ca, Fe, Zn, Rb, and Sr were detected in all 6 samples, either in the otolith or in the surrounding resin (Supplementary Information Figs. S1-S6). Cu and Pb were detected in 5 samples. Ba was detected in four samples, Br in three, and K and Mn in two. Hg was detected in one sample. Y was detected in all three samples analysed using appropriate incident beam energy to excite it. An additional two elements, Ga and W were detected on the 2 otoliths which had been marked by FIB, exclusively on the marks themselves (see Section 3.2). Ca and Sr consistently presented levels sufficient for reliable curve fitting. When weak spectral lines are superimposed with strong ones, accurate curve fitting can be more difficult. This is particularly the case for barium and rubidium. The Ba Lα 1 peak (4466 eV) is 454 eV from the very intense Ca Kβ peak (4013 eV) and is not resolvable on a per-pixel basis. The Rb Kα 1 peak (13 395 eV) is overlapped by the Sr Kα 1 (14 165 eV) and quantification has likely been impacted by peak overlaps: detected quantities of Rb are significantly higher than reported in literature (0.1 × 10 -3 -2 × 10 -3 µmol • g -1 (Geffen et al., 2013;Hicks et al., 2010;Curtis et al., 2014)). Relative quantification within a map of the other elements, in particular Sr, is expected to be reliable and absolute quantification of Sr accurate.

A combination of periodic and unique features can be seen in all elemental maps, which will be exemplified for the Micropogonias sp. sample QCD (Fig. 2a). In QCD, Ca, K, and Sr, in order of decreasing concentration, are present over 10 µmol • g -1 . K, Ca, Rb, Sr, and Ba are present only in the otolith. Only Sr exhibits a strong regular fluctuation, resembling parallel bands with values between 28 and 49 µmol • g -1 . Sr reaches its minimum concentration near the midpoint, at 1.5 mm (S1), and its maximum (S2) just before the outer edge. Other elements (K, Ca, Br, Rb) exhibit weak or very weak fluctuations. The Ca content is nearly homogeneous, exhibiting an overall variation of ca. 1% of its mean outside of artefacts from previous characterisation and preparation (see below). The otolith edge as seen in LM is coincident with the transition from high to no Ca (E). The transition from nearly homogeneous Ca content within the bulk to zero Ca in the resin is welldefined, occurring over just 1-2 pixels, or 10-20 µm. This edge position is identical in all low-Z elements up to transition metals. Examining the transition from significant to no Sr, the edge is no longer in the same position, but ca. 200 µm farther from the core (E). This second, lower edge is shared by Rb. The highest concentrations of Mn, Fe, Zn, and Cu can be found between the apparent edges in the Ca and Sr maps.

Surface relief on the sample produces effects that are visible in the elemental maps. A crack in the sample appears as a narrow thread, depleted in Ca, K, and to a lesser extent Sr can be seen on the left side of the map (C), in the same position as increased Zn. LA-ICP-MS craters produce a series of round depletions in Ca and Sr (L), with connected roughly semi-elliptical enhancements to the right. To the right of these, EPMA ablation results in a wide band of minor Ca depletion (A).

In and Figs. S2 and S3), 7 and 8 elements have been detected, respectively. K, Mn, Br, Ba, and Hg were undetectable in both samples, while Pb was detected only in 424. Like QCD, Ca, Sr, Ba, and Pb are only present within the otolith, while Fe, Zn, Cu, and Y are associated with the edge or resin. Contrary to QCD, low levels of Rb appear present in the resin as well as in the otolith. Both otoliths exhibit strong, regular fluctuations in Sr concentration. Rb exhibits very weak fluctuations. In Bayovar-1C, the Sr concentration fluctuations appear divided into two regimes: one near the core, in the range of 15-30 µmol • g -1 , and one toward the edge at 20-42 µmol • g -1 . [Sr] fluctuations in 424 are within a more restricted range of 14-22 µmol • g -1 . In both samples, there is no overall difference in the edge position between Ca and Sr, but 424 exhibits an indentation in the Ca map (E) which is smaller in the Sr map. An increased concentration of Ca is visible at the edge of both samples. A crack is visible near the core of Bayovar-1C (C2).

In the archaeological sample HP-12 (Fig. 3a and Fig. S4), taken as an example of G. peruvianus samples, 10 elements were detected. Ca, K, and Sr in order of decreasing concentration, were detected over 10 µmol • g -1 . K, Ca, Br, Rb, Sr, and Ba are detected within the otolith only. Like the Micropogonias sp. specimens, Sr exhibits strong, wellresolved fluctuations. Rb and Ca also show significant but more poorly-resolved fluctuations, while Br shows a very weak fluctuation. Sr fluctuations show a general trend of decreasing concentration range and increasing frequency from the core to the edge. The maximum Sr concentration, attained midway through the sample at 1.65 mm (S3), is 101 µmol • g -1 . This is the same position as the Rb maxi-mum and the Ca minimum. The minimum Sr concentration of 30 µmol • g -1 is attained at the outer edge. Mn, Fe, and Zn are significantly present in the resin, with low levels of Mn and Zn also in the otolith. The edge (E) is very uneven in the Ca and K maps, resulting from crystals removed during preparation. Ablation from EPMA (A) traces a vertical line across the sample which is depleted in Ca and Rb, and enhanced in K. Cracks (C3) are visible near the core in the K, Ca, and Sr maps.

A total of 9 elements were detected in GP-292 (Fig. 3b and Fig. S5). Like HP-12, Ca, Sr, and Ba were restricted to the otolith. Sr shows strong fluctuations in both G. peruvianus otoliths ranging from 20-55 µmol • g -1 . Rb shows relatively significant fluctuations, while Ca is largely homogeneous within the otolith. GP-292 exhibits a narrow Sr concentration spike (S4) similar to that in HP-12. Loss of crystals at the edge (E) is much more significant in these samples than HP-12, and shows variations in Ca and Sr concentrations at the edge and some variation between the apparent edge in the Ca and Sr maps. Fe, Zn, Mn, and Pb are primarily found in the resin with low levels present in the otolith, or near-zero in the case of Fe. The elements in the resin also show low levels within the otolith near the edges. A slight depletion of Ca near the core of GP-292 is present.

BH17 (Fig. 3c and Fig. S6), an archaeological Cathorops sp. specimen, exhibits 9 elements detected. Sr concentrations are within the ranges previously observed (25-57 µmol • g -1 ). Compared to other lapilli, the borders remain intact and the outer edges (E) are smooth. There is no difference in apparent edge observed between low and high Z elements. Higher concentrations of Fe and Zn can be seen near top and bottom borders, respectively. The resin contains detectable levels of Fe, Cu, Zn, and Y.

Unified coordinate marking

A series of tungsten marks were deposited on the samples using a FIB in selected locations near the intended analysis areas (Fig. 4a). Under LM, the deposits are highly reflective (Fig. 4b), particularly with grazing incident light, and opaque under transmitted light. An elemental and physical contrast is visible under FIB (Fig. 4c) or SEM imaging. The W deposits were easily detected by SR-µXRF (Fig. 4d) even though only approximately 90 pg/pixel were deposited; W Lα fluorescence yield is 0.255 and it is not naturally present in otoliths, producing a high SNR and a highly contrasted image. The motif, with features smaller than 50% of the spot size, provides a homogeneous appearance under XRF with micrometric lateral resolution. Integrating the W Lα peak over its FWHM at the points marked in Fig. 4d shows an average 435 counts/pixel within the mark compared to a background of 10 counts/pixel outside of the mark (Fig. 4e). The W marks, presenting both a chemical and physical change in the sample surface, are detectable by multiple probes. They were used to quickly calculate the relative position of any point on the otolith and later align SR-µXRF results with microstructure. In addition, they were valuable landmarks for positioning and alignment during the SR-µXRF experiment itself.

Discussion

Medium-energy synchrotron µXRF mapping

This work has yielded quantitative multielemental maps of archaeological otoliths with a good lateral resolution Table 2: Attenuation length (1/e) of characteristic lines of selected elements in pure aragonite (CaCO 3 , ρ=2.93 g • cm -3 ) and polyester ((C 10 H 8 O 4 )n, ρ=0.85 g • cm -3 ) at 45°incidence. Calculated using the CXRO X-ray Interaction with Matter database (Henke et al., 1993).

Element

Energy Attenuation lengths (µm) that reveals the biogenic signal along the growth axis with an ability to identify possible diagenetic alterations. It highlights the potential and limits of synchrotron µXRF mapping in relation to the specificity of the examination of otolith thin sections.

4.1.1. Information volume of medium-energy SR-µXRF in otolith thin section analysis As otoliths grow incrementally from the core, they form a three dimensional structure which can be compared to a "stack of dinner plates". Taking a thin section of an otolith, these plates are oriented approximately orthogonally to the surface. In the present work, a higher lateral resolution was in the vertical axis (On-sample spot FWHM 6.0 × 16 µm 2 V×H). As for any material studied by XRF, the precise lateral resolution attainable in this experiment is not a simple function of spot size, but is also dependent on the sample matrix (Janssens et al., 2000;Kanngießer et al., 2005). The effect on attainable lateral resolution can be largely decoupled on the horizontal and vertical axes. In the present work, the growth increments visible on the thin sections were oriented horizontally (corresponding to the X axis in Fig. 5) to maximise lateral resolution along the growth axis and definition between sequential growth increments.

(A) In the horizontal, a 16 200 eV incident beam at 45°to the sample surface has an attenuation length of 237 µm in pure aragonite. The incoming beam can thus be assumed to excite a volume of sample to this length. This is appreciably more than the attenuation length of the fluoresced Ca or Sr Kα rays (see Tab. 2), constraining the information volume to the escape length of sig-nal. Corresponding simulated XRF information volumes are shown in Fig. 5 for both Sr and Ca. The information volume is elongated and asymmetrical, with a slight majority occurring on the side toward the detector. This results from a slight difference in attenuation between the side of the volume closer to the detector compared to the opposite side. For Sr, 50% of the signal comes from the first 161 µm of sample depth, producing an averaged value across this distance. For Ca, 50% of the signal results from the topmost 30 µm. With our 45°incidence, the information volume is approximately as wide as it is deep, thus producing a mixed signal of this entire volume. Definition of a generic information depth is not possible -the information volume depends upon the nature of the sample, and where one defines the cutoff. It can be seen in Fig. 5 that the volume containing 50% of the information is significantly smaller than that containing 75%. The lateral resolution is dependent on the total information volume. In effect, the best attainable lateral resolution for Ca is smaller than that for Sr because the signal is recovered from a much more limited depth. The relative homogeneity of otoliths should produce a largely constant information volume across the sample. After the difference in information volumes was observed, the authors judged it inaccurate to present [Sr]:[Ca] ratios, and present absolute concentrations instead.

(B) In the vertical, the sample plane is positioned perpendicular to the incident beam, so there is no additional impact on lateral resolution. While this orientation relative to the X axis can be controlled, the internal slant of the increments (corresponding to the XZ plane) cannot be easily evaluated and could produce temporal averaging by measuring not only the visible surface, but also the subsequent growth underneath. A sharp peak in Sr content, for example, could be detected in several pixels at varying depth, increasing its apparent width.

To ensure the majority of the data in a single measurement originates from one isochronous volume, the angle to the XZ plane must be within a certain margin. For example, in sample QCD (Fig. 4) under light microscopy some subannual growth increments are homogeneous at lengths of approximately 40 µm. With an incident beam FWHM of 6.0 µm in the vertical axis, penetrating 161 µm, the angle between the increment and the "ideal" XZ plane must be less than 12°for the information volume. To perform two consecutive measurements within this increment, the angle must be less than 10°. Because the analysed thin sections have been taken including the core of the otolith, the slant of the growth increments relative to the surface should be minimised and this condition should be met in increments outside of the core. At or close to the core, the volume of aragonite representing one isochronous increment decreases and the curvature of each increment increases. It is possible to obtain a non-averaged measurement only if the information volume can fit within this increment. From the previous calculations for Sr, assuming a spherical core, this would not be possible for a radius (X×Y). The shaded plane represents the sample surface, the purple line representing the incident beam penetrates up to its 1/e attenuation length, and a point detector is situated 30 mm away, perpendicular to the incident beam, in the direction indicated by the teal line. For each X-ray emission line, the three shapes (blue, orange, and green) represent the 25 %, 50 %, and 75 % XRF information volumes.

at least 161 µm around the core, and most likely more depending on experimental geometry and thickness of these initial growth marks.

At the otolith edge, the information depth also produces effects. In some samples the apparent edge position depends on the element being examined. In QCD (Fig. 2a), the apparent edge is farther from the core in the Sr map than the Ca map; in Bayovar-1C (Fig. 2b), there is an indentation at the edge in the Ca map which is shallower in the Sr map. As previously noted, the information depth of the analysis can vary significantly between elements such as Ca or Sr. With a higher energy, the Sr Kα signal can escape from deeper in the sample than the Ca Kα (see Tab. 2), leading to such edge displacements where a subsurface structure exists within the resin. The Ca signal is reabsorbed by the intervening material, while the Sr signal is capable of escaping and evidencing this subsurface structure. In other cases, such as HP-12, there is an inversion of this geometry, where the otolith curves inward. It is visible on the surface by LM and in the Ca map, but not in the Sr map, because the smaller Ca information volume is filled with the otolith, but the larger Sr information volume is only partially occupied. The discon-tinuity in matrix between the otolith and the surrounding resin also has an influence on the chemical signal. More characteristic photons passing through the resin can reach the detector than the aragonite bulk (Tab. 2). Where the edge between otolith and resin is nearly perpendicular to the sample surface, the density change between aragonite and resin can produce reduced signal attenuation, as seen in Fig. 2b. The core and the edge of the otolith are areas of particular interest for environmental or palaeoenvironmental applications, including determination of natal origin or location of capture. These areas might be particularly affected by of a decreased temporal resolution or decreased analytical precision. For example, the first annual growth mark in otoliths of Micropogonias furnieri occurs ca. 900-1000 µm from the nucleus [START_REF] Albuquerque | Estuarine dependency in a marine fish evaluated with otolith chemistry[END_REF]. Using the µXRF technique described in the present work, the first 2-3 months of life may be poorlyresolved, but the remainder of the first year is measurable; in this species, migration occurs at 1-3 years of age, so determination of natal origin would be possible. The question of information depth and its interaction with internal morphology presents a point of reflection for all analytical techniques applied to otoliths. An appreciable information volume is not unique to SR-µXRF and can be observed in other techniques as well. Ablation is commonly observed in both LA-ICP-MS (Jones and Chen, 2003) and EPMA. The total integration volume in LA-ICP-MS has been shown to significantly impact the measured concentrations in a life history scan (Hoover and Jones, 2013). In addition, this ablation makes these techniques ultimately destructive, whereas SR-µXRF exhibits very limited RISE. Good alignment along the horizontal axis of the majority of the growth increments was achieved for all samples except Bayovar-1C and GP-292 (Figs. 2b and 3b). An equally sensitive approach permitting analysis of a more restricted sample volume, such as reducing the incident X-ray energy, or using PIXE may be of value.

Lateral resolution and data alignment in sclerochronological archives

The most common techniques applied to the elemental analysis of otoliths are LA-ICP-MS, EPMA and to a lesser extent PIXE. While all three techniques can be used for mapping (Hinz and Kohn, 2010;Huszank et al., 2009;Shirai et al., 2014), on otoliths they are generally used for transect analyses. Maps of otoliths from wild specimens by PIXE (Huszank et al., 2009;Limburg et al., 2003) and Srtagged specimens raised in tanks by SEM-EDX (Morales-Nin et al., 2012a) have been previously shown. SR-µXRF scanning on a macro scale is a recent and actively developing technique in the study of both modern and ancient materials (Bertrand et al., 2012;Lintern et al., 2009;Anné et al., 2014). Application of milli-to microbeam synchrotron XRF mapping to otoliths is in its early stages, with some environmental and ecological studies on modern samples (Nakai et al., 1999;Limburg et al., 2007;Lochet et al., 2010;Limburg et al., 2010a,b;Nakazawa et al., 2012;Limburg et al., 2015), and one on modern and archaeological otoliths using increased Mn content as an indicator of hypoxia (Limburg et al., 2011). The high incident flux and sensitive detectors found in state-of-the-art SR-µXRF setups permit collection of maps of whole otoliths or significant portions in reasonable time periods. In the present study, maps were collected with a step size as small as 4 × 10 µm 2 (V×H) across significant portions of otoliths to obtain a per-sample analysis time of a few hours, rather than possibly longer than a day (Limburg et al., 2007).

In sclerochronological archives such as otoliths and mollusk shells a high lateral resolution yields a high temporal resolution. Otolith analyses using the most common techniques (LA-ICP-MS, EPMA, and PIXE) are rarely carried out with a lateral resolution better than around 10 µm; higher lateral resolution is attainable, although sometimes requiring specially-designed apparatus (Shirai et al., 2014;Woodhead et al., 2005). Even if one measurement per season (warm/cold, dry/wet season) is sufficient to access the seasonality, more points permit better discrimination of the annual cycles and more accurate identification of the true extremes (Shirai et al., 2014). Maps of several years' growth of bivalve shells (ca. 100 µm per year) by EPMA have been previously presented, but only for a portion of a shell of the long living Arctica islandica, representing 15 years of the individual's life (Shirai et al., 2014). In our Micropogonias sp. otoliths annual growth increments are observed with widths of 70-140 µm. A 4 µm lateral resolution permits a monthly to biweekly period, while a lateral resolution of 10 µm typically permits a temporal resolution of 1-2 months (depending on growth rate that varies both ontogenically and seasonally). The resolution attained in our SR-µXRF analysis reveals subseasonal variations, observable, for example, in the high-frequency fluctuations in QCD just above the Sr minimum (Fig. 2a,S1) or in HP-12 between the core and the Sr maximum (Fig. 3a,S3).

Sample features, including impacts of previous experiments, aid in sample alignment. Precision of location by surface features, particularly in the Ca maps, can vary depending on the dimensions of the feature, with smooth features such as LA-ICP-MS craters presenting significantly less precision than steep ones such as cracks. Although rough sample positioning was readily possible on the synchrotron experiment both with and without an optical microscope, the pre-placed tungsten marks proved particularly helpful in precise positioning for specific zones, and indeed enabled alignment on precise areas of interest even in unfavourable visual observation conditions. We developed multi-technique on-sample coordinate marking in the present study, described for the first time on this type of samples. Placing marks a priori alleviates the difficulty of locating small analysis zones and further permits alignment of zones which exhibit limited landmark features. Due to the absence of any visible marks left by SR-µXRF characterisation, location of the analysed points a posteriori has previously been noted as more difficult than with techniques which leave a mark (Zumholz et al.,

2007

). The marks decrease the uncertainty in positioning to one pixel with minimal intervention on the sample surface. The marks were also efficient during later experiments using other techniques, both for repositioning and accurate intercomparison of maps. They enhance the ease and reliability of comparisons to sclerochronological data and to results obtained from other geochemical analyses.

Quantification process

Semi-quantitative XRF analysis relies upon accurate determination of the area of peaks at characteristic energies (Janssens et al., 2000). This work applied curve deconvolution using PyMca software to identify and quantify the constituent elements. However, the superposition of very strong peaks and weak peaks can produce difficulties, as seen for elements whose characteristic peaks fall close to the Ca K (Kα=3690 eV) or Sr K (Kα=14 165 eV) peaks. One particular example is that of Rb (Kα=13 395 eV), whose peak occurs relatively close to the Sr peak. In the Sr and Rb maps these elements appear to be correlated, which could suggest a tail effect from the large peak from orders of magnitude higher Sr concentrations. However, examining spectra both with and without excitation of Sr (Fig. 6) confirms the presence of an Rb signal. Furthermore, both the intense Sr Kα and weak Rb Kα peaks were successfully deconvoluted using PyMca (see Supplementary Information SI.4). Future work with an interest in Rb could benefit from a second analysis using an incident energy below the Sr K-edge (16 105 eV), eliminating the Sr Kα peak and any potential overlap.

Sample matrix effects are typically countered by calibration from known references of an identical or similar material to the sample, which was performed in this study using pellets of CRM22 certified reference material (Yoshinaga et al., 1999(Yoshinaga et al., , 2000) ) composed of ground otoliths. The standard and samples used in this work should be very similar and variations in matrix effects should therefore ganic) is shown on the inner labels. Molecular formula and sample density were varied in the PyMca fit setup to reflect changing matrix composition. The matrix characteristics of the organic fraction were approximated from otolin-1 (M=52 138 g • mol -1 , Murayama et al., 2002; ρ=1.43 g • cm -3 , Quillin and Matthews, 2000). Even a change from one extreme to the other in the 0-10 % range is insufficient to account for the Sr variation in the most homogeneous otolith examined here (Otolith 424, 14-22 µmol • g -1 i.e. a variation of 36 %).

be negligible. One potential variation that cannot be wellaccounted for is the organic content, the fraction of which could vary both between the reference and samples, as well as within each sample. To estimate the effect, data was extracted from a single pixel of the map of otolith QCD and fitted with varying matrix composition of 0-10 %wt. Over the full range of possible organic content, Sr concentration varies by less than 9 % and Ca by 5 % (Fig. 7). The detected variations in Sr are well beyond this range (minimum range in sample 424, 14-22 µmol • g -1 , variation of 36 %) so they cannot be artefacts resulting from fluctuating organic content. Our model assumes a homogeneous dispersion of organic and mineral fractions, which is known to be a simplification Dauphin and Dufour (2008). Variation in organic content with circadian rhythm has been long described in otoliths, although quantitative data remains sparse (Jolivet et al., 2013a). The true ultrastructure of otoliths includes nanometric organic envelopes and variation of organic/mineral ratios within growth increments, which are only beginning to be described and are very difficult to accurately model. Given the general absence of quantitative data on intra-otolith fluctuation of organic content, the further absence of specific knowledge of organic fraction for our samples, and the relatively minor impact of potential variations in organic content, we have elected to use a matrix pure aragonite.

Detected Sr content of all samples fell in the range of 14-101 µmol • g -1 . Quantification of other elements, however, is more problematic. The absolute Ca quantities measured in our otoliths were 6000-19 000 µmol • g -1 , while pure aragonite contains 9990 µmol • g -1 (40%wt).

We believe that the spectral resolution of the detector varying over the 10 000 eV range from Ca Kα to Sr Kα, and calibration on the single minor element (Sr) are responsible for this discrepancy. A linear correction factor was applied to the elements whose signals occur in the portion of the spectrum below 9650 eV (K, Ca, Mn, Fe, Cu, Zn, Ba), calculated to bring the median Ca concentration in line with that of the CRM22 reference (38.8%wt, or 9690 µmol • g -1 ). The absolute quantification of other elements may also be inaccurate as a result of poor SNR, due to their low concentrations and the short count time accepted as a compromise for faster mapping of Sr. Using longer count times and/or higher incident photon flux, quantitative maps of trace elements in otoliths by SR-µXRF, including Br, Ba, and Se have been reported in the literature (Limburg et al., 2007(Limburg et al., , 2010a,b;,b;Lochet et al., 2010;Limburg et al., 2011). SR-µXRF point analyses with 1000 s count times have successfully quantified Sr, Ba, and detected Y, Zr, and Nb in cuttlefish statoliths (Zumholz et al., 2007).

Insights into the reliability of the Sr signal

The set of otoliths in this work is part of a larger study of palaeoenvironmental and past human occupation conditions on the Pacific coast (Béarez et al., 2011;Dillehay et al., 2012;Aubert et al., 2012). The tropical Pacific Ocean is the center of action of the ENSO (El Niño Southern Oscillation), the strongest source of interannual climate variability on the planet (Webster and Palmer, 1997;Tudhope and Collins, 2003). Historical understanding of El Niño activity and marine conditions over the Holocene on the Peruvian coast in the past has often been limited by existing proxies (Tudhope and Collins, 2003).The development of new palaeoenvironmental proxies including otoliths offers new windows to the past, but the reliability of proxies is critical for accurate reconstructions. When performed, the identification of altered from unaltered specimens is sometimes limited to visual examination for morphological changes of the otoliths overall. Other criteria are occasionally used based on microstructural features: presence of daily and annual increments, crystal habit, and identification of CaCO 3 polymorphs (Dufour et al., 2000;Vanhove et al., 2011;Kern et al., 2012). Preservation of the microstructure and mineralogy is often taken to indicate the geochemistry therein is pristine, but absence of morphological and mineralogical change does not guarantee a pristine geochemical signal (Dufour et al., 2000). The development of new methodologies to better assess the fidelity of the geochemical signals recorded in palaeoproxies is therefore a meaningful step in their further exploitation.

Statistical distribution of Sr concentrations

Our SR-µXRF maps reveal for all specimens very regular major fluctuations with a thickness on the order of 100 µm, as expected for individuals several years of age (Kalish, 1991). More rapid fluctuations on the order of 1-2 pixels, or ca. 20 µm can be observed, for example shortly before the minimum in Sr content in QCD (S1, Fig. 2a) and near the outer edges of the G. peruvianus specimens Figs. 3a and 3b). Localised enrichment or depletion in Sr are observed along the growth axis in QCD, 424, HP-12, and GP-292. Inter-individual comparison of maps can be difficult whereas life history profiles provide convenient comparison between individuals and interpretation of life history chronology. Here, we will propose two approaches for treatment of the large data set which underline the complexity of the measured Sr signal, with the perspective of examining its reliability through time.

Mapping provides data beyond that required to produce an elemental profile. With a data set consisting of hundreds or thousands of points, statistical approaches become viable, for instance to examine the distribution of values. A kernel density plot is produced by placing a smooth distribution function (A Gaussian here) of a set width with its centre at each data point's value along the X-axis. The kernel density (Fig. 8) is the sum of all Gaussians over all data points. Compared to histograms, kernel density plots reduce the impact of bin width and eliminate the need to select an origin, both of which can alter the distribution's appearance (Silverman, 1998).

The kernel density plots reveal an additional dimen-sion beyond the total spread of the data, and the maps in Figs. 2 and3, by highlighting their distribution. HP-12 exhibits a wide distribution without an obvious primary mode, ranging from 21-101 µmol • g -1 , although 91% of values fall from 35-70 µmol • g -1 . The high Sr region (Fig. 8 inset) contains an excursion to 78 µmol • g -1 and a small population above 80 µmol • g -1 . This range of values exceeds typically-observed literature values for fishes from different origins (up to 48 µmol • g -1 , Campana, 1999;Nakai et al., 1999;[START_REF] Morales-Nin | Seasonal and ontogenic migrations of meagre (Argyrosomus regius) determined by otolith geochemical signatures[END_REF][START_REF] Albuquerque | Estuarine dependency in a marine fish evaluated with otolith chemistry[END_REF]Albuquerque et al., , 2010)), but similarly high concentrations have been reported (up to 137 µmol • g -1 , Munro et al., 2005;Lin et al., 2015). The distributions for the other two samples are narrower, extending over 14-22 µmol • g -1 in 424, and 27-51 µmol • g -1 in QCD. Sample 424 is bimodal, with a primary mode centred at ca. 16 µmol • g -1 and a secondary at ca. 20 µmol • g -1 . QCD exhibits a multimodal distribution, with a primary mode ca. 33 µmol • g -1 , and excursions to both higher (ca. 40 and 50 µmol • g -1 ) and lower (28 µmol • g -1 ) values. The two archaeological specimens present multiple modes of distribution, different from the modern one. Assuming these distributions reflect a biogenic signal, they can indicate that the fish lived in an environment with varying Sr levels, and/or in various environments which presented contrasts in Sr concentration.

Part of the distribution could also result from post-mortem alteration/contamination. Although the temporal (ontogenic) aspect of the data is lost, using kernel density plots, the distinction between different modes of life for different individuals could be clearly revealed and discussed. The Sr concentrations in archaeological Micropogonias sp. and Cathorops sp. otoliths measured in this study are mostly within, although some points are slightly above, commonly-observed literature biogenic values. In contrast, some of the Sr concentrations measured in HP-12 and GP-292 exceed the range observed in literature up to present, with the archaeological HP-12 exceeding in some portions the maximum concentration of its modern counterpart. The data analysis performed in this work (Figs. 8 and 9) supports that the obtained values are not analytical errors or the result of surface defects or contamination. The highest Sr concentrations measured in HP-12 and GP-292 exceed the 58 µmol • g -1 equilibrium solubility of Sr in bulk aragonite reported from crystallisation experiments (Plummer and Busenberg, 1987). Theoretical studies, however, indicate significantly higher values (up to 1100 µmol • g -1 ) (Ruiz-Hernandez et al., 2010), well above the highest Sr concentrations measured in this work. To the authors' knowledge no elemental data have been previously published for G. peruvianus. The Sr concentrations observed herein for a modern specimen suggest a need to expand the expected biogenic range of [Sr] values for otoliths of marine fish. Further measurements are necessary to more accurately determine an expected Sr concentration range for the species of the coast of northern Peru. Until such a corpus of data is available, it is difficult to interpret the highest Sr levels in HP-12 as biogenic or resulting from post-mortem alteration based on the concentration itself. The various excursions or modes in Sr concentration observed in the archaeological specimens of this study are within the defined biogenic range and are located at different positions along the growth axis within each otolith and not, as may be logically expected, only on the outer edge which is most susceptible to post-mortem alterations (Trueman, 2013). The enrichment is only truly at the edge in Bayovar-1C. In QCD the enrichment reaches its maximum some 100 µm from the actual edge and has a median absolute deviation (MAD) of 0.4 µmol • g -1 , which indicates high intra-band homogeneity. Neither of the archaeological lapilli (HP-12 and BH17) exhibit such near the edge, so this is not a constant among all the archaeological samples in this work, while the modern sagitta 424 and the modern lapillus GP-292 both exhibit similar increases in Sr concentration. Given these observations, the modes and excursions in Sr in the archaeological otoliths do not seem to evidence post-mortem alteration but might rather reflect variations in Sr content of habitats frequented by the fish over their lives. As such, the 28 µmol • g -1 mode measured in QCD (Figs. 8 and 9b) with an MAD of 0.2 µmol • g -1 may indicate an episode of life in freshwater as previously suggested by other proxies (Béarez et al., 2011;Aubert et al., 2012). In our work, we observe some samples which have a higher Sr concentration near the edge which decreases toward the centre, followed by fluctuations. The fluctuations are inconsistent with a typical diffusion profile, which would exhibit only a smooth transition from high to low concentration (or vice-versa). As such, we can conclude that even for those samples where post-mortem alteration cannot be excluded in the near-edge region, fluctuations beyond the first are most likely biogenic and the signal therein is intact. For example, although we are not able to define conclusively if the Sr concentration increase near the edge of QCD evidences post-mortem alteration, the lack of Sr enrichment beyond a few hundred micrometers deep seems to confirm the viability of the Sr signal as a palaeo-environmental indicator this and our other archaeological samples.

Integration to Sr life history profiles

Axial ('time') profiles of Sr concentration in archaeological otoliths HP-12 and QCD are depicted in Fig. 9. These curves were produced by integrating lines of 11 and 13 pixels (equivalent to 165 and 195 µm), respectively, perpendicular to the growth axis, as indicated by the saturated regions in the maps. The position of the integration bands (Fig. 9) were chosen to minimise the inclusion of obvious damage such as the EPMA transect (Fig. 3a, annotation A), while obtaining the full core to edge profile. The Sr concentration profile exhibits a close to constant MAD when normalised to the median concentration (median: 1.5%, Fig. 9). Localised areas of increased MAD can primarily be associated with physical features of the sample, such as the edge. The core represents a zone with a small volume, resulting in a decrease in temporal resolution and an increase in MAD. The low MAD confirms the apparent homogeneity of the band visible in the maps, which should correspond to isochrones, so heterogeneity within isochrones is minimal. The band of high- [Sr] values seen in the map of HP-12 (Fig. 9a) contains the small population of high values observed in the kernel density plot (Fig. 8) and appears as a spike reaching 100 µmol • g -1 along the growth axis in the integrated profile. The MAD increases only slightly, to 1.6 µmol • g -1 , at the maximum of the spike, compared to a mean of 1.2 µmol • g -1 , across the rest of the sample. This indicates that the integrated band is largely homogeneous and that the integration of neighbouring isochrones was negligible. The two-dimensional information collected from mapping indicates that the extreme values are not artefactual, nor are they related to a surface defect as revealed by LM and there were no surface defects included which were not visible a priori. In transect approaches, as widely used in geochemical studies of otoliths, outlier values could lead to erroneous datapoints. EPMA measurements of HP-12 indicate a maximum value of 60 µmol • g -1 at the same position on the profile. The discrepancy in absolute value between the two analytical methods may be explained by the sampling size. EPMA analysis was performed with a lateral resolution of approximately 50 µm. Taking a mean of 5 points around the highest value in the SR-µXRF profile yields a more consistent average value of 80 µmol • g -1 . The importance of sufficient lateral resolution to distinguish the true extremes has been previously demonstrated for the example of isotopic variations in mollusk shell by simulating a varying sampling width and comparing the extreme values identified to those of the simulated curve (Shirai et al., 2014). Using the pixels as a set of data permits examination of data reliability by revealing variations within isochrones, can reveal additional trends or extreme values, given sufficient lateral resolution, and therefore enhances confidence in measurements by providing multiple analogous data points.

Biogenic versus post-mortem signal

A full examination of fish life history and biological characteristics is beyond the scope of this article, but the capabilities of the technique used in this work, and the data treatments applied have implications for the examination of the quality of the otolith elemental signals. Different criteria have been used to assess the level of diagenetic alteration of ancient fish otoliths (Dufour et al., 2000;Disspain et al., 2011). The elemental signal itself can be considered a criterion to identify post-mortem alteration, assuming that elemental concentrations found in archaeological fish must be coherent with those found in modern specimens (Dufour et al., 2000;Disspain et al., 2012). In order to define a "fingerprint" of alteration, beyond Sr concentration, one can consider other elements which are either (a) not expected to be present in the otolith in significant quantities, or (b) regulated by the organism and expected to be present at certain levels.

Supporting evidence from other elements

Other than Sr, elements with Z > 20 are not typically present in otoliths at significant concentrations. Some heavier trace elements, which can be relatively common in the environment, can be present in otoliths (Campana, 1999). Y has been previously used as an indicator of diagenesis, as have rare earth elements (Brand and Veizer, 1980;Pucéat et al., 2004;[START_REF] Reynard | Trace elements and their isotopes in bones and teeth: Diet, environments, diagenesis, and dating of archaeological and paleontological samples. Palaeogeography[END_REF]; Fe, Cu, and Zn could be expected to serve similar roles as markers of the arrival of exogenous elements if the otolith examined exhibits relatively low mean levels coherent with those reported in literature. In this work, the concentrations of Fe, Cu, Zn, and Y were always higher in the resin than in the otolith within a given thin section, and decreased toward the inner otolith. Mn, Fe, Cu, and Zn were detected on the outer edges of archaeological samples QCD and Bayovar-1C, as well as modern sample 424 (Figs. 2a,2b,and 2c). Where present, these elements show a maximum at the otolith-resin interface with a generally smooth decrease to zero or near-zero over ca. 500 µm from the edge toward the core of the otolith. A similar presence on the edge of the modern otolith as the archaeological samples indicates that diagenetic alteration is not involved in the appearance of these elements at the interface between the samples and the resin. In three cases (QCD,, cracks in the samples also showed localised concentrations of at least one of Fe, Cu, and Zn. It therefore appears that Fe, Cu, Zn, and Y originate primarily from the resin, and either infiltrated during embedding, or were spread across the otolith surface during polishing, as described by Jolivet et al. (2013b) for otoliths of Merluccius merluccius. The resin used for embedding the otoliths was selected to have limited concentrations in trace and minor elements which may interfere with analysis. This was indeed the case for K, Ca, and Sr, but not for Fe, Cu, Zn, or Y, at least one of which was detected in the resin of every sample (Figs. 2 and 3). Resin contamination has been shown to impact analysis of the organic fraction of otoliths, and the potential impact of elemental impurities in the resin was noted (Jolivet et al., 2013b). Although contamination by the resin therefore made the use of Fe, Cu, Zn, or Y as indicators of diagenesis-related exogenous elements impossible in our samples, the contamination is generally limited to a few hundred micrometres. The sen-sitivity of SR-µXRF to the minute contaminations does bode well for detection of potential exogenous elements. As general advice, care must be taken in the selection of materials and preparation of thin sections to reduce alteration or contamination during preparation as much as possible for palaeoenvironmental proxies.

Na, S, Cl, and K are the only minor elements typically expected in otoliths at significant concentration other than Sr (Campana, 1999). They are, however, poorly accessible by in-air XRF because of the low energies of their characteristic fluorescence lines, and only K was detected in any samples in this work. Interestingly, where detected Figs. 2a and S4), we did not observe depletion in K at the sample periphery. It is generally expected that K is one of the more easily soluble elements in otoliths because it is presumed associated with the organic matrix rather than in the aragonite lattice (Gauldie et al., 1998;Proctor and Thresher, 1998). This is an indication of limited post-mortem alteration of these samples.

Conclusion

Synchrotron micro-X-ray fluorescence multi-elemental mapping at micrometric lateral resolution is a fast-developing technique. This work sought to assess its the potential and limits for application to archaeological otoliths and bring insight to examination of the elemental compositions to better distinguish biogenic signal from post-mortem and post-depositional sources of alteration. SR-µXRF using medium X-ray energies provided accurate maps of Sr concentration with semi-quantitative calibration. This mapping approach allows for the investigation of intra-and inter-incremental variations with a subseasonal temporal resolution, which was coupled to a coordinate marking strategy for increased precision in positioning. Analysis of zones, complementing that of transects, yields highly reliable data sets to which statistical approaches can be applied. The distribution of Sr concentrations facilitates intercomparison of palaeo-environmental conditions and modes of life. Including a representation of the data variability along an integrated profile facilitates detection and identification of potential post-mortem alteration and local artefacts, including from sample preparation and previous analyses. Limited contamination from sample preparation could be detected in some samples. No definitive evidence for diagenetic alteration was identified from the Sr signal or those of other elements. The random substitution of Sr for Ca in aragonite of otoliths was recently demonstrated in modern otoliths (Doubleday et al., 2014). The stability of this substitution over long time scales, if proven, would be another strong indicator of the preservation of the corresponding environmental signal. No alteration of the elemental signal was detected deeper than a few hundred micrometres into the otoliths, which supports their reliability for use in palaeo-environmental reconstructions. Sr concentrations detected in some otoliths extend the range of reported values from literature, indicating a need to expand the knowledge in Sr concentration range for distinct species and environments. In the future, performing X-ray absorption spectroscopy or X-ray diffraction on archaeological otoliths in tandem with SR-µXRF may reveal the mode of incorporation and local chemical environment of minor and trace constituents to better elucidate their endo-or exogenous origin. SR-µXRF presents some limitations in attainable lateral resolution as a result of the integrated information volume, comparable to that presented by LA-ICP-MS. Examining the Sr L fluorescence lines would significantly decrease the information volume and permit a further increase in lateral resolution, at the expense of a greater sensitivity to surface artefacts and heterogeneities. In this work a few core to edge maps were produced per day, which is a throughput comparable to common techniques such as LA-ICP-MS and EPMA. This rate can be increased by an order of magnitude or more using new high-throughput methods coupled to faster detection and recording while retaining a high sensitivity analysis (Medjoubi et al., 2013;Barberie et al., 2014). SR-µXRF mapping could therefore open new avenues to the nondestructive analysis of stratified biocarbonate palaeoproxies thanks to its high sensitivity, speed, and data reliability.

SI. Supplementary Information

SI.1. Techniques for elemental analysis of otoliths

There are multiple techniques available for the elemental analysis of otoliths. Among these, the most commonlyobserved are Laser Ablation Inductively-Coupled Plasma Mass Spectrometry (LA-ICP-MS), Electron Probe Microanalysis (EPMA), and Proton-Induced X-ray Emission (PIXE). Inter-laboratory and inter-technique comparisons have been undertaken to better understand the variability in results observed. These studies provide useful "baseline" values for comparing the speed and resolution of the synchrotronbased µXRF in the present work (Tab. S1). In general, these common techniques require at least an hour to perform a 2 mm long transect with step size equal to the spot size. Exceptions are noted for LA-1, LA-A, and LA-B, all of which have a large spot size (1-2 orders of magnitude more than the SR-µXRF in the present work) and thus requiring few points to complete the transect. The µXRF demonstrated in the present work is roughly an order of magnitude faster than other commonly-used approaches.

SI.2. Fitting of SR-µXRF spectra

The PyMca software fits an entire XRF spectrum using an algorithm which reduces the number of parameters needing optimisation. Parameters concerning the multichannel analyser (MCA) energy calibration, the detector response and the photon beam properties are fitted for the spectrum as a whole using a fundamental parameter approach, while the set of lines for each element are combined into a single equation using theoretical ratios and positions. This reductionist approach essentially requires fitting of only one parameter per element, alongside, in our case, 6 experimental parameters (Tab. S2).

The following parameters were used for curve deconvolution and quantification from fundamental parameters in PyMca. The five parameters corresponding to MCA energy calibration and detector response were fitted freely, first simultaneously for an initial estimate, then individually with the other 4 fixed for two cycles moving in order through the list to refine them, then a final cycle fitting all parameters simultaneously with a possible range of ±2%. Once determined from the reference, these parameters were maintained for all remaining fits.

Among the non-fitted parameters, the theoretical Fano factor for silicon (0.114 [START_REF] Fano | Ionization yield of radiations. II. The Fluctuations of the number of ions[END_REF]) was fixed, in the absence of an experimentally measured value. The experimental Fano factor is expected be close to its theoretical value. Pseudo-Voigt peak shapes were found to give a better fit than Hypermet peaks, so this function was selected. The background estimation and Savitzky-Golay smoothing were varied manually to obtain a reasonable continuum shape. The SNIP algorithm implemented in PyMca provides a satisfactory approximation with two orders of magnitude fewer iterations than the STRIP algorithm. The sample distance was set per sample, as the detector position was periodically adjusted between measurements; the distance was measured manually for each sample. The air thickness was set according to the sampledetector distance as the measurement was carried out in open air. A dead layer of 20 nm of silicon on the detector was accepted. The beryllium window thickness, detector thickness and active area were set to the detector specifications.

SI.3. SR-µXRF for all elements

In the main text (Section 3.1), a selected subset of elemental maps are presented. In the present section, all maps showing detectable levels of each element are shown. Annotations are described in the main text (Section 3.1), except for that of "FIB" on sample QCD (Fig. S1. The "FIB" annotation indicates four W marks that can be seen along the growth axis in the Zn map, resulting from a spectral overlap of W Lα and Zn Kα.

SI.4. Validation of Rb content and correlation with Sr

The Rb and Sr signals often appear to be correlated in the elemental maps (Figs. 2 and 3). The Rb Kα peak (Kα 1 =13 395 eV) is indeed close to the Sr Kα (Kα 1 =14 165 eV). With a sufficiently broad Sr peak, a contribution to the Rb peak may be expected. We conclude, however, that the Rb signals observed in the present work are not artefactual. Observing the spectra of several selected points on sample BH17 (Fig. S7) with varying Rb and Sr concentrations, 20

Introduction

Teleost otoliths ("ear stones") are biomineral accretions, three pairs of which are found in the inner ear of fish (Nolf, 1985). An organic template comprising 1-10 %wt of the otolith guides the deposition of the mineral phase (Degens et al., 1969). The mineral phase, comprising the remaining 90-99 %wt is composed of calcium carbonate (CaCO 3 ) (Degens et al., 1969) with some minor and trace elements. The CaCO 3 in otoliths is * Corresponding author Email address: loic.bertrand@synchrotron-soleil.fr (Loïc Bertrand) URL: http://ipanema.cnrs.fr (Loïc Bertrand) 1 Current address: Centre de recherche et conservation des collections, CNRS, Muséum national d'histoire naturelle, CP 21, 36 rue Geoffroy-Saint-Hilaire, F-75005 Paris, France.

The following abbreviations are used in the manuscript: EXAFS: Extended X-ray absorption fine structure; FIB: Focused ion beam; FT: Fourier transform; FWHM: Full-width at half maximum; IR: Infrared; LA-ICP-MS: Laser ablation inductively-coupled plasma mass spectrometry; LM: Light microscopy; MEE: Multi-electron excitation; SD: Standard deviation; SEM: Scanning electron microscopy; SNR: Signal-to-noise ratio; SR: Synchrotron radiation; SR-µXRF: Synchrotron micro-X-ray fluorescence.; XANES: X-ray absorption near edge structure; XAS: X-ray absorption spectroscopy; XRD: X-ray diffraction; XRF: X-ray fluorescence normally in the aragonite polymorph (Carlström, 1963), except for some species where one of the less-developed pairs can be calcite or vaterite (Gauldie, 1993) or in rare cases where pathology may lead to formation of calcite or vaterite (Béarez et al., 2005;Strong et al., 1986;Tomas and Geffen, 2003). Geochemical analyses -determination of elemental and isotopic compositions -of fish otoliths provide a wealth of information about the biology of fish and the environment frequented over the life of an individual. During accretional growth the otolith aragonite and the organic matter can incorporate essential and nonessential elements. Among the at least 27 minor and trace elements that have been reported in otoliths, Na, K, P, and Sr are typically found in the highest concentrations (Campana, 1999). Sr concentration profiles (normalised to Ca content) is the most widely used environmental tracer in fish and fisheries sciences.

In solutions, Sr incorporation in aragonite follows thermodynamic fractionation relations (Plummer and Busenberg, 1987). In fishes, incorporation of Sr into otoliths is dependent primarily on water chemistry, and on the ambient temperature (Elsdon andGillanders, 2004, 2005). Physiological effects and a possible impact of diet can be observed (Elsdon and Gillanders, 2003).

The elemental and isotopic signals from ancient otoliths are being applied more frequently for various types of studies (Campana, 1999(Campana, , 2005)): biological and environmental archives for the study of past fish migration or metabolism (Carpenter et al., 2003;Wurster and Patterson, 2003), palaeoenvironmental and palaeoclimatic conditions from the Jurassic to the Holocene (Aubert et al., 2012;Disspain et al., 2011;Long et al., 2014;Patterson, 1998;Vanhove et al., 2012), and fishing practices of past human populations (Carré and Dufour, 2012;Disspain et al., 2012;Hufthammer et al., 2010;Long et al., 2014). The species-specific shapes of otoliths and quality of morphology preservation over geologic timescales enable taxonomic identification of fish assemblages from archaeological excavations (Nolf, 2013). Identifying the species in such assemblages and comparing them to modern species with known behaviour and ecology enable biostratigraphic studies and reconstructions of palaeoecology and palaeoenvironment (Nolf, 1985(Nolf, , 2013)). Nevertheless, the reliability and permanence of the geochemical information encoded within ancient otoliths is critical to their use as information archives. Diagenesis might hamper such reconstructions from ancient samples, and the level of this alteration in the otoliths has to be estimated (Disspain et al., 2011;Dufour et al., 2000).

Examination of the integrity of the internal and external morphology and mineralogy of ancient otoliths is a prerequisite to use of their geochemical signals. As a standard practice, altered specimens are distinguished from unaltered ones by detection of microstructural alteration and/or change in the identity of calcium carbonate polymorph. An intact microstructure is defined by the presence of growth marks typical of otoliths (daily and yearly increments), long needle-like (prismatic) aragonite crystals, and the absence of recrystallised or neoformed minerals (Dufour et al., 2000;Hufthammer et al., 2010;Kern et al., 2012;Vanhove et al., 2011). Aragonite is metastable and can undergo a transformation to calcite over time (Plummer and Busenberg, 1987). Transformation to a crystal polymorph can be detected by X-ray diffraction (Béarez et al., 2005;Dufour et al., 2000;[START_REF] Geffen | Highlatitude climate variability and its effect on fisheries resources as revealed by fossil cod otoliths[END_REF]Patterson, 1998;Vanhove et al., 2011;Wurster and Patterson, 2001), vibrational spectroscopy (Kern et al., 2012), or cathodoluminescence (Béarez et al., 2005;[START_REF] Geffen | Highlatitude climate variability and its effect on fisheries resources as revealed by fossil cod otoliths[END_REF]Kern et al., 2012), as well as from the absence of apparent vein-like incursions in between the crystals at the millimetre and micrometre scales as viewed under light miscroscopy (LM) and scanning electron microscopy (SEM). The absence of calcite and vaterite is considered an indicator of geochemical preservation. The quality of preservation of the pristine geochemical signal measured in ancient otoliths can also be assessed through comparison to the otoliths of modern individuals of the same species or of a taxonomically close species which are expected to exhibit similar geochemical signatures (Dufour et al., 2000). Otoliths of taxa with different ecologies can be compared in order to reveal expected contrasts in different geochemical signatures (Vanhove et al., 2011).

However, these three parameters (mineralogy, microstructure, geochemistry) may not allow concluding on the low impact of diagenesis on the geochemical signal. For instance, diagenetic alteration may lead to an artifactual increase in ele-mental concentrations yet still within a realistic range for living fishes. Another example is the stability of elemental ratios in gastropod shells [START_REF] Wardlaw | Transformation of aragonite to calcite in a marine gasteropod[END_REF].

Central questions are whether: (a) samples with no evident alteration can be considered as intact at all length scales below a few micrometres, (b) atypical sample areas with significant elemental or isotopic excursions could show a specific behaviour, (c) outer surfaces of samples could be particularly affected. Working at the microscale is necessary as alteration could occur not only in the whole otolith, but also in localised zones (Disspain et al., 2011;Dufour et al., 2000). However, to our best knowledge, no previous work controls in detail for speciation or potential post-mortem microscale alteration of ancient fish otoliths. The outer zones of the otolith may be expected to be more susceptible to alteration as a result of their direct contact with the surrounding environment (Trueman, 2013), including fluids such as those from the decaying animal. Heterogeneous post-mortem alteration is more likely to occur on the outer surface in contact with the burial environment (Cochran et al., 2010), but is not restricted to this zone (Dufour et al., 2000).

Regarding Sr used as a palaeoenvironmental proxy, its local mode of incorporation and stability within the organic fraction of otoliths and/or in the aragonite biomineral are likely to influence crucially its long-term behaviour, and therefore its reliability as a palaeoenvironmental proxy. Elements bound within the aragonite lattice may be less susceptible to exchange than those adsorbed on the surface or incorporated in the organic material (Gauldie et al., 1998). Over the past years, synchrotronbased spectroscopy and imaging have appeared as particularly adapted to the examination of the Sr chemical environment in modern and ancient biominerals (Allison et al., 2001;Doubleday et al., 2014;Finch et al., 2003) Furthermore, development of microscale raster-scanning approaches with chemical contrast may allow monitoring of localisation of contamination by other elements. This work examines the chemical environment of strontium in the aragonite of ancient otoliths at the microscale as a possible approach to detect post-mortem alterations and determine the permanence of the geochemical signal. A microscale examination of the Sr chemical environment was performed on five archaeological and three modern otoliths from the Pacific coast of South America exhibiting a wide range of Sr concentrations from 7.1-94 µmol • g -1 . A synchrotron micro-beam permits analysis of a small selected sample volume. The lateral resolution afforded was used to select limited X-ray illumination areas with different Sr concentrations for analysis, such as the outer edge or a significant excursion. Millimetric area on an archaeological sample were examined using a fast speciation mapping approach. The Sr chemical environment detected was universally Sr in aragonite with no evidence for the presence of other polymorphs on the scale of these analyses. We discuss the palaeo-environmental and diagenetic implications of these results.

Materials and methods

Archaeological and modern fish otoliths

Archaeological otoliths from excavations at Pampa de los fósiles, Huaca Prieta, and Bayovar-1 (northern Peru), were chosen because of their potential for palaeoclimatic information (Béarez et al., 2011;Dillehay et al., 2012;Goepfert et al., 2014). They form part of a larger study of palaeoenvironmental conditions and past human occupation in a region that lacks palaeoclimatic proxies. Otolith specimens of five archaeological and three modern fishes from the tropical Pacific coast of South America have been examined (Tab. 1). The specimens all come from coastal marine species of Sciaenidae (Micropogonias sp., Micropogonias altipinnis, Micropogonias manni) and Ariidae (Galeichthys peruvianus). In these two families the most-developed otolith pair (of sagittae, lapilli, and asterisci) is different: Sciaenidae present more developed sagittae while Ariidae present more developed lapilli (Nolf, 2013).

Sections were prepared from otoliths embedded in resin. Polyester resin (Buehler) was used for HP-11, HP-12, GP-292, 111 and 424;epoxy resin (ref. K2020, Brot Technologies) was used for QCD, Bayovar-1C, and BH17. Embedded samples were then cut into frontal (sagittae) or transverse (lapilli) sections using a diamond disk or wire saw. Sections were then mounted to microscope slides using Crystalbond 509 (Structure Probe Inc., Paris, France) for QCD, Bayovar 1-C, and BH17, or Brot D200 glue (Brot Technologies) for HP-11, HP-12, GP-292, 111 and 424. The section of otolith QCD could be removed from the glass slide after polishing and analysed without other support, while it was not attempted for the other sections owing to their apparent fragility. Sections were carefully polished using progressively finer grit, first by hand using 13 µm then 3 µm silicon carbide suspensions (Brot Technologies), finishing by polishing machine with a 0.1 µm alumina suspension (Brot Technologies) on a velvet disk (Escil, Chassieu, France). Gentle polishing did not prevent detachment of some crystals at the edge of G. peruvianus otoliths. Internal stresses during the preparation process can also induce cracks in some samples. Micropogonias sp., G. peruvianus, and Cathorops sp. otoliths are typically large (several millimetres) and exhibit growth increments on the order of up to a few hundred micrometres (Béarez et al., 2005(Béarez et al., , 2011)).

After preparation, the otolith sections were examined for gross alteration using LM, SEM, Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD). FT-IR spectra were collected in reflection mode with a spot size of 350 µm using a Bruker Fourier Equinox 55 FT-IR spectrometer coupled to a Bruker A-590 microscope (PALEOPROXUS, IRD, Bondy). XRD data (not shown here) were collected from subsamples of otoliths with Cu Kα radiation by the Gandolfi method using a custom-built diffractometer where an Inel curved position-sensitive detector replaces one rotation axis (Laboratoire de Minéralogie, MNHN, Paris).

Reference materials

Three reference compounds were selected for comparison to assess the chemical environment of Sr in our samples: natural aragonite (orthorhombic CaCO 3 , Alfa Aesar, batch I15T027), natural calcite (rhombohedral CaCO 3 , Alfa Aesar, batch C08S004), and synthetic strontianite (orthorhombic SrCO 3 , 99.994% pure, Alfa Aesar, batch 23669). All references were prepared neat, except for strontianite, which was diluted with synthetic calcite (VWR, lot DBJ 150004) and with 6.8 %wt microcrystalline cellulose (Merck, batch K36832831 733) during grinding to 0.6 %wt SrCO 3 (equivalent to 0.4 %wt in Sr) in order to limit autoabsorption of fluoresced X-rays due to high Sr content in the pure material. Each material was prepared by grinding into a fine, homogeneous powder in a clean agate mortar and pestle for 5 minutes, then pressed into a pellet in a 13 mm die.

Synchrotron beamline configuration

Synchrotron

micro-X-ray fluorescence (SR-µXRF) and micro-X-ray absorption spectroscopy (µXAS) measurements were performed on all samples at the DiffAbs beamline (SOLEIL synchrotron, Gif-sur-Yvette, France, http://www.synchrotronsoleil.fr/Recherche/LignesLumiere/DIFFABS) using a microbeam configuration. A pair of bendable mirrors is used for primary vertical focusing and harmonic rejection together with a double-crystal monochromator for energy selection. Primary horizontal focussing is achieved using a bendable second crystal and a secondary one using an achromatic Kirkpatrick-Baez mirror setup (Kirkpatrick and Baez, 1948). The beam was focussed to a spot size of 6.0 × 11 µm 2 FWHM (V×H) with on-sample flux of ca. 3 × 10 9 photons/s. The sample was mounted in-air on a goniometer at 45°to both the incident beam and detector. This resulted in an effective spot size on the sample of 6.0 × 16.0 µm 2 . Samples were mounted with the growth axis parallel to the 6.0 µm axis of the beam to maximise the lateral resolution across isochronous increments. A Vortex SII monoelement silicon drift fluorescence detector (Hitachi) was placed in the same horizontal plane at 90°from the beam to minimise X-ray scattering contribution. When using the linearly horizontal plane polarized X-ray beam, Thomson scattering is eliminated, in theory, at this angular position. The sample-detector distance was optimised for each sample to maximise the detected signal but remain within the detector's range of linear response.

A focused beam of micrometric size is provided using achromatic Kirkpatrick-Baez mirrors. Measurements were performed using incident X-ray photons with energy of 15 900-17 000 eV. The on-sample full width at half maximum (FWHM) spot size is 6 × 16 µm 2 (vertical × horizontal) and the on-sample photon flux is ca. 3 × 10 9 photons/s. A wide-beam configuration (spot size: ca. 250 × 300 µm 2 FWHM, on-sample photon flux: ca. 5 × 10 12 photons/s) was used to record X-ray absorption near-edge spectroscopy (XANES) spectra of the references aragonite and strontianite, as well as of the QCD section.

Synchrotron micro-X-ray fluorescence (SR-µXRF)

SR-µXRF maps were collected to identify areas of interest for X-ray absorption measurements and estimate Sr concentrations and distribution. Maps were collected using an incident beam energy of 16 200 eV. Fluorescence from the sample in the 2000-16 000 eV range was detected using a Vortex SII silicon drift detector (Hitachi) with an active area of 50 mm 2 . The detector was mounted in the horizontal plane at a 90°angle to the beam, 27-40 mm from the sample surface. Sections were mounted vertically with a 45°angle to both the incident beam and detector. Data treatment was performed using PyMca (Solé et al., 2007) and 'R' software [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF], http://rproject.org). Elemental quantification was performed by deconvolution of the XRF spectra using the PyMca software (Solé et al., 2007). This software fits an entire XRF spectrum using an algorithm which reduces the number of parameters needing optimisation. Parameters concerning the multichannel analyser (MCA) energy calibration, the detector response and the photon beam properties are fitted for the spectrum as a whole using a fundamental parameter approach, while the set of lines for each element are combined into a single equation using theoretical ratios and positions. This reductionist approach essentially requires fitting of only one parameter per element, alongside, in our case, 6 experimental parameters. The fundamental parameters were experimentally determined from processing the mean spectrum collected as a 1 × 1 mm 2 grid of 10 × 10 points on a pellet of a fish otolith Certified Reference Material no. 22 (CRM22, National Institute for Environmental Studies, Tokyo, Japan) reference pellet (Yoshinaga et al., 1999(Yoshinaga et al., , 2000)).

Elemental quantification was calibrated on certified Sr and Ca contents of CRM22. After determining the basic fitting parameters, the photon flux was estimated manually beginning with an approximation of 1×10 9 ph/s, then adjusted manually using the reference's certified Sr mass fraction of 0.236% (±5%) (Yoshinaga et al., 1999(Yoshinaga et al., , 2000) ) to obtain the correct value of the total photon flux.

Synchrotron X-ray absorption spectroscopy (XAS)

X-ray absorption spectroscopy allows examination of the local chemical environment of a selected element in crystalline or amorphous materials [START_REF] Sayers | New technique for investigating noncrystalline structures: Fourier analysis of extended X-rayabsorption fine structure[END_REF]. This permits identification of the mode of incorporation of Sr by examining the nature, distances to neighbouring atoms and local geometry in the crystal lattice. All XAS spectra were collected around the Sr K-edge at 16 104.5 eV. When a wide X-ray beam was used, XANES measurements were recorded in transmission with nitrogen-filled ionisation chambers (200 V operating voltage) providing photon flux measurements before and after the sample. µXAS measurements were performed with fluorescence yield detection using the same configuration and detector as for SR-µXRF. Incident photon flux (I 0 ) was recorded by a 10 µm thick thin film Si diode (Hamamatsu Photonics) in transmission.

2.5.1. Synchrotron macro-and micro-X-ray absorption measurements Reference XANES spectra of Sr-containing aragonite and strontianite were recorded in transmission mode using a wide X-ray beam to limit the impact of microscale heterogeneity of the standards. These spectra were recorded with a count time of 3 s/point in three ranges: 15 902-16 030 eV in 2 eV steps, 16 032-16 132 eV in 1 eV steps, and 16 134-16 250 eV in 2 eV.

For micro-beam measurements, the µXAS signal was calculated by numerically integrating a 600 eV wide window centered on the Sr Kα fluorescence peak. The integrated signal was divided by the recorded I 0 from the thin film diode for normalisation.

µXANES spectra of otolith samples were recorded with a count time of 4 s/point in the 16 023-16 083 eV energy range in 2 eV steps, 16 084-16 134 eV in 1 eV steps, and 16 135-16 373 eV in 2 eV steps.

µEXAFS spectra (Fig. 5) were recorded with a count time of 8 s/point for 16 023-16 083 eV in 2 eV energy steps, 16 084-16 224 eV in 1 eV steps, and 16 225-16 773 eV in 2 eV steps.

XAS data processing

The spectra were processed using the iXAFS suite version 2.2 including ATHENA for data extraction and ARTEMIS for fitting of the spectra, alongside IFEFFIT for calculation of theoretical spectra (Ravel and Newville, 2005). Extrapolation of the edge jump and normalisation was performed using values in the pre-edge region, from 60-30 eV below the absorption edge (E 0 ), and in the post-edge region at 16-670 eV after E 0 region for µEXAFS. As oscillations extend in the whole XANES region, normalisation of XANES spectra was performed by adjusting the normalisation range to align the spectrum with similar EXAFS spectra from visual comparison. The data were normalised and post-edge region flattened using ATHENA with a E 0 approximated at the energy corresponding to half of the edge jump. An arctangent function was fitted to the pre-edge region, defined as points of normalised intensity below 0.45 of the edge jump, and post-oscillation region, defined as E ≥ 16 300 eV. The E 0 determined from the fitted arctangent function was used for the rest of the treatment. Spectrum background was removed by fitting with splines over the full energy range after E 0 , unless noted differently. In the processing of EXAFS data, a k 2weighting was used to amplify the spectrum in the mid-to highk range without excessively weighting the noise in the upper k range. A Hanning apodization window from 1-9 Å-1 with a taper of 1 Å-1 was applied before applying a Fourier transform to retrieve the pseudo-radial distribution with phase correction from FEFF calculations on the first O shell. EXAFS fitting was performed on the interval 1-6 Å in R-space. Occupancy of each shell was fixed at theoretical values 2.5.3. Multi-energy µXANES mapping µXANES mapping was performed using a reduced dataset on the section of QCD, detailed as follows. Five diagnostic energies were selected to provide significant discrimination between aragonite and strontianite based on the µXANES spectra of reference inorganic aragonite and strontianite (as identified in Fig. 2). A SR-µXRF map was recorded at each of these five energies. The data were processed using procedures written under the 'R' statistical environment [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF] on a per-pixel basis.

A model (Eqn. 1) was constructed based on the assumption that the X-ray absorption spectrum at any given pixel would result from a linear combination of the signals from Sr in aragonite and strontianite. Background removal and normalisation of both EXAFS reference spectra was performed in ATHENA. A linear energy-dependent term was included to correct for matrix-induced background variations; a linear decrease was found a good approximation in the restrained post-edge energy range considered.

I Exp (E) = β[a × I Arag (E) + b × I Stro (E)] + × E + δ (1)
The linear system was solved using four of the energy values (16 149, 16 170, 16 185

Results

X-ray absorption spectroscopy characterisation of stron-

tium in otoliths LM and SEM examination of the samples (Figs. S1-S3) indicate that none of them exhibit significant alteration. Subsamples of HP-11, HP-12, and GP-292 were confirmed to be aragonite by XRD. Samples QCD, Bayovar-1C, 111, and BH17 were examined by FT-IR spectroscopy at 3-11 points each, all of which were identified as aragonitic. All otoliths in this study were analysed for Sr elemental content by SR-µXRF to identify positions for µXAS analyses.

Sr fluctuations in the otolith sections of samples QCD, HP-12, Bayovar-1C, BH17, 424 and GP-292 are assumed to be biogenic, with some limited contamination by exogenous elements possible from preparation and/or burial assumed to extend no deeper than a few hundred micrometres/ The three maps of archaeological otolith sections presented in Figure 1 on the archaeological otolith sample QCD, and the inorganic strontianite and aragonite references were collected to provide reference spectra with a high signal-to-noise ratio while averaging possible microscopic fluctuations (Fig. 2). The vertical lines mark selected characteristic energies used for µXANES mapping.

The spectrum of the inorganic aragonite reference exhibits the following features, where energies are referred to as a shift relative to E 0 (ie. E 0 + n eV): (i) no evident pre-edge features; (ii) a white line with several inflection points observable in the spectrum derivative (Fig. S4); (iii) a white line maximum at +7.3 eV (16 112 eV), (iv) a low intensity absorption feature at ca. +20.8 eV, (v) oscillations with a first significant maximum at +43.4 eV preceded by a shoulder at ca. +30 eV, (vi) a shoulder at +65 eV, and (vii) a third somewhat sharp maximum at +94.1 eV. All features are shared by QCD. The spectrum of QCD also exhibits some similarity to that of the strontianite reference. However, characteristic differences are observed: the low-intensity feature at +20.8 eV (iv) and the shoulder at ca. +30 eV (vi) are absent, and the oscillation at +94.1 eV is very broad.

In four spectra -archaeological specimens Bayovar-1C ([Sr]=48.7 µmol • g -1 ; Fig. 3) and HP-12 ([Sr]=90.5 and 93.3 µmol • g -1 ; Fig. 4), and modern specimen 111 ([Sr]=12.2 µmol • g -1 ; Fig. 3) -we observed variations in the maximum intensity and position of the white line. This is attributed to a flattening of the peak when recording with a high count rate where the detector was operating at the limits of the linear range. Using the arctangent fitting approach, the E 0 value is consistent regardless of these variations, as are the positions of the post-peak oscillations.

Strontium K-edge µEXAFS spectra were recorded on 7 otolith sections (Tab. 1), and are compared to two inorganic Sr-containing reference materials: aragonite and strontianite. A spectrum from QCD is presented in Fig. 5 as a general represen- Archaeo.

Modern Inorganic tative of that from the archaeological otolith samples compared to the inorganic references. An asymmetric peak just above 2 Å is present in the FT magnitude of all three spectra.

Qualitatively, the FT of QCD closely resembles that of aragonite (Fig. 5). Fitting of the EXAFS was performed (Tab. S4) for both references and otolith samples. Reference natural aragonite and synthetic strontianite exhibit Sr-M (M: metal, ie. Ca or Sr) lengths of 3.980 and 4.155 Å, respectively. All otoliths exhibit Sr-M lengths in the range of 3.965-3.988 Å, which corresponds closely to that identified in the aragonite reference. These distances are also coherent with those reported in previous works on carbonate biominerals and strontianite chemistry (Doubleday et al., 2014;Finch et al., 2003;O'Day et al., 2000;Singer et al., 2008). Fits of the EXAFS spectra resulted in good agreement to the experimental data for the first O, C, and Ca shells up to 4.0 Å in aragonite and QCD, and to 4.5 Å in strontianite. Inclusion of additional O shells and multiple scattering paths was not attempted, as not requested for this work. This explains the absence of good fit of the contribution in the 4-5 Å range, also observed and not fitted by other authors (Doubleday et al., 2014).

Between one and 8 Sr K-edge µXANES spectra per sample were collected for all samples and references (Tab. 1) to permit analysis of a wide variety of zones and check for intra-sample homogeneity. Representative µXANES spectra of the samples are compared to that of the aragonite reference in Fig. 3. The characteristic features, including the small oscillation at +20.8 eV, oscillation and shoulder at +43.4 and +65 eV, and the oscillation at +94.1 eV are consistent for all otoliths (Fig. 3). Between 5 and 8 µXANES spectra were recorded on each of QCD, HP-11, and HP-12 at the locations depicted in Fig. 1, with an overall Sr concentration range of 27-95 µmol • g -1 . These spectra all show superimposable features (Fig. 4). No variation is observed in the position of E 0 nor any of the characteristic oscillations. The relative intensity and sharpness of the white line does vary slightly in some areas, as mentioned previously.

µXANES mapping of chemical environment

To assess the representativeness of point X-ray absorption measurements over large 'spatial dynamics' (Bertrand et al., 2013), µXANES mapping was carried out using five energies selected to correspond to characteristic features observed in the Sr K-edge XANES spectra of the aragonite and strontianite standards (marked by the dashed lines in Fig. 2). SR-µXRF maps were collected at these energies across areas up to 570 × 500 µm 2 on archaeological sample QCD (Fig. 6).

A transmission LM image (Fig. 6b) reveals variations in the luminosity, corresponding to approximately 1.5 years' growth. A crack in the otolith was observed in the Ca map and using LM, which was excluded from the analysis zone, leaving a final map width of 450 µm.

The map of aragonite fraction a derived from Eqn. 1 is shown in Fig. 6e. The map exhibits a high homogeneity [standard deviation (SD) of 0.072 in a unimodal distribution] across the full mapped area, centered on 91 % aragonite (Fig. 6f). The SD along the growth axis (0.075) -in the direction of Sr variations -is comparable to the SD along a growth increment (0.072), and the median integral covers a range of ±8 %.

The Ca concentration map (Fig. 6c) shows limited variation aside from an enhancement at the outer edge. The Sr concentration map (Fig. 6d) reveals a total range of 19-39 µmol • g -1 in the mapped zone, with a maximum value close to the edge. The maximum at the sample edge (Fig. 6g "Max") corresponds to the maximum observed on the entire otolith, while the minimum (Fig. 6g, "Min") is only a local minimum with lower concentrations elsewhere along the growth axis (Fig. 1). Some substructure can be seen in the transition between high and low Sr areas (Fig. 6g, "SS"). The aragonite fraction shows no relation to variation in Sr content.

Discussion: Contribution and open questions regarding the diagenesis of otoliths

Recovered in archaeological and paleontological contexts, ancient biominerals are key structures for reconstructing past human activity, and climatic and environmental conditions. The potential to use the geochemistry of ancient otoliths as information archives has long been recognized (Devereux, 1967) and is now fostering significant development in archaeology. In particular, otoliths have the capacity to elucidate fishing activities, interaction with aquatic ecosystems and seasonality of climates. Stable isotope analysis has suggested more humid conditions at the beginning of the Holocene for the arid northern coast of Peru (Béarez et al., 2011) and the winter settlement at different coastal Stone Age sites of Norway (Hufthammer et al., 2010). Oxygen isotope and Sr content analysis showed that Australian aboriginal settlement on the shores of Lake Mungo (Australia) correlates to the lake water levels at the time of the Last Glacial Maximum (Long et al., 2014). The type of habitat exploited by ancient Mid-to Late-Holocene fishermen were documented in two South Australian waterways by Sr and Ba content analysis (Disspain et al., 2011(Disspain et al., , 2012)). Elemental analyses also revealed the significant impact of human activities on hydrologic conditions of these waterways and provide a baseline reference dating before the arrival of Europeans in support of conservation and restoration efforts (Disspain et al., 2011(Disspain et al., , 2012)). Interpreting the geochemistry of ancient biominerals is, however, challenging, requiring an understanding of biological processes of incorporation (element partitioning or isotopic fractionation) and interpretation of post-mortem changes linked to pre-and post-burial environment. One of the major issues is that biominerals undergo post-mortem alteration that could impact the reliability of the biogenic geochemical information encoded. In this respect, otoliths have far less been studied than other biominerals such as bones (Collins et al., 2002;Hedges, 2002;Nelson et al., 1986;Trueman, 2013), enamels (Hinz and Kohn, 2010;Hoppe et al., 2003), shells (Buchardt and Weiner, 1981;Chappell and Polach, 1972;Cherns et al., 2011), or corals (James, 1974;Webb et al., 2009). No comprehensive study of otolith diagenesis has been conducted so far. The present paper constitutes a first effort to understand the long term behavior of Sr in archaeological otoliths by examining the chemical environment of this element.

Incorporation of secondary elements into carbonate biominerals can occur in one of the five main modes illustrated in Fig. 7 that can be broadly categorised into four mechanisms: (1) incorporation into the organic material, (2) adsorption onto the surface of micro-or nano-crystals of aragonite, intra-crystalline incorporation, comprising [START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] substitution for Ca and (4) incorporation into interstitial spaces, and (5) as discrete strontianite phases. Sr (or any other element) adsorbed on the surface or incorporated into the organic material is expected to be less stable than in the crystal lattice (Gauldie et al., 1998). In aragonite, Ca 2+ is coordinated to 9 oxygen ions (while coordination is 6 in calcite). Sr 2+ is assumed to substitute isomorphously for Ca 2+ owing to the similar charge and ionic radius of the two ions: 118 pm for Ca 2+ and 131 pm for Sr 2+ in 9-fold coordination (Tab. 2) [START_REF] De Villiers | Crystal structures of aragonite, strontianite, and witherite[END_REF]Shannon, 1976;Speer, 1983). Sr is not significantly leached out during handling or preparation (cleaning, sectioning, polishing, staining, and etching) of otoliths (Andrus and Crowe, 2002;Gauldie et al., 1998;Milton and Chenery, 1998;Proctor and Thresher, 1998). This has generally been interpreted as Sr incorporation into the aragonite lattice (Andrus and Crowe, 2002;Milton and Chenery, 1998) but has alternatively be interpreted as the presence of stable strontianite domains (Gauldie et al., 1998). Recently, in a study involving otolith samples from modern marine fishes, Doubleday et al. supported the hypothesis of Sr randomly substituting Ca in aragonite (Doubleday et al., 2014). If Sr in random substitution for Ca is confirmed as a general characteristic in modern otoliths, a different mode of incorporation in ancient otoliths would provide a direct identification of post-mortem alteration.

Micrometric point µXAS analyses ubiquitously indicates Sr in aragonite

In the present work, µXAS was used to examine the Sr chemical environment in 5 archaeological otoliths from the Holocene, and 3 modern counterparts at the microscale. Both Sr K-edge µXANES (for all specimens) and µEXAFS (for 7 specimens) analyses were performed. µEXAFS enables the precise measurements of inter-atomic distances while µXANES probes the overall chemical environment but is less time consuming. The combination of the two modalities allowed to performance a large number of point analyses (total of 32) in a reasonable timeframe (beamtime of ca. 1 week). Accurate selection of analysis points and data comparison at both interand intra-individual levels was achieved by collection of SR-µXRF elemental maps before µXAS analysis. SR-µXRF elemental mapping shows that the sections all have Sr distributed throughout their entirety with variations in concentration along the growth axis, as expected for otoliths of individuals several years of age (Kalish, 1989) Our XAS and µXAS results globally indicate Sr in an aragonitic environment within the limits of detection. Distinction from calcite and/or vaterite environments is relatively straightforward as calcite and vaterite crystallize in a significantly distinct mineralogical form (Tab. 2). Calcite and vaterite therefore exhibit a distinctly different µXANES spectrum than aragonite, with in particular a double peak at the white line (Finch et al., 2001;Hayakawa et al., 2008;O'Day et al., 2000); such a feature was never observed on spectra of the present work. We can therefore rule out the presence of calcite or vaterite as significant environment for Sr in any otolith.

Distinction of aragonite from strontianite is a more difficult task (Finch et al., 2003;Greegor et al., 1997). µXANES spectra of both carbonate compounds are relatively similar owing to their isostructurality (Tab. 2). However, XANES spectra did allow the identification of discriminating features (Fig. 2), attributed to the change in geometry and crystal lattice parameters, as well as Sr-Sr paths. Detectable differences are observed in the EXAFS Fourier transform (FT) (Fig. 5). In the strontianite reference, the Sr metal signal and peak occurs at 3.95 Å (Fig. 5). Inorganic aragonite exhibits a Sr-metal peak at ca. 3.90 Å, which is shared by all otolith samples. The positions of these peaks are coherent with those previously presented for Sr in aragonite (Doubleday et al., 2014;Finch et al., 2003). The imaginary part of the FT of the otolith µEXAFS signal distinctly differs from that of strontianite while it is in very good agreement with the inorganic aragonite standard (Fig. 5, bottom). Indeed, the imaginary part of the FT is more sensitive to a change in neighbouring element than the magnitude. The magnitude will reveal fine changes in radial distribution, but examination of the imaginary part permits a sensitive detection of phase shifts resulting from the exchange of the neighbouring atom Ca 2+ for Sr 2+ even associated to limited change in interatomic distances. Four species from two families for a total of eight individuals and two different types of otoliths (sagittae and lapilli) were studied here and Sr in an aragonitic environment was ubiquitously identified. Focusing first on modern samples, our results complement the previously-cited recent study that identified the random substitution of Sr 2+ for Ca 2+ in otoliths from ten modern individuals belonging to seven species from Australia, using wide-beam EXAFS (ca. 0.3 × 0.3 mm 2 ) (Doubleday et al., 2014). With the addition of our results, the random substitution of Sr 2+ for Ca 2+ in the otolith aragonite is now supported in a total of 6 teleost families for which either the sagittae or the lapilli are the most developed otoliths. The environments of the species studied in Doubleday et al. (2014) ranged from freshwater to hypersaline estuarine. Our corpus of samples brings a broader diversity in fish ecology, as we have examined coastal marine species that can enter freshwater systems such as estuaries and lagoons. This diversity in ecology and our methodology (quantification of Sr concentrations with SR-µXRF coupled to XAS measurements) allowed a simultaneous extension of the conditions of Sr speciation to a Sr content range of 7.1-94 µmol • g -1 (620-8200 µg • g -1 ). This strongly indicates random substitution of Sr for Ca, independent of concentration across a wide concentration range at both intra-and inter-individual scales in this extended range of fish families.

The majority of published works using XAS have observed no sign of calcite or strontianite formation in biomineral aragonites. The random substitution of Sr for Ca has been demonstrated in different species of corals, both modern (Allison et al., 2005(Allison et al., , 2001;;Doubleday et al., 2014;Finch and Allison, 2003;Finch et al., 2003) and ancient (Allison et al., 2001;Finch and Allison, 2003), the shell of one modern bivalve species (Foster et al., 2009), and statoliths or cuttlebones of two modern cephalopod species (Doubleday et al., 2014). However, one work identified strontianite domains within modern and ancient aragonitic corals (Greegor et al., 1997) by fitting the FT magnitude of wide-beam EXAFS analyses with linear combinations of pure references (strontianite and Sr in aragonite).

A macroscopic XAS analysis, as in Doubleday et al. (2014), typically produces an analysis volume at least 6.3 × 10 7 µm 3 , assuming a 250 × 250 µm 2 spot size in transmission and a 100 µm thick sample. Our work employed a Gaussian-shaped X-ray spot with FWHM dimensions of 6 × 11 µm 2 (V×H) on sample. At the energy range and geometry employed, this produces a stretched information volume along the beam axis, penetrating ca. 260 µm deep into the sample and across the horizontal. The corresponding information volume totals 3.7 × 10 4 µm 3 (for the volume producing 75 % of the signal). Taken as an example, the DiffAbs beamline provides 10 12 photon/s in wide-beam mode, and ca. 3 × 10 9 photon/s in micro-beam mode at the Sr K-edge. A greater photon flux in wide-beam mode results in an improved (lower) detection limit than in micro-beam mode. However, the smaller spot size in micro-beam mode results in a cleanly-shaped beam of a greater photon flux density (ca. 45 photon/s/nm 2 ) than in widebeam mode (ca. 13 photon/s/nm 2 ). In a homogeneous system, the higher signal-to-noise ratio (SNR) of macro-mode provides a higher sensitivity, but in our biomineral system which could present microstructural heterogeneities, increased lateral resolution yields an increased sensitivity to local alterations. Even assuming an order of magnitude higher detection limit for the microscale compared to macro-scale (eg. 10 000 ppm vs. 1000 ppm, respectively), a microscale analysis has the potential to reveal heterogeneous alteration 1-2 orders of magnitude smaller. The combination of µEXAFS and µXANES in the present study has permitted analysis of otoliths on the microscale with an increased sensitivity to small-scale alterations. No alternate phases were detected on the micrometric scale. Examination at a further increased lateral resolution, approaching that of the nanometric crystallites, would require increasingly complex and prohibitive sample preparation (Stangl et al., 2013). Kamhi (1963); [START_REF] Sass | Interatomic distances and thermal anisotropy in sodium nitrate and calcite[END_REF]; Swanson and Fuyat (1953) Series of µXAS points can provide information on Sr chemical environment at multiple locations across a sample. However, the overall representativity of microscale point analyses may be lower than that at millimetre scale because selection of the points could have, intentionally or not, avoided local singularities. In order to confirm the representativity of point analyses and trace potential high resolution spatial variations, the multi-energy µXANES mapping employed in this work examined a selected zone of 0.26 mm 2 of the archaeological sample QCD (Micropogonias sp.). The universal presence of Sr permitted microscale analysis at multiple points and at varying Sr concentrations.

The application of the linear model (Eqn. 1) produces a map of the sample composition, based on the assumption that each Sr K-edge XANES spectrum results from the linear combination of contributions from strontianite and aragonite domains. If this assumption was incorrect, the model is likely to produce incoherent results. In particular, significant variations in the values of β, , or δ may constitute such an indication. The β factor reflects the scaling for Sr concentration and all other fitted values are related to the concentration. The spatial distribution of β values (Fig. S9), closely resembles the Sr map (Fig. 1). This is a good indication that the model is accurate and that there are no tertiary phases. Indeed, normalising the β factor by the Sr concentration at each pixel produces a homogeneous narrow distribution (mean=1.8 × 10 -3 , SD=8.3 × 10 -5 ).

The estimated aragonite fraction a is largely homogeneous across the map (mean=91 %, SD=7.2 %) while the contribution from strontianite-type domains is estimated to be ca. 9 %. However this figure is similar to the observed noise, attributed to the limited Sr XRF counts per pixel, and we do not see any outlier hotspot of strontianite. Without completely ruling out the possible presence of strontianite domains as a minor component of the probed information volumes, a purely aragonitetype system where Sr would randomly substitute for Ca suffices to explain the observed data. The presence of Sr-Sr neighbours resulting from random substitution has been suggested as a possible source for strontianite-type contributions to XAS spectra in aragonite regions of higher Sr concentration (Doubleday et al., 2014). However, it appears relatively unlikely in our case. The expected probability to find Sr atoms with more than another Sr neighbour is very low -1.6 × 10 -5 for the highest Sr concentration observed in the mapped zone. Contributions from random Sr-Sr pairs would therefore be negligible in our absorption spectra. Although no constraints were applied to the model, the majority of points fell within the expected aragonite fraction range of a = (0, 1). Some outlier points (ca. 1.4 SD from the mean) occurred above 1.0, to a maximum of a=1.18. This further supports the model's accuracy.

In all otoliths analysed, a great attention was taken to orient the growth axis vertically to maximise the lateral resolution along this growth axis (see Fig. 6a). Annual growth increments in fish otoliths are perpendicularly oriented relative to the growth section and were observed to be 70-140 µm in width in QCD. As a result, the QCD maps crossed two sequential isochronous increments, and allowed measurements at high resolution within a single increment. The information volume for this analysis is identical to that previously described for point µXAS and is on the same order of size as the needle-like microcrystals composing individual increments which are visible under SEM (Fig. 7;Fig. 2 in Dauphin and Dufour (2008)).

Fast µXANES mapping at high resolution over a large area offers for the first time the ability to study Sr speciation over 'large spatial dynamics' (Bertrand et al., 2013) covering 2 orders of magnitude from ca. 10 µm to the millimetre range, in order to identify any heterogeneity. Ancient biominerals can exhibit heterogeneous alteration at submillimetric scales (eg. (Guzmán et al., 2009;Webb et al., 2009)). Mapping on the edge of QCD was chosen because, during burial, the outer surface of the otolith is expected to be the most vulnerable to alteration by being in direct contact with the decaying fish, as well as fluids and sediments from the environment (Trueman, 2013). µXRF showed that this zone exhibits significant variation in Sr content, including the largest variation for the whole specimen, that could alternatively be interpreted as resulting from postmortem alteration or an event during the life of the specimen. However, the mapping demonstrates that no heterogeneity in Sr speciation is present within the limits of detection.

At the length scale of the increment thickness and at the limit in terms of attainable lateral resolution, an intriguing and yet unexplained behavior was observed. The Ca content of the otoliths was observed to vary up to 3.5 % of the median in the core to edge profiles produced by SR-µXRF (Fig. 8). This fluctuation appears mostly anticorrelated to the Sr concentration. This is particularly noticeable for the most extreme Sr concentration peaks observed in the archaeological specimens QCD and HP-12.

It is unlikely that this anticorrelation results from improper XRF matrix correction as the attenuation length of the Ca signal is altered by no more than 0.25 % across the range of Sr concentrations found in otoliths (0.05-0.5 %at). The variation in Ca is too large to be accounted for purely through increased substitution by Sr. Ca-depleted regions are likely to correspond to zones richer in organic matter. However, in our samples, even in those zones, Sr appears to substitute randomly for Ca in an aragonitic chemical environment, with no evidence of Sr in another chemical environment. This anticorrelation may come unnoticed in measurements using Laser ablation-inductively coupled plasma-mass spectrometry or SR-XRF as Sr concentration is usually normalised to Ca concentration (eg. (Limburg et al., 2011)), and deserves further investigation.

We collected multi-energy µXANES maps to obtain spatially-resolved speciation information. XAS imaging is undergoing a significant development for ancient materials stud-ies (Bertrand et al., 2012). For such measurements, two general options are offered: using a full-field illumination or a scanning microbeam. Full-field approaches have been implemented in transmission geometry at several synchrotron beamlines (Fayard et al., 2013;Meirer et al., 2013). At an identical SNR (i.e. count per pixel), full-field imaging allows to decrease the flux per pixel by several orders of magnitude, and suppresses dead times due to sample movement. However, the attenuation length of Sr K-edge photons is ca. 330 µm. Collecting a signal in transmission would require thinning down samples to this typical thickness. Samples of very small areas could be self-supported, but larger samples would have to be fixed on a low X-ray absorbing support with attached risks of contamination. In both cases, this would contravene strongly to our aim to work as much as possible non-invasively on historical samples that were studied with complementary characterisation techniques. Use of fluorescence detection in a reflection geometry in a full-field mode is emerging but remains limited due to the low solide angle of detection of 2D XRF detectors in full-field mode (Tack et al., 2014). Using a scanning microbeam, data collection is also possible using fluorescence detection in a reflection geometry. Recording a full XAS spectrum at each pixel with such an approach requires both a very high flux at each beam impact position and a very long total acquisition time. High fluxes may increase radiation-induced side effects (Bertrand et al., 2015). Even a relatively fast acquisition of 5 min per XAS spectrum, would take over an unrealistic 19 days. The five-energy µXANES mapping approach employed in this work (570 × 500 µm 2 map with a step size of 5 × 10 µm 2 ) resulted in a map obtained in 10 h on a bending magnet beamline, while comparatively vastly reducing sample exposure to radiation. In our experiments, two scan strategies were possible: (i) positioning the sample and collecting successive XRF spectra, or (ii) setting successively each energy and collecting a full XRF map at each energy. This last option is usually preferred as it limits movements of the monochromator that may impose significant additional dead times. However, in this second option, the precision of the mechanical setup should be high enough to allow repositioning each energy map at the pixel level, while the stability of the synchrotron beam position is also essential. We have discussed the complexity of interpreting X-ray absorption data collected at a limited number of excitation energies, however, it provided a unique insight into the large-scale homogeneity of the Sr 2+ substitution in otolith aragonite in realistic experimental conditions applicable to schlerochronological and ecological studies.

Post-mortem alteration of Peruvian archaeological otoliths

According to the common criteria which are used to assess the lack of diagenetic alteration in ancient otoliths and other biocarbonates (Dufour et al., 2000;Guzmán et al., 2009), the five Holocene Peruvian specimens of this work can be considered as intact. As aragonite is not the most thermodynamically stable of the three pure CaCO 3 polymorphs under terrestrial surface conditions, aragonite (biogenic or abiogenic) tends to transform to calcite over time (Cherns et al., 2011). There was no calcite present in our samples from the macroscale (seen with XRD and FTIR) to the microscale of the Sr chemical environment (demonstrated with µXAS). Textural observations showed no evidence of recrystallization at the level of the needle-shaped microcrystals (ca. 10-100 µm long and 1 µm in diameter), as observed under SEM (Supplementary information, Figs. S1-S3).

Otolith texture presents further levels of organization that were not explored in the present work, but that may play a significant role during diagenesis. Microcrystals are polycrystalline, composed of crystallites on the order of several hundreds of nanometers embedded completely in an organic matrix (Dauphin and Dufour, 2008;Falini et al., 2005;Söllner et al., 2003). The crystallites themselves are composed of granules a few tens of nanometers in diameter and wrapped in organic envelopes (Dauphin and Dufour, 2008;Gauldie, 1999;Söllner et al., 2003). Even if this level of organization has only been described in sagitta, it is expected to be identical in lapilli (and asterisci) since at the higher level (microcrystal units) they share similar organization (Dauphin and Dufour, 2008). Alteration of the microstructures within the prismatic calcitic and crossed lamellar aragonitic layers in the shell of Concholepas concholepas has been recognized even if these layers appear well preserved (Guzmán et al., 2009).

A third criterion to assess diagenetic alteration is the examination of the geochemical signals themselves (Disspain et al., 2011(Disspain et al., , 2012;;Dufour et al., 2000). Elements that are known to be endogenous such as Na, P, K, and Sr can be studied to detect possible alteration on the basis of significant deviation from usual content. Exogenous elements diffusing into the otolith directly mark alteration. µXRF maps revealed that the Peruvian otoliths exhibit large variations in Sr concentrations (Fig. 1). While [Sr] values in specimens QCD, Bayovar-1C and BH-17 fall within the range reported in otoliths of modern fishes, a significant positive deviation is observed on two specimens of G. peruvianus ) that both exceed this usual range as well as that observed for the modern counterpart studied here (GP-292) (Figs. 1 and8). Significant increase in Sr content was identified in the outer hundreds of micrometers of several archaeological and modern otoliths. They could result from post-mortem alteration. Unambiguous attribution to sample handling and preparation, early or long-term diagenesis is difficult. Microscale Sr speciation was researched as a potential disambiguating approach. The present study supports that the chemical environment of Sr in the archaeological specimens is identical to that of modern otoliths independent of the element concentration, location in the otolith and species studied. There is no evidence of extra-crystalline Sr, presence in interstitial spaces, or discrete strontianite crystals. The large Sr content excursions observed in two Peruvian otoliths are not linked to a change in the mode of incorporation of Sr. A question that remains to be assessed is if this stability of the Sr chemical environment -exclusively in random substitution for Caguarantees the preservation of the original Sr content and accurate reconstructions based on µXRF mapping of our specimens.

A porous mineral organization that permits the circulation of fluids promotes mineralogical change and elemental exchange in abiogenic minerals and biominerals. The complex multiscalar organisation down to tens of nanometers increases the surface of contact with fluids. The organic matrix is present at all scales of crystalline organisation in otoliths (Dauphin and Dufour, 2008;Falini et al., 2005). Water diffusion in otoliths has been directly evidenced by mass increases up to 4.18 %wt after soaking otoliths in water for 48 h (Gauldie et al., 1998). This water is not bound to the matrix, but is mostly molecular water according to the characteristic IR peak, and is therefore available for ionic transport (Gauldie et al., 1998). Diffusion of ions and molecules into and out of the otoliths is also indirectly evidenced by the increased contrast of internal features when otoliths are soaked in water, oil, solvents, or dyed by large molecules to facilitate the reading of growth marks under light transmission (Gauldie and Coote, 1997). As a consequence, changes in elemental contents were noticed when using several analytical preparation and storage methods (Milton and Chenery, 1998;Proctor and Thresher, 1998;Rooker et al., 2001). This was reported for "labile elements" such as Na, K, S or Cl (Proctor and Thresher, 1998). On the contrary, the Ca and Sr contents of modern specimens, immersed in solutions, were noted as relatively stable. This observation was made in the limited timeframe of laboratory experiments -a few hours to several days, and may not correspond to the long-term behaviour of archaeological samples over thousands of years. After the fish death, decay of the organic matrix could further increase exchange of elements with the environment. According to (Guzmán et al., 2009), the dissimilarity in the diagenetic changes between the calcitic and aragonitic layers within shells of C. concholepas are related to the difference in quality and quantity of the preserved organic matrix.

The Peruvian otoliths studied here are likely to have experienced limited exposure to fluids after their initial taphonomy, due to the exceptional climatic conditions that prevail on the Pacific coast. Coastal fogs that form during the austral winter are the main source of humidity in the very arid environment while intense precipitation is mostly limited to El Niño events. Moreover, the otoliths in the present work originate from two sandy surface sites (Bayovar-1 and Pampa de los fósiles) deprived of pore water and from one stratified mound (Huaca Prieta) not susceptible to significant ground water exposure. Prevailing climatic conditions on the Peruvian and Chilean coasts have favored the conservation of Holocene bivalve or gasteropod shells (Carré and Dufour, 2012;Guzmán et al., 2009), limiting the diagenetic alteration, or on the contrary led to their rapid diagenesis, thereby precluding palaeoenvironmental reconstructions based on geochemical analyses (Labonne and Hillaire-Marcel, 2000). In the latter case, the shells originated from emerged marine terraces. Macroscopic examination showed that the aragonitic layer was frequently pulverulent. Early diagenesis during marine immersion is evidenced by a marine U/Th signal. Later contamination by sea spray could be present as an agent of alteration, but the effects are expected to be very limited compared to immersion (Labonne and Hillaire-Marcel, 2000). Samples from marine sites may undergo significant alteration as a result of the circulation of pore water in the surrounding sediment.

The diagenetic trajectory followed by our samples is more favorable for preservation as post-mortem contact with environmental fluids is minimal in a continental arid environment. The otoliths studied in this work have not been in direct contact with seawater or sediments with circulation of pore water, as the fish was caught and prepared, and the otoliths later discarded with the carcass in an arid continental context. They could however have been affected by cooking operations. Cooking and burning were shown to alter the mineralogy and elemental concentrations in otoliths (Andrus and Crowe, 2002). Even though cooking operations may not always leave recognisable evidence, we intentionally chose otoliths with no sign of burning or macroscopic alteration. The samples examined in the work appeared well-preserved by the common microstructural and mineralogical criteria; further work examining samples which exhibit visible alteration maybe valuable to expand knowledge of otolith taphonomy and reliability.

Conclusion

This work presents the first examination of the chemical environment of strontium in archaeological otoliths, which are increasingly used as environmental proxies for palaeoenvironmental reconstructions and anthropological studies. Five archaeological and three modern otoliths of fish from two families (Sciaenidae and Ariidae) were examined. Elemental mapping by synchrotron µXRF permitted a precise positioning of analyses on the otolith sections in relation to the sub-annual fluctuations of Sr. Multi-energy µXANES mapping was implemented to study the speciation homogeneity over a 480 × 550 µm 2 area. XAS is the optimal choice for targeted analyses of ion incorporation and speciation. The chemical environment of Sr(II) in archaeological and modern fish otoliths has been examined on the microscale using µXANES and µEXAFS. The chemical environment was confirmed in all cases as Sr(II) randomly substituted for Ca(II) in the aragonite lattice, independent of location on the otolith, Sr concentration, or archaeological age of the otolith. This mode of substitution shows no change from that reported on modern otoliths from teleost fishes in previous studies, as well as on modern counterparts studied here. In addition, it is identical to most other bioaragonitic systems including deep sea corals. This work reinforces the validity of fish otoliths as an invaluable biological and palaeoenvironmental archive. Acanthopterygii A superorder of teleost fish.

Ariidae

A family of ostariophysian catfish inhabiting warm or tropical waters.

Attenuation length

The distance at which a photon beam has been reduced to 1/e of its original intensity.

Bragg relation

The relation between X-ray energy, lattice spacing, and incident angle such that diffraction occurs. See Section 3.7.

Catadromous organism

An organism which is born at sea and migrates to freshwater during its lifespan.

Crystallites

The microscopic crystals which compose a polycrystalline material.

Diadromy

Movement between fresh and salt water.

Domain (crystallography)

A region with a uniform crystallographic phase, different from neighbouring regions.

Energysensitive detector

A detector in which both energy and number of photons are measured.

Environmental proxy

A characteristic (e.g. Sr concentration) which can be measured as a substitute for direct in situ measurement of environmental characteristics (e.g. temperature, salinity).

Escape peak A peak below the energy of the characteristic line, where a photon has been only partially absorbed by the detector. The energy difference between the characteristic line and the escape peak is characteristic of the detector material (e.g. 1.74 keV for a silicon detector).

Full Width at Half Maximum

The total width of a measured peak (e.g. spectral, diffraction) at half of its maximum intensity.

Hypermet function

A peak shape function consisting of a Gaussian function combined with a low-energy tail which decays exponentially [START_REF] Phillips | Automatic analysis of gamma-ray spectra from germanium detectors[END_REF].

Kernel density plot

A kernel density plot is produced by placing a kernel (a smooth, peaked function) on the X-axis where each point of data lies. The total area of all kernels is summed to produce the kernel density (Silverman, 1998).

Continued on following page.

holders were used to support the powder samples. Samples were rotated about the φ axis at 15 RPM during analysis. Diffractograms were recorded in the range 15° 2θ 65°with 0.02°steps. In all but one case, recording time was 1 s/step. For the 0.5 %wt strontianite pellet 10 s/step were used to increase the signal-to-noise ratio due to the low analyte concentration.

D C H A R A C T E R I S AT I O N O F R E F E R E N C E M AT E R I A L S

The reference materials (Sec. 2.3) were analysed using a benchtop XRD system (Sec. C). The system was designed with the intention of using fixed-height sample holders to maintain the geometry.

Although best efforts were made to place the the pellets at the correct height, there is still some shift in 2θ when intact pellets were measured. # I 0 i s a n o r m a l i s a t i o n f a c t o r f o r beam i n t e n s i t y # sigmaX and sigmaY a r e t h e beam shape parameters (SD ) on t h e x and y axes , r e s p e c t i v e l y # t h e t a and phi a r e t h e i n t e r s e c t i o n angle o f t h e beam with t h e normal o f sample s u r f a c e on t h e x and y p l a n e s r e s p e c t i v e l y # L1 , L2 , L3 a r e t h e a t t e n u a t i o n l e n g t h s i n um o f t h e incoming beam i n t h e m a t e r i a l ( L1 ) , e mi t te d photons i n t h e m a t e r i a l ( L2 ) and e mi t te d photons i n t h e atmosphere ( L3 ) # detectorCoord i s a 3-element v e c t o r d e s c r i b i n g t h e l o c a t i o n o f t h e d e t e c t o r r e l a t i v e t o t h e sample # beamX and beamY a r e where t h e beam i n t e r s e c t s t h e sample s u r f a c e i n t h e x and y axes # dmax i s a s a n i t y check on t h e maximum s i m u l a t i o n depth I . f l u o r e s c e d 3 D <-f u n c t i o n ( x , y =0 , z =0 , I 0 =1 , sigmaX , sigmaY , t h e t a = t h e t a , phi=phi , L1=L1 , L2=L2 , L3=L3 , detectorCoord=c ( 0 , 0 , -1 0 0 0 ) , beamX=0 , beamY=0 , dmax=1 e12 ) { # E r r o r t r a p p i n g # I f t h e f i n a l z i s going t o be over t h e l i m i t i f ( max ( z ) > dmax ) { c a t ( " E r r o r : y has exceeded dmax" ) ; r e t u r n (NULL) } #Check f o r t h e r i g h t format on each a x i s i f ( ! a l l ( i s . v e c t o r ( x ) , i s . v e c t o r ( y ) , i s . v e c t o r ( z ) ) ) { c a t ( " E r r o r : x , y , and z must be v e c t o r s or s i n g l e v a l u e s . " ) ; r e t u r n (NULL) }

# S e t up an empty a r r a y t o f i l l with t h e d e t e c t e d f l u o r e s c e d i n t e n s i t y f l u o S i g n a l <-a r r a y ( dim=c ( l e n g t h ( x ) , l e n g t h ( y ) , l e n g t h ( z ) ) )

# I n f o r m a t i o n progBar <-t x t P r o g r e s s B a r ( min=0 , max= l e n g t h ( z ) , s t y l e =3 , i n i t i a l =0) zlim= q u a n t i l e ( ( hdXRF$umol_g [ , , which ( hdXRF$ m a s s F r a c F i t s == " Sr L3 " ) ] ) , probs=c ( 0 . 1 , 0 . 9 9 5 ) , na . rm=T ) )

#
# S e l e c t curves . . . Loop u n t i l done zlim= q u a n t i l e ( ( hdXRF$umol_g [ , , which ( hdXRF$ m a s s F r a c F i t s == " Sr L3 " ) ] ) , probs=c ( 0 . 1 , 0 . 9 9 5 ) , na . rm=T ) )

#Redraw e x i s t i n g f o r ( i i n 1 : c u r v e B u i l d e r S t a t u s $ nCurves ) { ( theData ,c ( 2 ,[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] , mean , na . rm=T ) x r f P r o f i l e [ [ " median " ] ] <-apply ( theData ,c ( 2 ,[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] , median , na . rm =T )

x r f P r o f i l e [ [ " sd " ] ] <-apply ( theData ,c ( 2 ,[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] , sd , na . rm=T ) x r f P r o f i l e [ [ "max" ] ] <-apply ( theData ,c ( 2 ,[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] , max , na . rm=T )

x r f P r o f i l e [ [ " min " ] ] <-apply ( theData ,c ( 2 ,[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] , min , na . rm=T ) x r f P r o f i l e [ [ " m a s s F r a c F i t s " ] ] <-hdXRF$ m a s s F r a c F i t s

Listing 5: An algorithm to interactively, graphically define a series of "master curves" on an elemental map, define a series of curves between each pair of master curves, and extract the data from each pixel on the series of curves.

  Figure 1Location of otoliths and labyrinth systems within the fish. . . . . . . . . . . . . . . . . . . . . Figure2Structure of the labyrinth system and otosacs . . . Figure3Interior face of a whole otolith from a channel flatfish showing light and dark zones constituting annuli in transmitted light. . . . . . . Figure4Aragonite unit cell . . . . . . . . . . . . . . . . . . Figure5The structure of an otolith from atoms to macroscopic object . . . . . . . . . . . . . . . . . . Figure6Otolith sections before and after etching . . . . . . Figure7Secondary centres of calcification produce complex forms . . . . . . . . . . . . . . . . . . . . . Figure8A whole otolith viewed from multiple angles . . . Figure9Simplified phylogeny of species in this work . . . Figure10Map of sample origins . . . . . . . . . . . . . . . . Figure11Photos of archaeological sites . . . . . . . . . . . . Figure12Sectioning planes of typical sagitta . . . . . . . . . Figure13Photographs of otoliths as analysed . . . . . . . . Figure14Reference pellet mounted for analysis . . . . . . . Figure15A unified analytical methodology . . . . . . . . . Figure16Examples of previous works using SR-XRF for life history studies. . . . . . . . . . . . . . . . . . . Figure17Tungsten XRF signal within and outside of a FIB mark . . . . . . . . . . . . . . . . . . . . . . . . . . Figure18Atomic cross-sections . . . . . . . . . . . . . . . . . Figure19Excitation of a core electron by an X-ray photon . Figure20Regions of a XAS spectrum . . . . . . . . . . . . . Figure21Schematic of the components in a XAS experiment. Figure22A double crystal monochromator is configured with two mirrors, such that an X-ray of the desired energy (hν 1 ) passes through the exit slit. X-rays of a second, undesired energy (hν 2 ) do not follow the same path and are blocked. . . Figure23Experimental setup for µXRF on DiffAbs . . . . . Figure24The XRF process . . . . . . . . . . . . . . . . . . . Figure25Configuration for derivation of quantitative XRF formula . . . . . . . . . . . . . . . . . . . . . . . . . Figure26FF-XRF experimental setup . . . . . . . . . . . . . Figure27µPIXE experimental setup . . . . . . . . . . . . . . Figure28Illustration of Bragg's law . . . . . . . . . . . . . .

Figure 28

 28 Figure 1Location of otoliths and labyrinth systems within the fish. . . . . . . . . . . . . . . . . . . . . Figure2Structure of the labyrinth system and otosacs . . . Figure3Interior face of a whole otolith from a channel flatfish showing light and dark zones constituting annuli in transmitted light. . . . . . . Figure4Aragonite unit cell . . . . . . . . . . . . . . . . . . Figure5The structure of an otolith from atoms to macroscopic object . . . . . . . . . . . . . . . . . . Figure6Otolith sections before and after etching . . . . . . Figure7Secondary centres of calcification produce complex forms . . . . . . . . . . . . . . . . . . . . . Figure8A whole otolith viewed from multiple angles . . . Figure9Simplified phylogeny of species in this work . . . Figure10Map of sample origins . . . . . . . . . . . . . . . . Figure11Photos of archaeological sites . . . . . . . . . . . . Figure12Sectioning planes of typical sagitta . . . . . . . . . Figure13Photographs of otoliths as analysed . . . . . . . . Figure14Reference pellet mounted for analysis . . . . . . . Figure15A unified analytical methodology . . . . . . . . . Figure16Examples of previous works using SR-XRF for life history studies. . . . . . . . . . . . . . . . . . . Figure17Tungsten XRF signal within and outside of a FIB mark . . . . . . . . . . . . . . . . . . . . . . . . . . Figure18Atomic cross-sections . . . . . . . . . . . . . . . . . Figure19Excitation of a core electron by an X-ray photon . Figure20Regions of a XAS spectrum . . . . . . . . . . . . . Figure21Schematic of the components in a XAS experiment. Figure22A double crystal monochromator is configured with two mirrors, such that an X-ray of the desired energy (hν 1 ) passes through the exit slit. X-rays of a second, undesired energy (hν 2 ) do not follow the same path and are blocked. . . Figure23Experimental setup for µXRF on DiffAbs . . . . . Figure24The XRF process . . . . . . . . . . . . . . . . . . . Figure25Configuration for derivation of quantitative XRF formula . . . . . . . . . . . . . . . . . . . . . . . . . Figure26FF-XRF experimental setup . . . . . . . . . . . . . Figure27µPIXE experimental setup . . . . . . . . . . . . . . Figure28Illustration of Bragg's law . . . . . . . . . . . . . .

1. 4

 4 Otoliths as information archives . . . . . . . . . . . 1.4.1 Taxonomy . . . . . . . . . . . . . . . . . . . . 1.4.2 Age and growth . . . . . . . . . . . . . . . . 1.4.3 Environment and physiology . . . . . . . . . 1.4.4 Otoliths as archives for palaeontology and archaeology . . . . . . . . . . . . . . . . . . . 1.5 Reliability of otoliths: Effects of post-mortem alteration . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Potential alteration in modern otoliths . . . 1.5.2 Alteration in archaeological contexts . . . . 1.6 A need for further investigation: Questions addressed in the present work . . . . . . . . . . . . . .

  (a) Tomographic reconstruction of an Etroplus maculatus showing the head with the otoliths (red, purple, and yellow) in place. Adapted from Schulz-Mirbach et al.

Figure 3 :

 3 Figure 3: Interior face of a whole otolith from a channel flatfish showing light and dark zones constituting annuli in transmitted light.

Figure 4 :

 4 Figure 4: Aragonite unit cell based on structure determined by Caspi et al. (2005).

Figure 6 :

 6 Figure 6: Secondary electron images of modern Micropogonias otolith sections from a single individual under identical conditions before and after acid etching.Variation in structure must be revealed through etching.

Figure 7 :

 7 Figure 7: The centres of calcification change the orientation of the growth axis, producing a more complex form visible in a secondary electron image of a polished, etched, and gold-coated Myctophidae fossil otolith. (Courtesy E. Dufour, using Tescan)

Figure 8 :

 8 Figure 8: Images of the left otolith of a Micropogonias sp. recovered during an archaeological excavation related to the Paiján human group of Peru. The arrows represent the orientation of the fish.

  employed Sr:Ca and Ba:Ca profiles to identify an individual's occupancy of fresh-or salt water. Long et al. (2014) used a composite of Sr:Ca, Ba:Ca, and δ 18 O to detect water level fluctuations of a now-dry lake area which experienced periodic flooding. Understanding of fish movements and environment gained through geochemical analyses were then used for reconstruction of palaeoenvironment and palaeoecology

δ

  13 C remains more limited due to its more complex interpretation. It is often used alongside δ 18 O to highlight variations in metabolic activity and support interpretation of δ 18 O measurements. In their study of the extinct V. vulpes,Carpenter et al. (2003) interpret periodic depletions in δ 13 C as indicative of annual migrations or predation stress.1.5 reliability of otoliths: effects of post-mortem alterationThe reliability and permanence of the information encoded within otoliths is critical to their use as information archives. Evidence of post-mortem modifications of the biogenic elemental concentrations are provided by a number of studies carried out to quantify the effect of different observation, storage, or preparation methods used in the laboratory. The stability of the elemental and isotopic signals used for environmental reconstructions must remain stable for such reconstructions to be accurate, and indeed must remain so over thousands of years in the case of ancient samples.1.5.1 Potential alteration in modern otolithsIn most otolith studies for age estimation or chemical analysis, samples are prepared by embedding in resin, sectioning, and polishing.Jolivet et al. (2013) identified the transfer of resin from the surrounding block onto the surface of the otolith by this preparation.

  need for further investigation: questions addressed in the present work With growing use of ancient otoliths for palaeoenvironmental reconstruction, it becomes more important to determine the stability of mineral phase, and elemental composition and spatial distribution of otoliths in the face of alteration. Reliable reconstruction from ancient samples requires the ability to decouple the biogenic signal from potential post-mortem and post-depositional alterations.Detection of alteration via modification of expected elemental concentrations and/or spatial distributions, or the presence of exogenous elements -whether potentially biogenic or not -could be traced using a high lateral resolution, large-area, multielemental mapping approach. For ancient otoliths, alteration is highly likely to be heterogeneous throughout the otolith; an approach with good statistics over a wide area would decrease potential errors in analyses from point contamination. In the present work, semi-quantitative micro-X-ray fluorescence (µXRF) is used to fulfill these demands. The methodological requirements, implications, and limitations of µXRF are examined. With the arrival of nondestructive beam-based analyses, alignment of the temporal data from growth marks observed by microscopy and the geochemical life history profiles collected by one or more techniques can be difficult to correlate; a coordinate marking strategy has been elaborated that allows fast and precise alignment of large high-definition maps from different techniques. Although one macro-scale study has evidenced the random substitution of Sr for Ca in modern otoliths, the stability of this substitution at the microscale is yet untested. The present work provides an investigation into the microscale chemical environment of Sr in archaeological and modern otoliths across a wide range of Sr concentrations (two orders of magnitude) at many points across several samples. A fast speciation mapping approach is devised and implemented to comprehensively examine selected zones. The palaeo-environmental and diagenetic implications of alteration are discussed.

Figure 9 :

 9 Figure 9: Simplified phylogeny of the fish species considered in the present work.Approximately 96 % of all modern fish are teleosts, one-third of which are percifomes[START_REF] Nelson | Fishes of the World[END_REF].

Figure 10 :

 10 Figure 10: Maps of Peru and South America (inset) showing origin of archaeological and modern samples (red and green, respectively). Adapted from Central Intelligence Agency (2013).

  (a) Cupisnique Desert, reproduced from Béarez et al. (2011) with permission. (b) Huaca Prieta mound, courtesy E. Dufour.(c) Sechura Desert, courtesy N. Goepfert.

Figure 11 :

 11 Figure 11: Photos of archaeological sites where otoliths studied in this work were found.

Figure 12 :

 12 Figure 12: Sectioning planes of a sagitta from Micropogonias sp. recovered from archaeological exploration related to the Paiján human group (Sec. 2.2.1). The otolith is shown from the rear interior; arrows show the fish orientation relative to otolith.

Figure 13 :

 13 Figure13: Photographs of all mounted, polished otolith sections as analysed, grouped by species.All images are to the same scale with the inner face of the otolith oriented to the bottom of the page.

Figure 16 :

 16 Figure 16: Examples of previous works using SR-XRF for life history studies.

Figure 19 :

 19 Figure19: Excitation of a core electron by an X-ray photon: the effect is dependent on the photon's energy.

  references 1-6 therein). The energy is tuned using a monochromator to scan the desired energy range. A schematic double crystal monochromator is shown in Figure22. In a double crystal monochromator, a pair of crystals are set to an angle θ according to the Bragg relation 1 such that only a narrow band around the desired energy passes

  through the exit window. The incident photon flux is measured before the sample to determine I 0 . Two approaches to measurement of the sample absorption are transmission detection and fluorescence detection. Transmission detection places the detector behind the sample and directly measures the transmitted flux I 1 . Fluorescence detection occurs in front of the sample and does not directly measure the absorbed beam, but rather the emitted fluorescence of the element being probed. This positioning allows analysis of samples which are too thick or otherwise unsuitable for transmission mode.

Figure 23 :

 23 Figure 23: Experimental configuration on DiffAbs for µXRF. The incoming monochromatic X-ray beam is focussed onto the sample using Kickpatrick-Baez mirrors (Focusing optics). Incident flux (I 0 ) is measured using a thin film diode. The sample was mounted in air on a goniometer for positioning and scanning.

  heterogeneity of the standards. Reference measurements were recorded in transmission with nitrogen-filled ionisation chambers (200 V operating voltage) providing photon flux measurements before and after the sample (Fig. 21). These spectra were recorded with a count time of 3 s/point in three ranges: 15 902-16 030 eV in 2 eV steps, 16 032-16 132 eV in 1 eV steps, and 16 134-16 250 eV in 2 eV steps. µEXAFS spectra (Fig. 52) were recorded with a count time of 8 s/point for 16 023-16 083 eV in 2 eV energy steps, 16 084-16 224 eV in 1 eV steps, and 16 225-16 773 eV in 2 eV steps.

  of the edge jump and normalisation was performed using values in the pre-edge region, from 60-30 eV below the absorption edge (E 0 ), and in the postedge region at 16-670 eV after E 0 region for µEXAFS. As oscillations extend in the whole XANES region, normalisation of XANES spectra was performed by adjusting the normalisation range to align each spectrum with similar EXAFS spectra from visual comparison.Extraction of the EXAFS signal was performed as follows: (i) The data were normalised. (ii) Using ATHENA with E 0 initially approximated at the energy corresponding to half of the edge jump, the pre-edge matrix absorption was removed using a Victoreen function and postedge Sr atomic contribution was flattened using fitted splines.(iii) An arctangent function was fitted to the pre-edge region, defined as points of normalised intensity below 0.45 of the edge jump, and post-oscillation region, defined as E 16 300 eV. The E 0 determined from the fitted arctangent function was used for the rest of the treatment. (iv) Spectrum background was removed by fitting with splines over the full energy range after E 0 , unless noted differently. In the processing of EXAFS data, a k 2 -weighting was used to amplify the spectrum in the mid-to high-k range without excessively weighting the noise in the upper k range. (v) A Hanning apodization window from 1-9 Å -1 with a taper of 1 Å -1 was applied before applying a Fourier transform to retrieve the pseudo-radial distribution with phase correction from FEFF calculations on the first O shell.(vi)

  on a per-pixel basis. A model (Eqn. 4) was constructed based on the assumption that the Sr K-edge X-ray absorption spectrum at any given pixel would result from a linear combination of the signals from Sr in aragonite and strontianite. A linear energy-dependent term was included to correct for matrix-induced background variations at each pixel; a linear decrease was found a good approximation of the Victoreen in the restrained 52 eV post-edge energy range considered. In Equation 4, I Exp (E) represents the measured fluorescence intensity for one point at energy E. The model depends on the known fluorescence intensity values of the reference spectra I Arag (E) and I Stro (E) at the same energy E; the fractions of aragonite and strontianite, a and b, respectively; a scaling factor β is proportional to the Sr concentration at the pixel; and a linear approximation × E + δ for the differential post-edge slope, where and δ are unknown. This linear model leaves four unknowns (βa, βb, , and δ) with the minimum set of four experimental measurements (four energies) recorded to calculate them. The model assumes that the sample is a mixture of aragonite and strontianite only , so a + b = 1. Using this, β can be determined, and isolated values for a and b calculated.

Fig

  Fig.49).

Figure 24 :

 24 Figure 24: The two-step XRF process.

  6) energy E 0 ; C z , the mass fraction of the analyte element z in the sample; p z , the probability of a photon interacting with an atom of element z; ω z , the fluorescence yield of the excited shell of element z; ψ , the angle of incidence of the incoming photon flux I 0 ; Ω, the solid angle of the detector, γ(E F ), the detector efficiency at energy E F ; and µ * , the total linear attenuation coefficient(Eqn. 6). The total linear attenuation coefficient (Eqn. 6) comprises the linear attenuation coefficient of the incident photons (µ 0 ) with energy E 0 and their angle of incidence to the sample surface (ψ ), the linear attenuation coefficient of the characteristic fluorescence photons (µ F ) with energy E F , the angle of the detector relative to the sample surface (ψ ), and the sample thickness T . It is possible to increase the accuracy of µ * by including other attenuators, such as air. Accurate simulation of the XRF from a given sample requires precise knowledge of the matrix composition. This model does not account for secondary excitation, where one of the matrix elements is excited by absorbing the fluoresced photons. Modelling from fundamental parameters can, nonetheless be used to quantify the composition of a given sample, as inLimburg et al. (2007).This gure has been removed due to copyright restrictions.It can be found in the referenced text or in the full version of this work at the Université Paris-Sud library.

Figure 25 :

 25 Figure 25: The configuration to derive the quantitative XRF formula depictsan infinitesimal thickness (dx) within the sample (distance x). The incident X-ray beam (I 0 ) intersects the flat sample surface at an angle of (ψ ). The resulting characteristic photons (I F ) exit at an angle (ψ ) toward the detector with a given surface area (Ω). Adapted from[START_REF] Mantler | Quantitative analysis[END_REF].

  8 shows the correction used to obtain the real concentration (C Real ), using the PyMca calculated value (C Calc ). The correction is broken into three factors for clarity. The first factor corrects the incident intensity I 0 of the i th pixel to equal the mean over the entire sample (I Sample 0

) 3 . 5 . 3 Figure 26 :

 35326 Figure26: The FF-XRF setup employed a full-sample illumination with collimated monochromatic X-rays on the BAMLine. The sample was placed at a 45°angle to the beam and the detector. The FF-XRF detector employs glass capillaries to guide the characteristic photons coming below the angle of incidence onto the detector surface and reject those coming from off-axis, providing a laterally-resolved image.

Figure 27 :

 27 Figure27: A cutaway view of the scanning ion microprobe endstation at ATOMKI. All apparatus is mounted inside a vacuum chamber(a).The proton beam (b) arrives on the sample (c) which is mounted on a motorised stage for positioning (not shown). Two Si(Li) detectors(d, e) are used to measure the characteristic photons over energy ranges. A video microscope (f) is used to observe the position of the sample and a rotating beam chopper (g) is used to measure the beam current during the experiment.

  ring depends on the experimental configuration and the Bragg angle: for this work, using a 39 mm sample-detector distance, the first strong reflections (ca. 2θ=12°) have a radius of 4 mm on the X-ray hybrid Pixel Area Detector (XPAD) surface. A series of diffraction cones and therefore Debye rings is produced as a result of the many points in the single-crystal diffraction pattern. In a perfectly isotropic sample, a straight line from the centre of the Debye rings (incident beam) will produce a profile of reflection intensity, corresponding to the powder diffractogram of the crystal. In the case of a non-isotropic powder sample, in which the crystals are oriented in some way, the distribution of individual beams is no longer homogeneous, but A sample containing oriented crystals produces incomplete rings as the crystals fulfill the Bragg conditions only for certain portions of the rings.

Figure 29 :

 29 Figure 29: Illustration of diffraction cones produced by crystalline powder samples and the Debye rings where the cones intersect a plane (detector or film) as observed in powder X-ray diffraction.

Figure 30 :

 30 Figure30: Diagram of the XRD experimental setup; schematic is not to scale. The monochromatic X-ray beam is focused onto the sample in the horizontal and vertical axes (x and z, resp.) by Kirckpatrick-Baez mirrors. The sample was mounted with a 6°tilt in ω (around the x axis) in order to position the main reflection of interest to meet the Bragg condition. The sample surface normal was rotated 25°in φ to face the fluorescence detector while minimising the increase in the thickness of sample contributing to XRD images. The XRD detector was positioned at 300 mm downstream of the sample and raised 12.5°in δ (twice the ω rotation so as the center of the beam is in a θ-2θ configuration) to remove the detector from the transmitted beam axis. This configuration provided a detection range on the XRD detector of over 20°in 2θ and up to 110°in ψ, as seen in Fig.31. The XRF detector was mounted with distance d Fluo =39 mm at 90°to the beam path in the xy plane to minimise Compton scattering.

Figure 31 :

 31 Figure 31: The process of transformation of raw XPAD image data to Cartesian (2θ, ψ) coordinates. The raw image (a) is corrected for the physical layout of the detector, including gaps between neighbouring chips (b), then transformed into Cartesian (2θ, ψ) coordinates (c) for direct integration.

Figure 32 :

 32 Figure 32: Observation of tungsten marks used for accurate sample repositioning on the polished thin section of archaeological otolith QCD. (a) Transmitted light image of a frontal thin section of archaeological otolith QCD with opaque and translucent marks of annual increments visible from the core (*) to the edge along the growth axis. FIB mark positions are indicated with arrowheads. The SR-µXRF analysis area in Fig. 33 is denoted by the dotted grey box. (b) Observation of a FIB mark in reflected light microscopy. (c) ROI integration map of the W Kα SR-µXRF signal on the same sector as in (a), recorded with hν=16 200 eV, count time 1 s/pixel, and step size of 4 × 2 µm 2 (V×H). (d) FIB secondary electron image of a second mark. Image has been corrected for geometric distortion due to coincidence angle. (e) The comparison of average XRF spectra from the white rectangles printed in (b) shows the presence of W as well as gallium used in deposition (zone A in c), while these signals are completely absent 30 µm away (zone B). Each spectrum results from averaging data on four adjacent pixels.

Figure 33 :

 33 Figures 33-38 present the quantified SR-µXRF maps, recorded with incident energy of 16 200-17 200 eV, count time 1 s/pixel, and effective on-sample FWHM spot size of 6 × 15 µm 2 (V×H) for the 6 analysed otoliths. In the maps, the otolith core is always oriented to the top with the outer edge and embedding resin visible at the bottom. In the Cathorops sp. and some G. peruvianus samples, resin can be seen at the top of the map as well. The size of the maps varied with the otolith, from (3000-4250)×(400-1000) µm 2 (V×H), with total acquisition time varying consequently. The largest map required 8 h.

FeFigure 35 :

 35 Figure 35: Elemental maps of modern M. manni sample 424 obtained from SR-µXRF mapping (hν=17 200 eV, 1 s/pixel). All concentrations in µmol • g -1 .The core is not visible, but the symmetry axis of the otolith is indicated by the line of . K, Mn, Br, Ba, and Hg were not detected. See text for explanation of marks.

Figure 36 :Figure 37 :Figure 38 :

 363738 Figure 36: Elemental maps of archaeological G. peruvianus otolith HP-12 obtained from SR-µXRF mapping (hν=16 200 eV, 1 s/pixel). All concentrations in µmol • g -1 . Core is marked with . Cu and Hg were not detected. Y was not excited by the incident beam. See text for explanation of marks.

  , and Ba are detected within the otolith only. Like the Micropogonias sp. specimens, Sr exhibits strong, well-resolved fluctuations. Rb and Ca also show significant but more poorlyresolved fluctuations, while Br shows a very weak fluctuation. Sr fluctuations show a general trend of decreasing concentration range and increasing frequency from the core to the edge. The maximum Sr concentration, attained midway through the sample at 1.65 mm (S3), is 101 µmol • g -1 . This is the same position as the Rb maximum and the Ca minimum. The minimum Sr concentration of 30 µmol • g -1 is attained at the outer edge. Mn, Fe, and Zn are significantly present in the resin, with low levels of Mn and Zn also in the otolith. The edge (E) is very uneven in the Ca and K maps, resulting from crystals removed during preparation. Ablation from EPMA (A) traces a vertical line across the sample which is depleted in Ca and Rb, and enhanced in K. Cracks (C3) are visible near the core in the K, Ca, and Sr maps.A total of 9 elements were detected in GP-292 (Fig.37). Like HP-12, Ca, Sr, and Ba were restricted to the otolith. Sr shows strong fluctuations in both G. peruvianus otoliths ranging from 20-55 µmol • g -1 . Rb shows relatively significant fluctuations, while Ca is largely homogeneous within the otolith. GP-292 exhibits a narrow Sr concentration spike (S4) similar to that in HP-12. Loss of crystals at the edge (E) is much more significant in these samples than HP-12, and shows variations in Ca and Sr concentrations at the edge and some variation between the apparent edge in the Ca and Sr maps.Fe, Zn, Mn, and Pb are primarily found in the resin with low levels present in the otolith, or near-zero in the case of Fe. The elements in the resin also show low levels within the otolith near the edges. A slight depletion of Ca near the core of GP-292 is present. BH17 (Fig.38), an archaeological Cathorops sp. specimen, exhibits 9 elements detected. Sr concentrations are within the ranges previously observed (25-57 µmol • g -1 ). Compared to other lapilli, the borders remain intact and the outer edges (E) are smooth. There is no difference in apparent edge observed between low and high Z elements. Higher concentrations of Fe and Zn can be seen near top and bottom borders, respectively. The resin contains detectable levels of Fe, Cu, Zn, and Y.

  Figure 40 shows the ROI integrated XRF signal for calcium and strontium on a secondary sample of otolith 424 (thin section L4). The XRF signals of Ca and Sr are observed within the otolith and not outside. They are recorded within an XRF 'snapshot' of the whole sample without the need for raster-scanning. There is a faint pattern of circles visible in the images resulting from the polycapillary optics. This variation

Figure 40 :

 40 Figure 40: Calcium (a) and strontium (b) elemental images of otolith 424L4 collected using synchrotron full-field X-ray fluorescence with a colour camera. A slight contrast is observed between the centre of the otolith and surroundings in the strontium map. The images have been corrected for geometric aberrations.

  Fig. 41) to maximise lateral resolution along the growth axis and definition between sequential growth increments. (A) In the horizontal, a 16 200 eV incident beam at 45°to the sample surface has an attenuation length of 237 µm in pure aragonite. The incoming beam can thus be assumed to excite a volume of sample to this length. This is appreciably more than the attenuation length of the fluoresced Ca or Sr Kα rays (see Tab. 9), constraining the information volume to the escape length of signal. Corresponding simulated XRF information volumes are shown in Fig. 41 for both Sr and Ca. The information volume is elongated and asymmetrical, with a slight majority occurring on the side toward the detector. This results from a slight difference in attenuation between the side of the volume closer to the detector compared to the opposite side. For Sr, 50% of the signal comes from the first 161 µm of sample depth, producing an averaged value across this distance. For Ca, 50% of the signal results from the topmost 30 µm. With our 45°incidence, the information volume is approximately as wide as it is deep, thus producing a mixed signal of this entire volume. Definition of a generic information depth is not possible -the information volume depends upon the nature of the sample, and where one defines the cutoff.

Figure 41 :

 41 Figure 41: Simulated XRF information volume for Sr Kα and Ca Kα lines in a pure aragonite matrix with a 16 200 eV incident beam. The incident beam intersects the surface at 45°and has transverse FWHM dimensions of 11 × 6 µm 2 (X×Y). The grey plane represents the sample surface, the line representing the incident beam penetrates up to its 1/e attenuation length, and a point detector is situated 30 mm away, perpendicular to the incident beam, in the direction indicated by the blue line. For each X-ray emission line, the three shapes represent the associated 25% (blue), 50% (red), and 75% (green) XRF information volumes.

Fig. 33 )Figure 42 :

 3342 Fig. 33) and near the outer edges of the G. peruvianus specimens (HP-12, GP-292; Figs 36 and 37). Localised enrichment or depletion in Sr are observed along the growth axis in QCD, 424, HP-12, and GP-292. Inter-individual comparison of maps can be difficult whereas life history profiles provide convenient comparison between individuals and interpretation of life history chronology. Here, we will propose two approaches for treatment of the large data set which underline the complexity of the measured Sr signal, with the perspective of examining its reliability through time.

  Figs 33-38, by highlighting their distribution. HP-12 exhibits a wide distribution without an obvious primary mode, ranging from 21-101 µmol • g -1 , although 91% of values fall from 35-70 µmol • g -1 . The high Sr region (Fig. 42 inset) contains an excursion to 78 µmol • g -1 and a small population above 80 µmol • g -1 . This range of values exceeds the majority of that compiled from literature data for fishes from different origins (typical max. 51 µmol • g -1 , few examples reaching 145 µmol • g -1 )

  Figure 43: [Sr] maps (top) and median profiles (bottom) for archaeological G. peruvianus sample HP-12 (a) and Micropogonias sp. sample QCD (b). The median is plotted in red and the minimum and maximum values of the integrated pixels are plotted in grey. The saturated area in the Sr maps designate the integration zones 11 and 13 pixels wide, respectively, used to produce the profiles. Selected areas of the median Sr profiles (red) are magnified in the insets with the data for all integrated pixels in grey.

  yields highly reliable data sets to which statistical approaches can be applied. The distribution of Sr concentrations facilitates intercomparison of palaeo-environmental conditions and modes of life. Including a representation of the data variability along an integrated profile facilitates detection and identification of potential post-mortem alteration and local artefacts, including from sample preparation and previous analyses. Limited contamination from sample preparation could be detected in some samples. No definitive evidence for diagenetic alteration was identified from the Sr signal or those of other elements. The random substitution of Sr for

5C

  H E M I C A L E N V I R O N M E N T O F S T R O N T I U M A N D I M P L I C AT I O N F O R D I A G E N E S I S S T U D I E S 5.1 Chapter introduction . . . . . . . . . . . . . . . . . . 111 5.2 Samples examined in this chapter . . . . . . . . . . 114 5.3 X-ray absorption spectroscopy characterisation of strontium in otoliths . . . . . . . . . . . . . . . . . . 119 5.3.1 Strontium environment analysis by point measurements . . . . . . . . . . . . . . . . . . 119 5.3.2 µXANES mapping of chemical environment 127 5.4 Contribution and open questions regarding the diagenesis of otoliths . . . . . . . . . . . . . . . . . . 128 5.4.1 Micrometric point µXAS analyses ubiquitously indicates Sr in aragonite . . . . . . . . 131 5.4.2 Chemical environment of strontium from the micro-to millimetre scale . . . . . . . . . 139 5.4.3 Post-mortem alteration of Peruvian archaeological otoliths . . . . . . . . . . . . . . . . . 143

  , development of microscale raster-scanning approaches with chemical contrast may allow monitoring of localisation of contamination by other elements. This work examines the chemical environment of strontium in the aragonite of ancient otoliths at the microscale as a possible approach to detect post-mortem alterations and determine the permanence of the geochemical signal. A microscale examination of the Sr chemical environment was performed on five archaeological and three modern otoliths from the Pacific coast of South America exhibiting a wide range of Sr concentrations from 7.1-94 µmol • g -1 .

Figure 44 :Figure 45 :Figure 46 :

 444546 Figure 44: Secondary electron images of HP-11 (G. peruvianus, Huaca Prieta). Courtesy E. Dufour.

  following features, where energies are referred to as a shift relative to E 0 (ie. E 0 + n eV): (i) no evident pre-edge features; (ii) a white line with several inflection points observable in the spectrum derivative (Fig.49); (iii) a white line maximum at +7.3 eV (16 112 eV), (iv) a low intensity absorption feature at ca. +20.8 eV, (v) oscillations with a first significant maximum at +43.4 eV preceded by a shoulder at ca. +30 eV, (vi) a shoulder at +65 eV, and (vii) a third somewhat sharp maximum at +94.1 eV. All features are shared by QCD.

Figure 47 :Figure 48 :

 4748 Figure 47: SR-µXRF Sr distribution maps of archaeological samples of Micropogonias sp. (QCD, Pampa de los fósiles, Peru) and G. peruvianus (HP-11 and HP-12). ♦ marks points analysed by µXANES, except QCD-D and HP-11-C which are µEXAFS analysis points. Otolith cores are marked with *.

Figure 49 :Figure 50 :Figure 51 :

 495051 Figure 49: First derivatives of XANES spectra in Fig. 50 illustrating the multiple inflection points observed.

Figure 52 :

 52 Figure 52: Magnitude and imaginary part of the phase-corrected Fourier transforms of the Sr K-edge µEXAFS (beam size FWHM 6 × 16 µm 2 ) of archaeological Micropogonias sp. otolith QCD (Pampa de los fósiles, Peru, [Sr]=36.2 µmol • g -1 ) compared to those of inorganic reference materials aragonite and strontianite, and the fitted spectrum. The FT of QCD is typical for all otolith samples.

Fig. 53e .

 53e Fig.53e. The map exhibits a high homogeneity [standard deviation(SD) of 0.072 in a unimodal distribution] across the full mapped area, centered on 91 % aragonite (Fig.53f). The SD along the growth axis (0.075) -in the direction of Sr variations -is comparable to the SD along a growth increment (0.072), and the median integral covers a range of ±8 %.The Ca concentration map (Fig.53c) shows limited variation aside from an enhancement at the outer edge. The Sr concentration map (Fig.53d) reveals a total range of 19-39 µmol • g -1 in the mapped zone, with a maximum value close to the edge. The maximum at the sample edge (Fig.53g"Max") corresponds to the maximum observed on the entire otolith, while the minimum (Fig.53g, "Min") is only a local minimum with lower concentrations elsewhere along the growth axis (Fig.47). Some substructure can be seen in the transition between high and low Sr areas (Fig.53g, "SS"). The aragonite fraction shows no relation to variation in Sr content. Figures 53 to 63 represent all fitted values for the linear system in Equation 4 and those resulting from calculation. The first four values

Figure 53 :Figure 53 (

 5353 Figure 53: Continued on page 131.

Figure 54 :Figure 55 :

 5455 Figure 54: Map (left) and histogram (right) of bβ

Figure 56 :Figure 57 :Figure 59 :Figure 60 :Figure 61 :Figure 62 :Figure 64 :

 56575960616264 Figure 56: Map (left) and histogram (right) of δ

  , the imaginary part of the FT is more sensitive to a change in neighbouring element than the magnitude. The magnitude will reveal fine changes in radial distribution, but examination of the imaginary part permits a sensitive detection of phase shifts resulting from the exchange of the neighbouring atom Ca 2+ for Sr 2+ even associated to limited change in interatomic distances. Four species from two families for a total of eight individuals and two different types of otoliths (sagittae and lapilli) were studied here and Sr in an aragonitic environment was ubiquitously identified.Focusing first on modern samples, our results complement the previously-cited recent study that identified the random substitution of Sr 2+ for Ca 2+ in otoliths from ten modern individuals belonging to seven species from Australia, using wide-beam EXAFS (ca. 0.3 × 0.3 mm 2 )(Doubleday et al., 2014). With the addition of our results, the random substitution of Sr 2+ for Ca 2+ in the otolith aragonite is now supported in a total of 6 teleost families for which either the sagittae or the lapilli are the most developed otoliths. The environments of the species studied inDoubleday et al. (2014) ranged from freshwater to hypersaline estuarine. Our corpus of samples brings a broader diversity in fish ecology, as we have examined coastal marine species that can enter freshwater systems such as estuaries and lagoons. This diversity in ecology and our methodology (quantification of Sr concentrations with SR-µXRF coupled to XAS measurements) allowed a simultaneous extension of the conditions of Sr speciation to a Sr content range of 7.1-94 µmol • g -1 (620-8200 µg • g -1 ). This strongly indicates random substitution of Sr for Ca, independent of concentration across a wide concentration range at both intra-and inter-individual scales in this extended range of fish families.

5. 4 . 2

 42 Chemical environment of strontium from the micro-to millimetre scale Series of µXAS points can provide information on Sr chemical environment at multiple locations across a sample. However, the overall representativity of microscale point analyses may be lower than that at millimetre scale because selection of the points could have, intentionally or not, avoided local singularities. In order to confirm the representativity of point analyses and trace potential high resolution spatial variations, the multi-energy µXANES mapping employed in this work examined a selected zone of 0.26 mm 2 of the archaeological sample QCD (Micropogonias sp.). The universal presence of Sr permitted microscale analysis at multiple points and at varying Sr concentrations. The application of the linear model (Eqn. 4) produces a map of the sample composition, based on the assumption that each Sr K-edge XANES spectrum results from the linear combination of contributions from strontianite and aragonite domains. If this assumption was incorrect, the model is likely to produce incoherent results. In particular, significant variations in the values of β, , or δ may constitute such an indication. The β factor reflects the scaling for Sr concentration and all other fitted values are related to the concentration. The spatial distribution of β values (Fig. 57),

Figure 65 :

 65 Figure 65: 'Time' profiles for Sr and Ca concentrations in archaeological samples from the Pacific coast of Peru: QCD (Micropogonias sp., Pampa de los fósiles, n=13, a) and HP-12 (G. peruvianus, Huaca Prieta, n=11, b), and modern reference sample GP-292 (G. peruvianus, n=11, c). Profiles were obtained by integrating bands of n pixels perpendicular to the growth axis on splines defined visually according to Sr fluctuations to conform to the otolith's curvature. * marks the presence of physical cracks.

  values in specimens QCD, Bayovar-1C and BH-17 fall within the range reported in otoliths of modern fishes, a significant positive deviation is observed on two specimens of G. peruvianus) that both exceed this usual range as well as that observed for the modern counterpart studied here (GP-292)(Figs. 47 and 65). Significant increase in Sr content was identified in the outer hundreds of micrometers of several archaeological and modern otoliths. They could result from post-mortem alteration. Unambiguous attribution to sample handling and preparation, early or long-term diagenesis is difficult. Microscale Sr speciation was researched as a potential disambiguating approach.The present study supports that the chemical environment of Sr in the archaeological specimens is identical to that of modern otoliths independent of the element concentration, location in the otolith and species studied. There is no evidence of extra-crystalline Sr, presence in interstitial spaces, or discrete strontianite crystals. The large Sr content excursions observed in two Peruvian otoliths are not linked to a change in the mode of incorporation of Sr. A question that remains to be assessed is if this stability of the Sr chemical environment -exclusively in random substitution for Ca -guarantees the preservation of the original Sr content and accurate reconstructions based on µXRF mapping of our specimens.

Figure 67 :

 67 Figure 67: XPAD images after geometric correction and transformation to Cartesian (2θ, ψ) coordinates show the visible space in a single image. Diffraction images from three points on the otolith QCD (1, 2, 3) display variation in the position of maximum intensity on ψ depending on position within the otolith. For example, the 111 reflection (marked with an arrow) exhibits a maximum intensity near 15°in ψ at point 1, 50°at point 2, and -40°at point 3. This indicates not only non-isotropic distribution of crystalline units, but also a correlation between orientation and position on the sample. 2θ is given in real angle as recorded at 17.2 keV.

200 + 102

 102 because it is the sum of these two peaks, which are unresolved because they have very close d-spacing (d 102 =2.485 17 Å, d 002 =2.480 85 Å, Caspi et al., 2005). The calculated relative intensities are 175/1000 for the 200 reflection and 265/1000 for the 102 reflection.

  , the intensity of the 021 reflection varies along the growth axis. Between the two arrows, regions of various reflection intensities can be discerned (Fig 70c, f): one region of slightly heterogeneous high intensity (1), one of homogeneous low-medium intensity (2), one of heterogeneous high-medium intensity[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF], and one of heterogeneous low intensity (4). These regions appear to be correlated with varying optical and elemental properties as well.

Figure 68 :

 68 Figure 68: Maps of reflection intensities in sample QCD for 012 (left, red), 021 (centre left, green), and 112 (centre right, blue) planes integrated over 10°intervals of ψ with a superposition of all three reflections ("Total", right). A light microscopy image (LM, lower right) displays the centre of the otolith marked with * and an arrow indicating a visible discontinuity in the growth marks.In mounting the sample, it was assumed that the sample is essentially planar, with the c axis running in the plane between the centre and the edge. Arrangement around the c axis was assumed to be anisotropic. Note the apparent "sweep" of each given reflection around the centre of the otolith with varying ψ. The discontinuity visible in the LM image also appears in the orientation maps as an orientation distinct from the overall tendency of regular variation in ψ.

Figure 69 :

 69 Figure 69: Maps of reflection intensities in sample QCD for 002 (left, red), 020 (centre left, green), and 200 + 102 (centre right, blue) planes integrated over 10°intervals of ψ with a superposition of all three reflections ("Total", right). A light microscopy image (LM, lower right) displays the centre of the otolith marked with * and an arrow indicating a visible discontinuity in the growth marks.

Figure 70 :

 70 Figure 70: XRF and XRD signals were measured simultaneously, illustrated here for modern otolith section 424. The transmitted light image (a) can be compared to the XRF map of strontium content (b) and the intensity of the 021 reflection (c). A detail view of the map's lower left quadrant (d, e, f) better reveals the appearance of the annuli. Annotations 1-4 are described in the text (Sec. 6.2.1).

Figure 71 :

 71 Figure 71: Localisation and identification of isolated pixels bearing non-aragonitic phases on otolith QCD. The locations of aragonite peaks are marked by solid vertical lines and those of calcite are marked by dashed lines. The pixels exhibiting reflections not accounted for by aragonite were identified and the diffractogram was plotted for the most intense pixel at the selected 2θ(as indicated in each plot). Inset intensity maps of the selected reflection highlight the sources of non-aragonite peaks (calcite or unknown), which are all contained to single pixels. The non-aragonitic pixels in the two top plots are calcite; those in the bottom pair are unidentified.

Figure 72 :

 72 Figure 72: Intensity of the 111 Reflection of otolith 424 showing spotty appearance (arrows) in different zones of the section.

For a single

  reflection (eg. 012, Fig. 68), the reflection intensity varies with position on the sample and ψ. Different regions of the sample fulfill the Bragg conditions at different ψ, revealing the highly oriented nature of the otolith biomineral structure. The apparent rotation of these regions about the nucleus suggests a stacked radial arrangement of crystals. However, within the regions of high reflection intensity, there are inhomogeneities which indicate that the crystals are not all homogeneously organised.

  Fig. 73. There are 12 % of pixels with negative Δ FWHM , which does not have physical significance in the proposed model, which were excluded. The negative values are spread throughout the map, with a higher concentration in the lower right quadrant. This concentration may be linked to the discontinuity in orientation seen in Figs. 68 and 69. The median Δ FWHM of all non-negative points on sample QCD is ca. 0.15°; applying the model of Suzuki et al. (2012), this corresponds to a twinning rate of 6 %.

Figure 73 :

 73 Figure 73: A map of the twinning rate in QCD based on the model from Suzuki et al. (2012) using Δ 012-111 to estimate twinning. 12 % of pixels (Plotted in black) had negative Δ 012-111 and were excluded. The twinning rate is lowest near the nucleus and toward the exterior region of the section (top of the map).Twinning rate varies both along the growth axis and within isochronous increments. A lower twinning rate near the nucleus is counter to what may be expected, as this region exhibits faster growth and more narrow growth increments, which would be expected to imply more frequent twinning.

Suzuki

  et al.(2012) employed a benchtop powder diffractometer with 2θ resolution of ca. 0.04°and instrument broadening of 0.081°at 2θ=28.47°. In the present work the experiment was optimised for a broad coveragein (2θ, ψ) rather than high resolution determination of peak shapes. The focused beam had a divergence of ca. 0.1°in the vertical axis as a result of the KB mirrors, and each pixel of the XPAD as mounted covered ca. 0.025°, both of which contribute to instrumental broadening. As noted in Sec. 6.3.2.3, instrument broadening was not quantified, nor was ultimate 2θ resolution sought in this work, but the experiment configuration could be easily improved. With an experimental setup optimised for this purpose and a sufficient sample corpus, it may be possible in the future to use twinning to detect, for example, defects or periods of stress in the fish's life, alteration of archaeological samples, or individual growth rate.6.3.4 Toward rapid crystal orientation measurementRapid recording of diffraction data opens new avenues for materials analyses. In this work, crystal orientation mapping was carried out with a synchrotron microbeam coupled to a dual-detector system comprised of a pixel area detector and an XRF detector. The area detector collected a large range in 2θ and ψ (ca. 50°and 100°, respectively), thus permitting analyses of crystal orientation from single frames. The use of pixel area detectors in crystallography is becoming increasingly common and permits a faster collection throughput than classic point or strip detectors. By examining a significant portion of the (2θ, ψ) space in a single exposure, it is possible to rapidly assess crystallite orientation. In this work, the measurement is close to permitting full texture reconstruction using the appropriate reflections. Gaps between detector chips in the XPAD (visible as the grids Fig.67) produce dead zones of ca. 1°in 2θ, but in this work it was demonstrated that orientation can nonetheless be accurately determined. Determination of the orientation of at least three (preferably orthogonal) reflection planes in principle permits computation of full texture, although the geometrical transformations are not straightforward.Orientation mapping with a similar approach has been previously demonstrated with a slower acquisition rate and lower lateral resolution, where it was used to observe the crystal orientation on sections of teeth[START_REF] Al-Jawad | 2d mapping of texture and lattice parameters of dental enamel[END_REF]. Exposure time in the present work was 1000 ms/pixel, requiring ca. 4 h for a map of 160×76 pixels. In the step-scan mode currently employed, it is not realistically possible to further accelerate the acquisition due to physical limitations of motor acceleration. The development of flyscan continuous scanning relieves this constraint, and a similar map has recently been acquired in 20 min. Continued development has decreased the acquisition time to under 5 min per diffraction image. At such high acquisition rates, a full pole figure map, consisting of ca. 20 diffraction images for each pixel could be acquired in an hour to permit full texture mapping of centimetre-scale samples with micrometric lateral resolution.The simultaneous XRF collection provides elemental information with no additional cost in experimental time. In the present work, the XRF was not quantified, but quantitative measurements are possible with appropriate calibration and data treatment(See Ch. 4).

Figure 74 :Figure 75 :

 7475 Figure 74: Orientation of the maximum intensity in ψ of 002, 020, and 200+102 (top, middle, and bottom, resp.) in QCD represented by the sticks illustrates the highly oriented nature of the otolith. The ratio of the maximum intensity to the background is represented by the stick colour. A minimum I Max /I Background ratio of 1.25 was used to ensure only pixels with sufficient signal were examined. The bottom 200+102 plot is a shows both similar-intensity reflections that cannot be resolved. The intensity curves of the 002 (top) reflection in ψ have been extracted between the points marked by arrows and plotted in Fig. 75.

7. 3

 3 (2012), indicating an average twinning rate of 6 %. The ability to map crystal twinning could prove invaluable for studies of biominerals and engineered materials. With the further upcoming developments of continuous-scan collection regimes, analyses such as those in this work promise to drop from not just hours to minutes, enabling thorough crystallographic examination on large areas with even micrometric lateral resolution. D I S C U S S I O N , C O N C L U S I O N S , A N D P E R S P E C T I V E S 7.1 Evaluation of potential alteration . . . . . . . . . . . 170 7.2 Observations on otolith biomineralisation . . . . . . 173 Methodological contributions . . . . . . . . . . . . . 174 7.4 Perspectives . . . . . . . . . . . . . . . . . . . . . . . 177The elemental and stable isotopic composition of fish otoliths are widely used as environmental proxies for estimation of an individual's living conditions. The incremental temporallyresolved structure of otoliths, often compared to the rings in a tree trunk, gives them great utility as life history archives capable of recording an individual's movements over lifespans of a few years to decades. The conditions experienced within and around an individual -physiology, ambient temperature (including seasonality), and ambient water chemistry (salinity, chemical, and/or isotopic composition) -are reflected by the elemental and stable isotopic composition of the otolith, with variations in these recorded in successive increments. In particular, strontium is a commonlyused environmental proxy as it reflects the environmental chemistry experienced by the individual and is the most easily-accessed proxying element with modern analytical techniques. Ancient otoliths are being used with increasing frequency as palaeoproxies to provide understanding of the environment, ecology, and biology of ancient fish, which can in turn be used to better understand the practices of the ancient humans who caught them. With increasing use of ancient otoliths, it is important to understand the potential alterations which may occur, and the elemental markers thereof. This work aimed to provide new methodologies for the analysis of otoliths, particularly for the study of ancient samples, while further investigating the fidelity of the information encoded within, and testing unverified assumptions. In total, sections of six archaeological and five modern otoliths of fish in two families (Sciaenidae and Ariidae) from the Pacific coast of South America were examined.

7. 1

 1 evaluation of the potential alteration of chemical composition in ancient otoliths used as palaeoproxies temporal fluctuations of strontium The SR-µXRF maps in the present work reveal regular fluctuations of Sr concentration along the growth axis for all specimens, as is expected for fish which have experienced several years of seasonal cycles. These fluctuations occur over length scales on the order of 100 µm. More rapid fluctuations on the order of 20 µm are also visible, with variations on single pixels -a few micrometres -detectable thanks to the high contrast afforded by µXRF. The length scale of these short fluctuations is at or below the lateral resolution afforded by currentlycommon techniques for the elemental analysis of otoliths: routinely, a few tens of micrometres in LA-ICP-MS, and a minimum interaction volume diameter of 7.4 µm for EPMA at 30 kV. The observation of fluctuations in the present work and the homogeneity within isochronous layers is a positive indicator for the good preservation and good reliability of the chemistry of the samples studied.

  have shown to be possible in the presence of water), post-mortem alteration may also have occurred in the elements (e.g.Sr, Mn, Ba) whose concentrations are used as palaeoenvironmental proxies. The concentration of these contaminants was observed to decrease toward the otolith core, typically reaching undetectable levels after a few hundred micrometres. This may be used as an indicator of reliability: the otolith elemental composition is unaltered in the uncontaminated zones. The resins used in the present work were free of Sr, selected based on best practices at the time of preparation. However, the resin was observed to contain detectable levels of Mn, Fe, Zn, and Cu, and may have played a role in their presence, so it is not clear whether the elements detected in the present work are exclusively a result of intrusion, or if the spread of resin during sample preparation as shown by Jolivet et al. (2013) is a factor. While it is not possible to conclude at present, this is a positive indicator for the capacity to detect potential alteration. Further attention to the selection of materials used in preparation and handling of samples to avoid elements such as Mn, Fe, Cu, and Zn could permit the use of these elements as indicators of alteration in future imaging work. strontium chemical environment The chemical environment of Sr 2+ in five archaeological and three modern fish otoliths has been examined on the microscale using µXANES and µEXAFS. Elemental mapping by synchrotron µXRF permitted a precise positioning of analyses on the otolith sections in relation to the subannual fluctuations of Sr. Multi-energy µXANES mapping was implemented to study the speciation homogeneity over a 480 × 550 µm 2 area. The chemical environment was confirmed in all cases as Sr 2+

  of the crystal lattice by fast X-ray diffraction. The maps of crystal orientation obtained on one modern and one archaeological otolith illustrate the well-known nucleus-oriented nature of otolith growth. While orientation in most cases varies smoothly about the nucleus, some areas exhibit sudden changes in orientation which are not represented in the literature and may be relevant in the study of otolith growth. The nanogranule-based growth of otoliths is wellsupported by the experiments in this work. Using the Scherrer equation, an upper limit of ca. 200 nm can be placed on individual domains, coherent with observations by atomic force microscopy

  many hundreds or thousands of data points it becomes possible to apply statistical approaches, such as the kernel density plots used in the present work, for quantitative discrimination of life history regimes. For example, otolith 424 exhibits an essentially bimodal distribution, consistent with the hypothesis that the individual had limited exposure to varying water chemistry, with seasonality being the primary source of Sr fluctuations. In contrast, otolith HP-12 exhibits a wide, multimodal distribution, with excursions to very high Sr concentrations, suggesting the individual lived in a variety of brackish to saltwater environments, and experienced some exceptional environmental or physiological event.fast xanes mappingThe fast XANES mapping in the present work provides a valuable approach to distinguish fluctuations in chemical environment over small areas without needing to record a full spectrum for each point. The maps presented in this work, covering 0.25 mm 2 with a resolution of 6 × 10 µm 2 required several hours of recording time, instead of several hundred, by reducing the data needed to four energies and using a multivariate model to quantify the composition.crystal mappingThe present work demonstrates the first application of large-area fast XRD mapping on the DiffAbs beamline.

Using 16 200

 16 eV X-rays to probe the Sr Kα signal produces 75 % of the information from a volume of 37 011 µm 3 , penetrating 259 µm into the sample. Using tender 2228 eV X-rays to probe the Sr Lα signal results in a volume of just 70 µm 3 and a penetration of

Figure 78 :

 78 Figure 78: A map of Sr concentration in an interior zone of otolith 424 collected on LUCIA using tender X-rays. A 2228 eV beam with 3 × 3 µm 2 on-sample spot size was used with 1.5 × 1.5 µm 2 steps.The growth axis is in the horizontal from right to left. Indications of aragonite microfibres parallel to the growth axis are visible through inhomogeneities within the isochronous increments (along the vertical here).

Figure 79 :

 79 Figure 79: Comparison of the information volume. Medium-energy X-rays (volumes 1 and 2) produce a relatively large information volume in the sample, integrating tens of thousands of cubic micrometres. Tender Xrays (volumes 3 and 4) produce a much smaller volume, on the order of tens or hundreds of cubic micrometres. Note that volumes 3 and 4 use different excitation energies in order to excite both the Sr L-and Ca Klines with good efficiency (Sec. 3.5). The line indicating each incident beam ends within the sample at the incident beam's attenuation length (1/e of initial photon flux).

Figure 1 :

 1 Figure1: Experimental setup on the DiffAbs beamline. The incoming monochromatic X-ray beam (intensity I 0 ) was focused onto the sample surface. The sample was mounted on a goniometer in air at an angle φ=45°to the incident beam, producing an effective spot size of 6.0 × 16 µm 2 (y ×x) projected on the sample surface. Samples were mounted with the growth axis parallel to the y-axis to maximise the lateral resolution across isochronous increments. A Vortex SII monoelement silicon drift fluorescence detector (Hitachi) was placed in the same y plane at 90°to the incident beam to minimise X-ray scattering contribution. When using the linearly horizontal plane polarized X-ray beam, Compton scattering is eliminated, in theory, at this angular position. The sample-detector distance was optimised for each sample to maximise the detected signal but remain within the detector's range of linear response.

  Archaeological Micropogonias sp. otolithQCD (hν=16 200 eV). Otolith core is marked with .

  Archaeological Micropogonias sp. sample Bayovar-1C (hν=17 200 eV). Otolith core is marked with . Modern M. manni sample 424 (hν=17 200 eV). Otolith core is not visible, but the symmetry axis of the otolith is indicated by the line of .

Figure 2 :

 2 Figure 2: Elemental maps of Micropogonias sp. specimens obtained with SR-µXRF mapping. All measurements were recorded for 1 s/pixel (real time); incident energy used is noted per sample. All concentrations in µmol • g -1 . Annotations are described in text (Section 3.1).

  Archaeological Cathorops sp. sample BH17 (hν=17 200 eV).

Figure 3 :

 3 Figure 3: Elemental maps of Ariidae specimens obtained from SR-µXRF mapping. All measurements were recorded for 1 s/pixel (real time); incident energy used is noted per sample. All concentrations in µmol • g -1 . Otolith cores are marked with . Annotations are described in text (Section 3.1).

Figure 4 :

 4 Figure 4: Observation of tungsten marks used for accurate sample repositioning on the polished thin section of archaeological otolith QCD. (a) Transmitted light image of a frontal thin section of archaeological otolith QCD with opaque and translucent marks of annual increments visible from the core (*) to the edge along the growth axis. FIB mark positions are indicated with arrowheads. The SR-µXRF analysis area in Fig. 2a is denoted by the dotted grey box. (b) Observation of a FIB mark in reflected light microscopy. (c) FIB secondary electron image of a second mark. Image has been corrected for geometric distortion due to coincidence angle. (d) ROI integration map of the W Kα SR-µXRF signal on the same sector as in (a), recorded with hν=16 200 eV, count time 1 s/pixel, and step size of 4 × 2 µm 2 (V×H). (e) The comparison of average XRF spectra from the white rectangles printed in (b) shows the presence of W as well as gallium used in deposition (zone A in d), while these signals are completely absent 30 µm away (zone B). Each spectrum results from averaging data on four adjacent pixels.

Figure 5 :

 5 Figure5: Simulated XRF information volume for Sr Kα and Ca Kα lines in a pure aragonite matrix with a 16 200 eV incident beam under the geometry describe in Fig.1. The incident beam intersects the surface at 45°and has transverse FWHM dimensions of 11 × 6 µm 2 (X×Y). The shaded plane represents the sample surface, the purple line representing the incident beam penetrates up to its 1/e attenuation length, and a point detector is situated 30 mm away, perpendicular to the incident beam, in the direction indicated by the teal line. For each X-ray emission line, the three shapes (blue, orange, and green) represent the 25 %, 50 %, and 75 % XRF information volumes.

Figure 6 :

 6 Figure 6: Spectra from incident energies above and below the Sr Kedge (16 105 eV) on otolith Bayovar1-C. The Rb Kα signal is present independent of excitation of Sr.

Figure 7 :

 7 Figure7: The potential effect of variations in organic fraction is shown for Sr and Ca. Calculated concentrations are given on the outer Y-axis labels while change relative to the maximum (0 % organic) is shown on the inner labels. Molecular formula and sample density were varied in the PyMca fit setup to reflect changing matrix composition. The matrix characteristics of the organic fraction were approximated from otolin-1 (M=52 138 g • mol -1 ,Murayama et al., 2002; ρ=1.43 g • cm -3 , Quillin and Matthews, 2000). Even a change from one extreme to the other in the 0-10 % range is insufficient to account for the Sr variation in the most homogeneous otolith examined here (Otolith 424, 14-22 µmol • g -1 i.e. a variation of 36 %).

Figure 8 :

 8 Figure 8: Kernel density plots of Sr concentrations in the maps of archaeological otoliths QCD and HP-12, and modern sample 424 determined from SR-µXRF. The number of pixels used for calculation is indicated by n in legend. The bandwidth of the Gaussian distribution function is 0.25 µmol • g -1 for all three samples. Inset: Zoom on high Sr content range for HP-12.

  Figure 9: [Sr] maps (top) and median profiles (bottom) for archaeological G. peruvianus sample HP-12 (a) and Micropogonias sp. sample QCD (b). The median is plotted in red and the minimum and maximum values of the integrated pixels are plotted in grey. The saturated area in the Sr maps designate the integration zones 11 and 13 pixels wide, respectively, used to produce the profiles. Selected areas of the median Sr profiles (red) are magnified in the insets with the data for all integrated pixels in grey.

  and 16 201 eV), with the map at 16 112 eV excluded because of the observation of significant variability of the white line shape. In Equation 1, I Exp (E) represents the measured fluorescence intensity for one point at energy E, while I Arag (E) and I Stro (E) represent the values of the reference spectra at the same energy. The remaining parameters are unknown. a and b are the fractions of aragonite and stron-tianite, respectively, multiplied by the scaling factor β which is proportional to the Sr concentration at the pixel. × E + δ is a linear approximation for the differential post-edge slope, where and δ must be determined. This linear model leaves four unknowns (βa, βb, , and δ) with four experimental measurements to calculate them. Assuming that the sample is a mixture of aragonite and strontianite only (ie. a + b = 1), β can be determined and isolated values for a and b calculated.

Figure 1 :

 1 Figure 1: SR-µXRF Sr distribution maps of archaeological samples of Micropogonias sp. (QCD, Pampa de los fósiles, Peru) and G. peruvianus (HP-11 and HP-12). ♦ marks points analysed by µXANES, except QCD-D and HP-11-C which are µEXAFS analysis points. Otolith cores are marked with *.

Figure 2 :

 2 Figure 2: Sr K-edge macro-XANES spectra of archaeological Micropogonias sp. sample QCD (Pampa de los fósiles, Peru) compared to inorganic aragonite and strontianite. Spectral features (i-vii) are described in the text (Sec. 3.1).The vertical lines mark selected characteristic energies used for µXANES mapping.

Figure 3 :

 3 Figure3: Sr K-edge µXANES spectra of 8 otoliths from four species at three archaeological site and three modern locations compared to that of Sr in the reference of inorganic aragonite. Spectra have been shifted vertically for easier intercomparison. The Sr concentration at the point of analysis in µmol • g -1 is indicated next to each sample name.

Figure 4 :

 4 Figure 4: Variation of Sr K-edge µXANES spectra at varying Sr concentrations on the archaeological otoliths QCD (Micropogonias sp., Pampa de los fósiles, Peru), Huaca Prieta, Peru). The Sr content determined by SR-µXRF is marked in µmol • g -1 above each curve. Spectra have been shifted for easier intercomparison.

Figure 5 :

 5 Figure 5: Magnitude and imaginary part of the phase-corrected Fourier transforms of the Sr K-edge µEXAFS (beam size FWHM 6 × 16 µm 2 ) of archaeological Micropogonias sp. otolith QCD (Pampa de los fósiles, Peru, [Sr]=36.2 µmol • g -1 ) compared to those of inorganic reference materials aragonite and strontianite, and the fitted spectrum. The FT of QCD is typical for all otolith samples.

Figure 6 :

 6 Figure 6: Assessment of the homogeneity of the speciation of strontium in a zone from the archaeological Micropogonias sp. otolith QCD section (Pampa de los fósiles, Peru). (a) LM image of sample QCD with positions of analyses indicated. (b) Magnified LM image of the analysed zone. The dashed line indicates the otolith edge. (c,d) Concentration maps of Ca in mmol • g -1 (c) and Sr in µmol • g -1 (d). (e) Map of the fraction of aragonite a in each pixel. (f) Histogram of aragonite fraction a. The pixels outside of the otolith (black in map c) were excluded from the histogram. The values for pure strontianite and pure aragonite are marked with vertical lines. (g) Profiles along the growth axis integrated along isochronous increments for elemental concentrations (Ca, Sr) and aragonite fraction a: median (solid line), range (shaded areas). Arrows indicate features in the [Sr] curve (see text).

  Figure 7: (a) Otoliths present a multiscalar organisation. Annual growth marks in the tens to hundreds of micrometers are visible across the otolith, composed of daily increments containing prismatic microcrystals aligned along the growth axis. The microcrystals exhibit further organisation on the scale of tens of nanometres(Dauphin and Dufour, 2008) but the present work is not sensitive to that level. (b) Within this ordered structure, five main modes of incorporation for strontium are envisaged: (1) incorporated into the organic matter, (2) adsorbed onto the surface of aragonite, incorporated within the aragonite lattice[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] in substitution for Ca or (4) in interstitial spaces, or (5) as discrete strontianite domains.

  Figure 8: 'Time' profiles for Sr and Ca concentrations in archaeological samples from the Pacific coast of Peru: QCD (Micropogonias sp., Pampa de los fósiles, n=13, a) and HP-12 (G. peruvianus, Huaca Prieta, n=11, b), and modern reference sample GP-292 (G. peruvianus, n=11, c). Profiles were obtained by integrating bands of n pixels perpendicular to the growth axis on splines defined visually according to Sr fluctuations to conform to the otolith's curvature. * marks the presence of physical cracks.

Figure S4 :

 S4 Figure S4: First derivatives of XANES spectra in Fig. 3 illustrating the multiple inflection points observed.

  56e-02 Med: 4.25e-02 SD: 1.08e-02

Figure S5 :Figure S11 :

 S5S11 Figure S5: Map (left) and histogram (right) of aβ

  Figure S12: Map (left) and histogram (right) of normalised to β

Figure S13 :

 S13 Figure S13: Map (left) and histogram (right) of δ normalised to β

  strontianite and aragonite pellets, as well as the background resulting from the cellulose used as binder and the polyethylene sample support. Both aragonite and strontianite match reference values. The strontianite signal has been divided by 10 to compensate for the 10 s/point recording time, compared to 1 s/point for other samples. diffractograms of pure strontianite powder, cellulose used as binder/diluent, and two pellets with different concentrations of strontianite. The strontianite remained intact through two stages of grinding to produce the 0.5 % pellet.

F

  ' R ' C O D E P R O D U C E D F O R M O D E L L I N G A N D D ATA T R E AT M E N T Listing 1 A function to calculate the intensity of detected fluorescence signal from a sample in 3 dimensions273 Listing 2 A function to find the iso-information level by recursive elimination of half the candidates . . . . 275 Listing 3 Calculation of the volume enclosed by an isosurface . . . . . . . . . . . . . . . . . . . . . . . 276 Listing 4 Calculation of the multivariate fit of few-energy XANES measurements . . . . . . . . . . . . . . . . 277 Listing 5 An algorithm to interactively, graphically define a series of "master curves" on an elemental map, define a series of curves between each pair of master curves, and extract the data from each pixel on the series of curves. . . . . . . . . . . . . . 278 # C a l c u l a t i o n s assume a l l u n i t s a r e i n micrometres # The d e t e c t o r i s assumed t o be a p o i n t d e t e c t o r c a p a b l e o f d e t e c t i n g a l l photons from t h e sample e q u a l l y , r e g a r d l e s s o f angle ( i e . no shade , c o l l a r , e t c . ) # # V a r i a b l e s ## # x , y , z a r e v e c t o r s r e p r e s e n t i n g t h e dimensions o f t h e sample , with t h e s u r f a c e i n t h e xy plane and sample depth i n z

3 , 3 ) 3 :#

 333 Loop though z s l i c e s f o r ( i i n 1 : l e n g t h ( z ) ) { # C a l c u l a t e t h e d e t e c t o r d i s t a n c e i n 3D d e t e c t o r D i s t a n c e <-distance3D( x , y , z [ i ] , detectorCoord [ 1 ] , detectorCoord [ 2 ] , detectorCoord[START_REF] Cochran | Effect of diagenesis on the Sr, O, and C isotope composition of late Cretaceous mollusks from the Western Interior Seaway of North America[END_REF] ) # C a l c u l a t e t h e d i s t a n c e t r a v e l l e d i n t h e atmosphere between t h e sample s u r f a c e and d e t e c t o r distAtmo <-abs( detectorCoord [ 3 ] ) / abs( detectorCoord [3] z [ i ] ) * d e t e c t o r D i s t a n c e [ , , 1 ] #The t o t a l d i s t a n c e minus atmospheric d i s t a n c e i s t h e insample t r a v e l distSample <-d e t e c t o r D i s t a n c e [ , , 1 ] -distAtmo # C a l c u l a t e t h e f l u o r e s c e n c e s i g n a l f l u o S i g n a l [ , , i ] <-I 0 * # Incoming beam o u t e r ( # Outer product o f v e c t o r f u n c t i o n s r e p r e s e n t i n g beam shape i n X and Y t o produce a matrix dnorm ( x=x , mean=z [ i ] * tan ( t h e t a * p i _ sur _ 1 8 0 ) +beamX , sd=sigmaX/cos ( t h e t a * p i _ sur _ 1 8 0 ) ) , # Gaussian on X dnorm ( x=y , mean=z [ i ] * tan ( phi * p i _ sur _ 1 8 0 ) +beamY , sd= sigmaY/cos ( phi * p i _ sur _ 1 8 0 ) ) # Gaussian on Y ) * # Multiply by a t t e n u a t i o n i n d i f f e r e n t media exp ( -( z [ i ] /cos ( t h e t a * p i _ sur _ 1 8 0 ) /cos ( phi * p i _ sur _ 1 8 0 ) /L1 ) ) * # A t t e n u a t i o n o f incoming beam exp(-distSample/L2 ) * # A t t e n u a t i o n o f photons e s c a p i n g sample exp(-distAtmo/L3 ) # A t t e n u a t i o n o f photons i n atmosphere s e t T x t P r o g r e s s B a r ( progBar , i ) } #End o f loop # Return t h e computed cube o f d e t e c t e d s i g n a l r e t u r n ( f l u o S i g n a l ) } Listing 1: A function to calculate the intensity of detected fluorescence signal from a sample in 3 dimensions # ## A f u n c t i o n t o f i n d t h e i s o -i n f o r m a t i o n l e v e l by r e c u r s i v e e l i m i n a t i o n o f h a l f t h e c a n d i d a t e s # # V a r i a b l e s # x i s t h e s e t o f v a l u e s t o be examined # f r a c t i o n i s t h e i n f o r m a t i o n f r a c t i o n d e s i r e d # p r e s o r t e d i s TRUE i f t h e v a l u e s i n x a r e a l r e a d y s o r t e d i n descending order f i n d I s o I n f o L e v e l <-f u n c t i o n ( x , f r a c t i o n = 0 . 5 , p r e s o r t e d =F , s i l e n t =T ) { i f ( ! p r e s o r t e d ) x <-s o r t ( x , d e c r e a s i n g =T ) # S o r t x i f i t i s not a l r e a d y s o r t e d t o t a l <-sum ( x ) # C a l c u l a t e t h e t o t a l o f a l v a l u e s i n x # I n i t i a l i s e t h e v a r i a b l e s t o check xmin <-1 xmax <-l e n g t h ( x ) #Loop u n t i l we converge on one value while ( xmin ! = xmax ) { # C a l c u l a t e t h e midpoint f o r t h e range t o check xmid <-round ( ( xmax-xmin ) / 2 ) + xmin i f ( ! s i l e n t ) c a t ( xmin , " -" , xmid , " -" , xmax , " \n " ) #Check where t h e c o r r e c t value l i e s i f ( sum ( x [ 1 : xmid ] ) / t o t a l == f r a c t i o n ) { # I f t h e xmid i s e x a c t l y t h e r i g h t p o i n t r e t u r n ( x [ xmid ] ) } e l s e i f ( sum ( x [ 1 : xmid ] ) / t o t a l > f r a c t i o n ) { # I f t h e sum up t o xmid i s g r e a t e r than t h e d e s i r e d f r a c t i o n xmax <-xmid # The p o i n t s above xmax a r e t h e wrong ones } e l s e { # Else , t h e sum up t o xmid i s too s m a l l and we need t o check t h e h i g h e r v a l u e s xmin <-xmid + 1 } } r e t u r n ( x [ xmid ] ) } Listing 2: A function to find the iso-information level by recursive elimination of half the candidates isosurfVolume <-f u n c t i o n ( t h e i s o s u r f a c e ) { # Define an " o r i g i n p o i n t " t o which a l l volumes w i l l be r e f e r e n c e d # Chose a p o i n t i n t h e middle o f where t h e beam i n t e r s e c t s t h e sample d <-c ( median ( t h e i s o s u r f a c e [ which ( t h e i s o s u r f a c e [ , 3 ] == 0 ) , 1 ] ) , #x median ( t h e i s o s u r f a c e [ which ( t h e i s o s u r f a c e [ , 3 ] == 0 ) , 0 # I n i t i a l i s e a sum t o 0 #Loop through each s e t o f t h r e e p o i n t s f o r ( i i n 0 : ( dim ( t h e i s o s u r f a c e ) [ 1 ] /3 -1) ) { # 3 c o n s e c u t i v e rows i n s u r f r e p r e s e n t t h e 3 p o i n t s d e f i n i n g one t r i a n g l e o f t h e i s o s u r f a c e # Load a s e t o f p o i n t s from s u r f , apply t h e s h i f t t o move them i n t o p o s i t i v e space , and s u b t r a c t d t o produce a s e t o f v e c t o r s A <-t h e i s o s u r f a c e [ ( 1 + 3 * i ) , ] B <-t h e i s o s u r f a c e [ ( 2 + 3 * i ) , ] C <-t h e i s o s u r f a c e [ ( 3 + 3 * i ) , ] volumeSum <-volumeSum + det ( a r r a y ( data=c (A-d , B-d , C-d ) , dim=c ( Calculation of the volume enclosed by an isosurface #Load t h e r e f e r e n c e s p e c t r a . . . This b l o c k l o a d s f l a t t e n e d , normalised XANES s p e c t r a { r e f d a t a <-as . matrix ( read . t a b l e ( " r e f e r e n c e s . norm " ) ) arag <-s p l i n e f u n ( x= r e f d a t a [ , 1 ] , y= r e f d a t a [ , 2 ] ) s t r o <-s p l i n e f u n ( x= r e f d a t a [ , 1 ] , y= r e f d a t a [ , 3 ] ) } # P o s i t i o n s o f p i x e l s i n t h e map x <-seq ( from =0 , by=getSpa ( XRFMaps$ x16112eV $map) [ 1 ] , l e n g t h . out=getNx ( XRFMaps$ x16112eV $map) ) y <-seq ( from =0 , by=getSpa ( XRFMaps$ x16112eV $map) [ 2 ] , l e n g t h . out=getNy ( XRFMaps$ x16112eV $map) ) # Get t h e e n e r g i e s and c a l c u l a t e ( i n t e r p o l a t e ) r e f e r e n c e v a l u Experimental v a l u e s I _Exp <-a r r a y ( dim=c ( l e n g t h ( x ) , l e n g t h ( y ) , l e n g t h ( E ) ) ) I _Exp [ , , 1 ] <-getData ( XRFMaps$ x16149eV $map) I _Exp [ , , 2 ] <-getData ( XRFMaps$ x16170eV $map) I _Exp [ , , 3 ] <-getData ( XRFMaps$ x16185eV $map) I _Exp [ , , 4 ] <-getData ( XRFMaps$ x16201eV $map) r e s u l t <-a r r a y ( dim=c ( 4 6 , 1 1 5 , 4 ) ) pbar <-t x t P r o g r e s s B a r ( min=1 ,max=46) # C a l c u l a t e a f i t f o r each p i x e l and save t h e c o e f f i c i e n t s f o r ( i i n 1 : 4 6 ) { f o r ( j i n 1 : 1 1 5 ) { model <-lm ( I _Exp [ i , j , ] ~I _Ar + I _ S t + E ) r e s u l t [ i , j , ] <-model [ [ " c o e f f i c i e n t s " ] ] } s e t T x t P r o g r e s s B a r ( pbar , i ) } I _ 0 <-r e s u l t [ , , 1 ] alpha <-r e s u l t [ , , 4 ] aBeta <-r e s u l t [ , , 2 ] bBeta <-r e s u l t [ , , 3 ] Beta <-aBeta+bBeta a <-aBeta/Beta b <-bBeta/Beta Listing 4: Calculation of the multivariate fit of few-energy XANES measurements #Load an hdXRF map load ( 'HP12_ massFrac . Rdata ' ) # Define/ r e i n i t i a l i s e b a s i c v a r i a b l e s { c u r v e B u i l d e r S t a t u s <-l i s t ( " c o n t i n u e " = T , " nCurves " = 0 , " xlim " = c ( 0 , dim ( hdXRF$umol_g ) [ 2 ] ) , " ylim " = c ( 0 , dim ( hdXRF$umol_g ) [ 2 ] ) , " xdim " = dim ( hdXRF$umol_g ) [ 2 ] , " ydim " = dim ( hdXRF$umol_g ) [ 1 ] , " redraw " = F ) p i c k e d P o i n t s <-l i s t ( ) pickedCurves <-l i s t ( ) } #Draw t h e f i r s t segment o f t h e map img ( x = 1 : dim ( hdXRF$umol_g ) [ 1 ] , y = 1 : dim ( hdXRF$umol_g ) [ 2 ] , z =(hdXRF$umol_g [ , , which ( hdXRF$ m a s s F r a c F i t s == " Sr L3 " ) ] ) , xlim= c u r v e B u i l d e r S t a t u s $ ylim , ylim= c u r v e B u i l d e r S t a t u s $ xlim , c o l = c o l o r P a l e t t e ( 1 0 0 ) ,

  while ( c u r v e B u i l d e r S t a t u s $ c o n t i n u e ) { #Redraw only i f needed i f ( c u r v e B u i l d e r S t a t u s $ redraw ) { img ( x = 1 : dim ( hdXRF$umol_g ) [ 1 ] , y = 1 : dim ( hdXRF$umol_g ) [ 2 ] , z =(hdXRF$umol_g [ , , which ( hdXRF$ m a s s F r a c F i t s == " Sr L3 " ) ] ) , xlim= c u r v e B u i l d e r S t a t u s $ ylim , ylim= c u r v e B u i l d e r S t a t u s $ xlim , c o l = c o l o r P a l e t t e ( 1 0 0 ) ,

  ( " S e l e c t 3 p o i n t s t o d e f i n e an increment\n " ) c u r v e B u i l d e r S t a t u s $ nCurves <-c u r v e B u i l d e r S t a t u s $ nCurves+1 p i c k e d P o i n t s [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] <-l o c a t o r( 3 , t = ' o ' ) # C r e a t e a s p l i n e f u n c t i o n using t h e s e l e c t e d p o i n t s , swap X and Y because o f r o t a t i o n curveFun <-s p l i n e f u n ( x = p i c k e d P o i n t s [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] $y , y = p i c k e d P o i n t s [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] $x ) # C a l c u l a t e curves , oversampling by 2 pickedCurves [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] <-l i s t ( " x " = seq ( from =1 , t o = c u r v e B u i l d e r S t a t u s $xdim , by = 0 . 5 ) , " y " = curveFun ( seq ( from =1 , t o = c u r v e B u i l d e r S t a t u s $xdim , by = 0 . 5 ) ) ) #Draw t h e curve on t h e image , draw a t h i c k b l a c k l i n e covered by a t h i n white one t o ensure v i s i b i l i t y l i n e s ( x=pickedCurves [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] $y , y=pickedCurves [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] $x , c o l = " b l a c k " , lwd=2 ) l i n e s ( x=pickedCurves [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] $y , y=pickedCurves [ [ c u r v e B u i l d e r S t a t u s $ nCurves ] ] $x , c o l = " white " , lwd=1 ) c u r v e B u i l d e r S t a t u s <-getNextAction ( c u r v e B u i l d e r S t a t u s ) # , s i l e n t =F ) } x r f P r o f i l e <-l i s t ( " p i c k e d P o i n t s " = p i c k e d P o i n t s , " masterCurves " = pickedCurves ) # ### Now b u i l d t h e p o i n t e r a r r a y s and add them t o t h e x r f P r o f i l e l i s t { # These w i l l r e p r e s e n t p o i n t e r s t o s i n g l e i n c r e m e n t s as determined by f o l l o w i n g a l i n e a r combination o f two master curves # Using p o i n t e r s w i l l allow l a t e r e x t e n s i o n ( eg . t o a combination o f t h e p i x e l s surrounding a given p o i n t ) # S t a r t by determining t h e space b e f o r e and a f t e r t h e master curves on each end o f t h e image #We w i l l use t h i s t o extend t h e f i r s t / l a s t curves u n t i l t h e image ends # Get t h e f i r s t determined Y value f i r s t Y <-min ( x r f P r o f i l e $ masterCurves [ [ 1 ] ] $y ) # I t i s p o s s i b l e t o s e l e c t p o i n t s o u t s i d e o f t h e image . In c a s e t h i s has happened , s h i f t t h e whole curve u n t i l t h e f i r s t Y w i l l round t o 1 t o avoid t r y i n g t o a c c e s s n o n e x i s t e n t p i x e l s while (f i r s t Y < 0 . 5 ) { x r f P r o f i l e $ masterCurves [ [ 1 ] ] $y <-x r f P r o f i l e $ masterCurves [ [ 1 ] ] $y + 0 . 5 f i r s t Y <-min ( x r f P r o f i l e $ masterCurves [ [ 1 ] ] $y ) } #Same p r o c e s s f o r t h e l a s t Y value l a s t Y <-max ( t a i l ( x r f P r o f i l e $ masterCurves , n=1) [ [ 1 ] ] [ [ " y " ] ] )# Ensure t h a t t h e l a s t l i n e w i l l round t o t h e l e n g t h o f t h e data a r r a y while ( l a s t Y > l e n g t h ( hdXRF$ t r a j e c t o r y _x + 0 . 5) ) { x r f P r o f i l e $ masterCurves [ [ l e n g t h ( x r f P r o f i l e $ masterCurves ) ] ] [ [ " y " ] ] <-t a i l ( x r f P r o f i l e $ masterCurves , n=1) [ [ 1 ] ] [ [ " y " ] ] -0 . 5 l a s t Y <-max ( t a i l ( x r f P r o f i l e $ masterCurves , n=1) [ [ 1 ] ] [ [ " y " ] ] ) } # S t a r t a t t h e f i r s t Y p i x e l y P o s i t i o n <-min ( 1 , f i r s t Y ) # I d e n t i f y which index o f t h e X a x i s we should use t o f o l l o w t h e a c t u a l Y p o s i t i o n yMonitor <-which ( x r f P r o f i l e $ masterCurves [ [ 1 ] ] $y == f i r s t Y ) # S t or e t h e nominal Y p i x e l s where we have e x t r a c t e d i n c r e m e n t s yAxisInPx <-c ( ) # Arrays t o hold t h e p o i n t e r s t o X and Y p o s i t i o n s # F i r s t dimension w i l l be p o s i t i o n along Y # Second dimension w i l l be p o s i t i o n along X p o i n t e r s X <-a r r a y ( ) p o i n t e r s Y <-a r r a y ( ) # The s i z e o f s t e p s f o r sampling -read from t h e X a x i s t o have i d e n t i c a l X and Y sampling s t e p s sampleStep <-d i f f ( head ( x r f P r of i l e $ masterCurves [ [ 1 ] ] [ [ " x " ] ] ,n=2) ) # S t a r t with t h e out-of-sample zone , i f i t e x i s t s i f ( y P o s i t i o n < round ( f i r s t Y ) ) { # C r e a t e a sequence o f Y s t e p s from t h e s t a r t i n g t o ending Ys seqY <-seq ( from =1 , t o =round (f i r s t Y ) , by=sampleStep ) yAxisInPx <-seqY f o r ( i i n 1 : l e n g t h ( seqY ) ) { p o i n t e r s X <-cbind ( pointersX , x r f P r o f i l e $ masterCurves [ [ 1 ] ] [ [ " x " ] ] ) p o i n t e r s Y <-cbind ( pointersY , x r f P r o f i l e $ masterCurves [ [ 1 ] ] [ [ " y " ] ]f i r s t Y + seqY [ i ] )} } e l s e { # I f t h e f i r s t master curve i s r i g h t a t t h e beginning o f t h e map , save i t d i r e c t l y # c a t ( " No pre-zone " ) p o i n t e r s X <-x r f P r o f i l e $masterCurves [ [ 1 ] ] [ [ " x " ] ] p o i n t e r s Y <-x r f P r o f i l e $ masterCurves [ [ 1 ] ] [ [ " y " ] ]} #A p r o g r e s s bar progBar <-t x t P r o g r e s s B a r ( min=1 , max=( l e n g t h ( x r f P r o f i l e $ masterCurves ) -1) , i n i t i a l =1)#Ready t o s t a r t looping through a l l t h e d e f i n e d master curves f o r ( curveIndex i n 1 : ( l e n g t h ( x r f P r o f i l e $ masterCurves ) -1) ) { s e t T x t P r o g r e s s B a r ( progBar , curveIndex ) # Get t h e Y v a l u e s f o r t h e two curves curve1 <-x r f P r o f i l e $ masterCurves [ [ curveIndex ] ] [ [ " y " ] ] curve2 <-x r f P r o f i l e $ masterCurves [ [ curveIndex + 1 ] ] [ [ " y " ] ] # C r e a t e a sequence o f Y a x i s p o i n t s t o use f o r t h e r e f e r e n c e p o s i t i o n seqY <-seq ( from=curve1 [ yMonitor ]+ sampleStep , t o =curve2 [ yMonitor ] , by=sampleStep ) yAxisInPx <-c ( yAxisInPx , seqY ) f o r ( i i n 1 : l e n g t h ( seqY ) ) { # For now , X p o i n t e r s a r e always t h e same p o i n t e r s X <-cbind ( pointersX , x r f P r o f i l e $ masterCurves [ [ 1 ] ] [ [ " x " ] ] ) # c r e a t e a l i n e a r combination o f t h e two curves and s t o r e i t p o i n t e r s Y <-cbind ( pointersY , ( 1-i / l e n g t h ( seqY ) ) * curve1 + ( i / l e n g t h ( seqY ) ) * curve2 ) } } #We have f i n i s h e d a l l but t h e post-sample s e c t i o n #We need t o s h i f t our monitoring p o i n t t o t h e h i g h e s t Y-value i n t h e l a s t master curve so i t w i l l st o p a t t h e edge o f t h e data yEndMonitor <-which ( t a i l ( x r f P r o f i l e $ masterCurves , n=1) [ [ 1 ] ] $ y == l a s t Y ) #How much w i l l be added t o t h e master curve p o s i t i o n s yExtend <-sampleStep while ( round ( ( t a i l ( x r f P r o f i l e $ masterCurves , n=1) [ [ 1 ] ] $y + yExtend ) [ yEndMonitor ] ) <= l e n g t h ( hdXRF$ t r a j e c t o r y _x ) ) { #Add a p o i n t t o t h e Y a x i s p o s i t o n t r a c k e r . Use t h e max value s i n c e we don ' t have t h e same p o s i t i o n r e f e r e n c e anymore , and we ' r e extending by a f i x e d sampleStep amount yAxisInPx <-c ( yAxisInPx , max ( yAxisInPx ) +sampleStep ) #Add t h e X p o s i t i o n s p o i n t e r s X <-cbind ( pointersX , x r f P r o f i l e $ masterCurves [ [ 1 ] ] [ [ " x " ] ] ) #Add t h e next Y curve , s h i f t e d by yExtend p o i n t e r s Y <-cbind ( pointersY , t a i l ( x r f P r o f i l e $ masterCurves , n=1) [ [ 1 ] ] [ [ " y " ] ] +yExtend ) # I n c r e a s e yExtend f o r t h e next i t e r a t i o n ( or t o break out ) yExtend <-yExtend + sampleStep } #Trim t h e f i r s t column , which i s j u s t NA a r t i f a c t s from t h e i n i t i a l dimensioning o f t h e a r r a y s p o i n t e r s X <-p o i n t e r s X [ , 2 : dim ( p o i n t e r s X ) [ 2 ] ] p o i n t e r s Y <-p o i n t e r s Y [ , 2 : dim ( p o i n t e r s Y ) [ 2 ] ] #Add t h e p o i n t e r s t o t h e x r f P r o f i l e data l i s t x r f P r o f i l e [ [ " p o i n t e r Y " ] ] <-p o i n t e r s X x r f P r o f i l e [ [ " p o i n t e r X " ] ] <-p o i n t e r s Y #Add t h e Y a x i s by c r e a t i n g a f u n c t i o n t o i n t e r p o l a t e Y p o s i t i o n s a t p a r t i a l p i x e l s . . Keep i n mind t h e X and Y axes a r e swapped due t o image( ) being a s i l l y p l a c e x r f P r o f i l e [ [ " yAxisReal " ] ] <-s p l i n e f u n ( 1 : l e n g t h ( hdXRF$ t r a j e c t o r y _x ) , hdXRF$ t r a j e c t o r y _x ) ( yAxisInPx ) x r f P r o f i l e [ [ " y A x i s P i x e l s " ] ] <-yAxisInPx } #Draw t h e e x t r a c t i o n l i n e s on t h e map t o check them { img ( x = 1 : dim ( hdXRF$umol_g ) [ 1 ] , y = 1 : dim ( hdXRF$umol_g ) [ 2 ] , z =(hdXRF$umol_g [ , , which ( hdXRF$ m a s s F r a c F i t s == " Sr L3 " ) ] ) , c o l = c o l o r P a l e t t e B ( 1 0 0 ) , zlim= q u a n t i l e ( hdXRF$umol_g [ , , which ( hdXRF$ m a s s F r a c F i t s == " Sr L3 " ) ] , c ( 0 . 0 1 , 0 . 9 9 ) , na . rm=T ) ) f o r ( i i n seq ( from =1 , t o =dim ( p o i n t e r s X ) [ 2 ] , by =5) ) { l i n e s ( x= p o i n t e r s Y [ , i n a l l y , c r e a t e a data cube c o n t a i n i n g t h e r e a l data as d e s c r i b e d by t h e p o i n t e r s theData <-a r r a y ( dim=c ( dim ( p o i n t e r s X ) , dim ( hdXRF [ [ " umol_g " ] ] ) [ 3 ] ) ) maskCube <-o u t e r ( hdXRF [ [ " mask " ] ] , a r r a y ( 1 , 2 4 ) ) progBar <-t x t P r o g r e s s B a r ( min=1 , max=dim ( p o i n t e r s X ) [ 1 ] , i n i t i a l =1) f o r ( i i n 1 : dim ( p o i n t e r s X ) [ 1 ] ) { s e t T x t P r o g r e s s B a r ( progBar , i ) f o r ( j i n 1 : dim ( p o i n t e r s X ) [ 2 ] ) { theData [ i , j , ] <-hdXRF [ [ " umol_g " ] ] [ round ( p o i n t e r s Y [ i , j ] ) , round ( p o i n t e r s X [ i , j ] ) , ] * hdXRF [ [ " mask " ] ] [ round ( p o i n t e r s Y [ i , j ] ) , round ( p o i n t e r s X [ i , j ] ) ] } } x r f P r o f i l e [ [ " umol_g " ] ] <-theData x r f P r o f i l e [ [ " mean " ] ] <-apply

  Cook. xi Work for this project was carried out on the IPANEMA European research platform on ancient materials (USR 3461 CNRS, MCC) in close collaboration with the Muséum nationale d'Histoire naturelle Archéozoologie, archéobotanique (UMR 7209 CNRS, MNHN), and was supported financially by Synchrotron SOLEIL. Beamtime for experiments was provided by Synchrotron SOLEIL and the Transnational Access to Research Infrastructures CHARISMA project. "Ear stones": Anatomy, function, and growth . . . . . . . 1.2 Organisation of an otolith from atoms to a macroscopic object . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Aragonite: the mineral in biomineral . . . . . . . . 1.2.1.1 Crystal structure of aragonite . . . . . . . 1.2.1.2 Crystal twinning . . . . . . . . . . . . . . . 1.2.1.3 Incorporation of elements by solid solution 1.2.2 Vaterite and calcite can be observed in otoliths . . 1.2.3 Biomineral architecture . . . . . . . . . . . . . . . . 1.3 Geochemistry of otoliths . . . . . . . . . . . . . . . . . . . Otoliths as information archives . . . . . . . . . . . . . . . Alteration in archaeological contexts . . . . . . . . 1.6 A need for further investigation: Questions addressed in the present work . . . . . . . . . . . . . . . . . . . . . . Rationale of sample selection . . . . . . . . . . . . . . . . . 2.2 Description of selected otolith samples . . . . . . . . . . . 2.2.1 Archaeological interest and geographical context . 2.2.2 Archaeological otoliths . . . . . . . . . . . . . . . . 2.2.2.1 Paiján . . . . . . . . . . . . . . . . . . . . . 2.2.2.2 Huaca Prieta . . . . . . . . . . . . . . . . . 2.2.2.3 Bayovar . . . . . . . . . . . . . . . . . . . . 2.2.3 Modern otoliths . . . . . . . . . . . . . . . . . . . . 2.2.4 Sample preparation . . . . . . . . . . . . . . . . . .

	C O N T E N T S
	Résumé	v
	Abstract	vii
	List of publications	viii
	Acknowledgements	xi
	List of Figures	xvi
	List of Tables	xix
	0 introduction
	1 otoliths: life history archives
	1.1 2 samples and sample preparation
	2.1
	xii

1.3.1 Elemental composition and controls . . . . . . . . . 1.3.2 Stable isotopic composition and controls . . . . . . 1.4 1.4.1 Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2 Age and growth . . . . . . . . . . . . . . . . . . . . 1.4.3 Environment and physiology . . . . . . . . . . . . . 1.4.4 Otoliths as archives for palaeontology and archaeology . . . . . . . . . . . . . . . . . . . . . . . 1.5 Reliability of otoliths: Effects of post-mortem alteration . 1.5.1 Potential alteration in modern otoliths . . . . . . . 1.5.2

Table 10 Characteristics

 10

	xix

of the samples examined in this chapter . . . . . . . . . . . . . . . . . . . . . . . . . Table 11 EXAFS fitting results . . . . . . . . . . . . . . . . .

  .2 Organisation of an otolith from atoms to a macroscopic object . . . . . . . . . . . . . . . . . . . Vaterite and calcite can be observed in otoliths 1.2.3 Biomineral architecture . . . . . . . . . . . .

	1.2.1 Aragonite: the mineral in biomineral . . . .
	1.2.2

1.3 Geochemistry of otoliths . . . . . . . . . . . . . . . . 1.3.1 Elemental composition and controls . . . . . 1.3.2 Stable isotopic composition and controls . .

  [START_REF] Bevan | On the structure of aragonite -lawrence bragg revisited[END_REF]) Caspi et al. (2005)

	Unit cell Orthorhombic	Orthorhombic	Triclinic	Orthorhombic
	Space group	Pmcn	Pmcn	P1	Pmcn
	a (Å)	4.94	4.961 4( 3)	5.739 4( 4)	4.961 83 (1 )
	b (Å)	7.94	7.967 1( 4)	4.961 6( 2)	7.969 14 (2 )
	c (Å)	5.72	5.740 4( 4)	7.970 0( 5)	5.742 85 (2 )
	α (°)	90	90	90.004 (4 )	90
	β (°)	90	90	90.012 (5 )	90
	γ (°)	90	90	90.001 (4 )	90
	Unit cell
	volume (Å 3 )	224.36	226.9064	226.959 (4 )	227.081 (1 )

Table 3 :

 3 Typical concentration range (or mean values), and corresponding type of regulation, of the elements reported in otolith studies. Where multiple publications are sourced, the extreme values are in bold. Only references presenting absolute quantification of elemental content are presented. *No references were found listing mass concentrations of H, C, O, or N, but the isotopic ratios of these elements have been measured. C and O are present as both inorganic (presumably in proportion to Ca) and organic material; N is presumed present in the organic material, and H is presumed present in organic material and possibly as water of hydration.

	Element	Range	Physiological	Influencing environmental
	Z Sym. (µmol • g -1 ) H No references found*	control	parameters
	Li	0.007 -0.15

  Sr 2+ , Ba 2+ ) in the aragonite matrix. A set of various experimental protocols (direct or delayed extraction, dry storage vs. in ethanol) revealed significant effects on elemental concentrations of K, Na, and

Table 4 :

 4 Studies on ancient otoliths and methods used for verification of potential alteration.

		Species	Geological	Visual observation	Mineralogy	Geochemistry
	Study	N o List	age	Macro Micro SEM		St. iso.*	Elemental
	Patterson (1998)	Aplodinotus grunniens	Fossil	X	-	-	XRD	-	-
	Dufour et al. (2000)	Diaphus sp., Gadiculus argentus,	Archaeol.	X	X	X	XRD	X	X
		Gadiculus labiatus, Gobius guerini,
		Hygophum hygomi, Lampanyctus pho-
		tonus, unidentified (genus "Gobi-
		idarum")
	Wurster and Patterson (2001)	A. grunniens	Fossil	-	-	-	XRD	-	-
	Wurster and Patterson (2003)	A. grunniens	Fossil	See Wurster and Patterson (2001)
	Béarez et al. (2005)	Cilus gilberti, Sciaena deliciosa	Archaeol.	X	X	X	XRD, CL*	X	-
	Hufthammer et al. (2010)	Gadus morhua	Archaeol.	X	X	-	-	-	-
	Vanhove et al. (2011)	Paraconger sauvagei, Paraconger	Fossil	X	-	X	XRD, CL*	X	-
		papointi, "Neobythitinorum" sub-
		regularis, Glyptophidium polli
	Geffen et al. (2011)	G. morhua	Archaeol.	X	-	-	XRD	-	-
	Disspain et al. (2011)	Argyrosomus japonicus, Acanthopa-	Fossil	X	X	-	-	-	X
		grus butcheri
	Limburg et al. (2011)	G. morhua	Archaeol.	-	-	-	-	-	-
	Kern et al. (2012)	Umbrina subcirrhosa	Fossil	-	-	-	CL*, Raman	-	-
	Vanhove et al. (2012)	Platycephalus janeti, P. papointi, "N."	Fossil	See Vanhove et al.
		subregularis

  Archaeological interest and geographical context . . . . . . . . . . . . . . . . . . . . . . 34 2.2.2 Archaeological otoliths . . . . . . . . . . . . 36 2.2.3 Modern otoliths . . . . . . . . . . . . . . . . . 40

	Micropogonias altipinnis	Micropogonias manni	Galeichthys peruvianus	Species
	diffraction on subsamples of otoliths to check for different mineral
	phases. A fast XRD mapping technique is implemented in the present Micropogonias Galeichthyinae Cathorops Genus
	work using a pixel-area detector to scan entire otolith sections with S A M P L E S A N D S A M P L E P R E PA R AT I O N
	micro-scale lateral resolution within a reasonable experimental time Sciaenidae Ariidae Family
	frame. Further investigations into crystal texture are enabled by this Perciformes Siluriformes Order
	approach and are investigated. Fast texture analysis is a promising
	endeavour not only for archaeological otoliths, but also for other 2.1 Rationale of sample selection . . . . . . . . . . . . . 33 Superorder Ostariophysi Acanthopterygii
	biominerals and for materials scientists. 2.2 Description of selected otolith samples . . . . . . . 34
	Chordata Actinopergyii 2.2.1 Animalia Teleostei	Kingdom Phylum Class Infraclass

2.2.4 Sample preparation . . . . . . . . . . . . . . 41 2.3 Reference materials . . . . . . . . . . . . . . . . . . . 44 2.3.1 Elemental reference . . . . . . . . . . . . . . 44 2.3.2 Chemical environment references . . . . . . 45

2.1 rationale of sample selection

The archaeological samples in the present work were selected on the basis of existing knowledge of the samples and archaeological interest. Knowledge of the samples was gained through taxonomic identification, stable isotope analysis, SEM, and FTIR analyses, previously performed by Philippe Béarez, Elise Dufour, and Olivier Tombret. All samples form part of a larger corpus employed for the study of ancient humans in northern Peru, as well as the palaeoclimate of the region. A diversity of sample characteristics was sought, with samples of four species belonging to two orders (Fig.

9

) with various modes of life, as well as originating from different burial environments. Modern otoliths were selected with the objective of providing appropriate counterparts for the archaeological corpus. Reference materials for elemental concentration and chemical environment were selected according to known and anticipated observations, in line with common usage in the literature.

Table 5 :

 5 List of samples studied.

	Name	Species	Otolith	Site	Locality	Period	Date of collection / capture
	Archaeological
	QCD	Micropogonias sp.	Sagitta	Pampa de los fósiles	Cupisnique Desert, Peru	Preceramic	06/2000
	BH17	Cathorops sp.	Sagitta	Pampa de los fósiles	Cupisnique Desert, Peru	Preceramic	06/2000
	HP-1	Galeichthys peruvianus	Lapillus	Huaca Prieta	Chicama Valley, Peru	Preceramic	2006
	HP-11	Galeichthys peruvianus	Lapillus	Huaca Prieta	Chicama Valley, Peru	Preceramic	2006
	HP-12	Galeichthys peruvianus	Lapillus	Huaca Prieta	Chicama Valley, Peru	Preceramic	2006
	Bayovar-1C	Micropogonias sp.	Sagitta	Bayovar-1	Sechura Desert, Peru	Early Intermediate	2012
	Modern
	111	Micropogonias altipin-	Sagitta	-	Puerto Lopez, Ecuador	Modern	14/06/2000
		nis
	119	Micropogonias altipin-	Sagitta	-	Oncocito, Ecuador	Modern	20/06/2000
		nis
	424	Micropogonias manni	Sagitta	-	Budi, Chile	Modern	17/11/2005
	409	Galeichthys peruvianus	Lapillus	-	Salaverry, Peru	Modern	23/05/2005
	GP-292	Galeichthys peruvianus	Lapillus	-	Chorillos, Peru	Modern	26/03/2002

Table 6 :

 6 Sample preparation parameters: otolith sectioning plane, thickness of final polished section, resin used for enrobing, glue used for fixing sample to slide, and laboratory where preparation was carried out.

	Thickness	Preparation

Table 7 :

 7 General characteristics of beam-based elemental analysis techniques.

	Campana et al. (1994); Gemperline et al. (2002); Elsdon and Gillanders
	(2004); Long et al. (2014)). However, LA-ICP-MS necessarily ablates
	a certain sample volume, which produces an averaging of the
	volume, and may preclude repeat or coupled analyses. All of the
	previously-mentioned techniques are rediscussed in a 2002 reference

Table 8 :

 8 Compilation of synchrotron X-ray fluorescence studies on otoliths for life history analysis

	Study	Elements	Objectives	Beamline and experimental con-	Main conclusion
		probed		ditions
	Nakai et al.	Sr, Ca, P	Geographic reference by SR-XRF	Photon Factory BL4A; 17 keV	SR-XRF elemental maps can be used to
	(1999)		trace element map	3 s/step 100 × 100 µm 2 ; 100 µm steps,	track individual movements
	Limburg	Ca, Sr, Zn, Ba,	Investigate potential of SR-µXRF for	CHESS D; 8-10 & 16-20 keV	SR-µXRF can be used for multielemental
	et al. (2007)	Mn, Se	life history analysis	20 µm diam.; 25-120 µm steps,	life history analyses
				6-11 s/point
	Yamauchi	Fe, Zn, Mn	Composition comparison between	Photon Factory 4A; not reported Composition of different otoliths in the
	et al. (2008)		species		same fish varies otolith size varies with
					migration
	Limburg	Br	Geographic tracing by Br composi-	CHESS F3; 16.1 keV 15-30 µm;	Br along different growth axes is useful for
	et al. (2010a)		tion	1-10 s/point	stock identification
	Limburg	Se, Hg, Sr	Geographic tracing by elemental	See Limburg et al. (2007) and	Se can serve as a tracer for exposure to
	et al. (2010b)		composition	Limburg et al. (2010a)	heavy metal pollution
	Limburg	Ca, Mn, Sr	Tracking historical hypoxia	See Limburg et al. (2010a)	Mn in archaeological otoliths can serve as
	et al. (2011)				a proxy for hypoxia
	Limburg	Ca, Mn, Fe, Zn,	Identification of elemental tracers	See Limburg et al. (2010a)	Mn is a promising tracer for hypoxia, Zn
	et al. (2015)	Cu, Br, Sr, I	for hypoxia		and I show potential

1

  Chapter introduction . . . . . . . . . . . . . . . . . . 81 4.2 Results of elemental mapping by SR-µXRF . . . . . 84 4.2.1 Unified coordinate marking . . . . . . . . . . 85 4.2.2 Micrometric fluctuation in elemental composition revealed by SR-µXRF mapping . . . 85

4.3 Discussion and interpretations . . . . . . . . . . . . 94 4.3.1 Medium-energy synchrotron µXRF mapping 94 4.3.2 Insights into the reliability of the Sr signal . 101 4.4 Intermediate conclusion . . . . . . . . . . . . . . . . 108

  Z elements up to transition metals. Examining the transition from significant to no Sr, the edge is no longer in the same position, but ca. 200 µm farther from the core (E). This second, lower edge is shared by Rb. The highest concentrations of Mn, Fe, Zn, and Cu can be found between the apparent edges in the Ca and Sr maps.

Sr, and Ba are present (i.e

. not in the resin). Only Sr exhibits a strong regular fluctuation, resembling parallel bands with values between 28 and 49 µmol • g -1 . Sr reaches its minimum concentration near the midpoint, at 1.5 mm (S1), and its maximum (S2) just before the outer edge. Other elements (K, Ca, Br, Rb) exhibit weak or very weak fluctuations. The Ca content is nearly homogeneous, exhibiting an overall variation of ca. 1% of its mean outside of artefacts from previous characterisation and preparation

(see below)

. The otolith edge as seen in LM is coincident with the transition from high to no Ca (E). The transition from nearly homogeneous Ca content within the bulk to zero Ca in the resin is well-defined, occurring over just 1-2 pixels, or 10-20 µm. This edge position is identical in all low-

Table 9 :

 9 Attenuation length (1/e) of characteristic lines of selected elements in pure aragonite (CaCO 3 , ρ=2.93 g • cm -3 ) and polyester((C 10 H 8 O 4 ) n , ρ=0.85 g • cm -3 ) at 45°incidence. Calculated using the CXRO X-ray Interaction with Matter database(Henke et al., 1993).

	Element	Energy	Attenuation lengths (µm) in
	line	(eV)	aragonite	polyester
	K Kα 1	3313.8	14.4	85.9
	Ca Kα 1	3691.7	19.5	118.6
	Mn Kα 1	5898.6	14.3	495.8
	Fe Kα 1	6403.8	17.2	639.0
	Cu Kα 1	8047.8	32.4	1297.1
	Zn Kα 1	8638.9	39.5	1615.4
	Br Kα 1	11 924.2	98.3	4322.5
	Rb Kα 1	13 395.3	137.1	6081.1
	Sr Kα 1	14 165	160.9	7129.2
	Ba Lα 1	4466.3	6.5	211.2
	Hg Lα 1	9988.8	59.5	2527.1
	Pb Lα 1	10 551.5	69.5	2988.2
	-	16 200	236.6	10 268.1
	-	17 200	281.0	11 964.6

could be corrected with a white field measurement. However, the level of contrast of Sr is barely noticeable in the collected images. This is attributed to the fact that the contrast is limited in this sample with a dynamic range lower than 2.0. Other images were collected using the 1:6 magnification lens and varying count times up to 8 hours, but no significant contrast could be retrieved. Running for longer collection times and/or higher incident flux could present better statistics, permitting a distinction of the strontium variations. While the full-field XRF camera does not currently appear as appropriate for measurement of this relatively limited concentration variation with a low mean value, is is undoubtedly a promising development for reliable intercomparison of XRF data across large sample areas.

Table 10 :

 10 Characteristics of the five archaeological and three modern otolith samples examined in this chapter. A µEXAFS spectrum was recorded at the analysis points marked with *. Letter annotations in the far right column correspond to points in Fig.47.

	Name QCD	Species Micropogonias sp.	Sample age Preceramic (Paíjan)	Origin Pampa de los fósiles, Cupisnique Desert, Peru	[Sr] range (µmol • g -1 ) 27-51	[Sr] at analysis points (µmol • g -1 ) 36.6 (A), 35.0 (B), 39.7 (C), 36.2* (D), 27.2 (E), 50.7 (F), 40.4 (G)
	HP-11 42.HP-12 Galeichthys peruvianus Preceramic Huaca Prieta, Chicama Valley, Peru 21-185 Galeichthys peruvianus Preceramic Huaca Prieta, Chicama Valley, Peru 21-101 60.Bayovar-1C Micropogonias sp. Late Interme-diate Period Bayovar-1, Sechura Desert, Peru 17-50 18.4, 48.7*
	BH17	Cathorops sp.	Preceramic (Paíjan)	Pampa de los fósiles, Cupisnique Desert, Peru	17-60	54.1*
	424	Micropogonias manni	17/11/2005	Budi, Chile	14-22	14.9, 15.7, 16.8, 21.5, 22.2*
	111	Micropogonias altipinnis 14/06/2000	Puerto Lopez, Ecuador	7-12	7.1, 12.2*
	GP-292	Galeichthys peruvianus	26/03/2012	Chorillos, Peru	15-52	29.1, 51.1*

1 (A), 48.5 (B), 62.5 (C), 58.5 (D), 53.1 (E) 0 (A), 79.5 (B), 93.3* (C), 90.5 (D), 93.9 (E), 74.9 (F), 64.5 (G), 49.0 (H)

Table 11 :

 11 EXAFS fitting results. Sr-M corresponds to a Ca shell in all samples except strontianite, where it is Sr.

	Section	R Sr-O (Å)	R Sr-C (Å)	R Sr-M (Å)	σ 2 Sr-O	σ 2 Sr-C	σ 2 Sr-M	∆E 0 (eV)
	Aragonite Strontianite 2.601 ± 0.015 2.565 ± 0.020 QCD 2.579 ± 0.025 HP-12 2.583 ± 0.026 Bayovar-1C 2.553 ± 0.027 BH17 2.571 ± 0.023 424 2.584 ± 0.024 111 2.568 ± 0.034 GP-292 2.583 ± 0.021	2.931 ± 0.087 2.982 ± 0.116 2.957 ± 0.141 3.010 ± 0.106 2.854 ± 0.150 2.958 ± 0.099 2.983 ± 0.100 2.933 ± 0.074 2.971 ± 0.076	3.980 ± 0.036 4.155 ± 0.042 3.975 ± 0.053 3.966 ± 0.053 3.965 ± 0.057 3.984 ± 0.048 3.983 ± 0.055 3.963 ± 0.068 3.988 ± 0.044	0.011 ± 0.002 0.011 ± 0.001 0.009 ± 0.002 0.009 ± 0.002 0.013 ± 0.004 0.011 ± 0.002 0.009 ± 0.002 0.010 ± 0.003 0.010 ± 0.002	0.041 ± 0.032 0.059 ± 0.047 0.046 ± 0.062 0.039 ± 0.046 0.031 ± 0.032 0.037 ± 0.037 0.037 ± 0.040 0.030 ± 0.029 0.030 ± 0.024	0.012 ± 0.004 0.010 ± 0.012 0.013 ± 0.006 0.013 ± 0.006 0.016 ± 0.007 0.014 ± 0.014 0.015 ± 0.067 0.013 ± 0.008 0.015 ± 0.006	4.201 ± 1.266 4.915 ± 1.404 4.201 ± 2.452 4.610 ± 2.255 3.810 ± 2.340 5.039 ± 2.021 5.039 ± 2.067 4.201 ± 1.257 5.720 ± 1.611

  Elemental mapping . . . . . . . . . . . . . . . 153 6.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . 156

	H S
	6.1 Chapter introduction . . . . . . . . . . . . . . . . . . 147
	6.2 Development of fast crystal orientation mapping
	of aragonitic otoliths . . . . . . . . . . . . . . . . . . 151
	6.2.1

6.3.1 Relation between structure and properties in fish otoliths . . . . . . . . . . . . . . . . . . 157 6.3.2 Crystalline orientation . . . . . . . . . . . . . 159 6.3.3 Heterogeneity of otolith crystal orientations 162 6.3.4 Toward rapid crystal orientation measurement . . . . . . . . . . . . . . . . . . . . . . . 164 6.3.5 Toward full texture measurement . . . . . . 167 6.4 Intermediate conclusion . . . . . . . . . . . . . . . . 167

  Figure77: The addition of essential information, stable isotopic ratios, to the unified methodology would provide another dimension of data but brings new challenges in spatial correlation.

	Elemental techniques -Sr/Ca -Ba/Ca -Mg/Ca ...	Chemical structure techniques -Neighbours of minor elements -Mineral phase
	Microscopy techniques -Microstructure -Ultrastructure	Isotopic techniques -δ 13 C, δ 18 O -88 Sr/ 87 Sr, 87 Sr/ 86 Sr -44 Ca/ 43 Ca, 44 Ca/ 40 Ca
	Uni ed analytical methodology
	-Sample preparation
	-Damage from analyses
	-Sequence of analyses
	-Spatial referencing
	-Correlated vs complementary analyses

12 th International Symposium on Biomineralization

	Freiberg, Germany, 27-30 August 2013.
	"Study of otoliths by synchrotron X-ray absorption and fluores-
	cence micro-spectrometries: Contributions and constraints"
	• Colloque de Sclérochronologie
	Rennes, France, 2-4 July 2013.
	"Analyse microchimique d'otolithes modernes et archéologiques
	par micro-spectrométrie de fluorescence des rayons X au
	synchrotron : contributions et contraintes." Delivered by M.-
	A. Languille.
	• Journées de l'École doctorale des ondes et matière
	Palaiseau, France, 25-26 February 2013.
	"Comparative analysis of biogeochemical signals in stratified
	carbonate systems studied by synchrotron radiation and
	isotopic mass spectrometry"
	• 13 th SOLEIL Users' Meeting
	Palaiseau, France, 23-24 January 2013.
	"Coupling X-Ray Speciation and Isotopic Methods to Obtain
	Subseasonal Resolution Analysis of Archaeological Sciaenid
	Fish Otoliths from Peru"
	Poster presentations
	• Synchrotron Radiation in Art and Archaeology
	Paris, France, 9-12 September 2014.
	"Micro-X-ray fluorescence evidences elemental fluctuation on a
	short time scale in otoliths"
	• 3 rd International Sclerochronology Conference
	Caernarfon, United Kingdom, 18-22 May 2013.
	"Study of modern and archaeological otoliths by synchrotron
	X-ray absorption and fluorescence micro-spectrometries: Con-
	tributions and constraints" Delivered by E. Dufour.
	• Colloque d'Archéométrie du GMPCA
	Caen, France, 22-26 April 2013.
	"Coupling X-Ray speciation and isotopic data to obtain
	subseasonal resolution analysis of archaeological Sciaenid fish
	otoliths from Peru"
	• 13 e Journées de la Matière Condensée
	Montpellier, France, 28-31 August 2012.
	"Comparative analysis of biogeochemical tracer signals in
	stratified calcium carbonate systems studied using synchrotron
	radiation and isotope mass spectrometry"

Table 1 :

 1 Names and details on the otolith samples studied. Geological ages correspond to the age of the stratum in which an otolith was discovered.

	Name	Species	Otolith Site	Locality	Geological age / Date of capture	Section thickness
	QCD	Micropogonias sp.	Sagitta	Pampa de los fósiles	Cupisnique Desert, Peru (7.55°S, 79.32°W)	1994) Preceramic (Paíjan) 11 000-8000 BP (Chauchat and Pélegrin,	710 µm
	Bayovar-1C	Micropogonias sp.	Sagitta Bayovar-1	Sechura Desert, Peru (6.10°S, 80.89°W)	Late Intermediate Period 2600-1360 BP (Goepfert et al., 2014)	490 µm
	424	Micropogonias manni	Sagitta -	Lake (38.89°S, 73.28°W) Budi, Chile	17/11/2005	290 µm
	HP-12	Galeichthys peruvianus	Lapillus Huaca Prieta	Chicama Valley, Peru (7.92°S, 79.31°W)	Preceramic 6641±49 BP (Dille-hay et al., 2012)	130 µm
	GP-292	Galeichthys peruvianus	Lapillus -	Chorillos, Peru (12.16°S, 77.03°W)	26/03/2012	160 µm
	BH17	Cathorops sp.	Sagitta	Pampa de los fósiles	Cupisnique Desert, Peru (7.55°S, 79.32°W)	Preceramic (Paíjan) 11 000-8000 BP

Table 1 :

 1 Characteristics of the five archaeological and three modern otolith samples. A µEXAFS spectrum was recorded at the analysis points marked with *. Letter annotations in the far right column correspond to points in Fig.1.

	Name QCD HP-11 HP-12 Bayovar-1C BH17 424	Species Micropogonias sp. Galeichthys peruvianus Galeichthys peruvianus Micropogonias sp. Cathorops sp. Micropogonias manni	Sample age Preceramic (Paíjan) Preceramic Preceramic Late Intermediate Period Preceramic (Paíjan) 17/11/2005	Origin Pampa de los fósiles, Cu-pisnique Desert, Peru Huaca Prieta, Chicama Valley, Peru Huaca Prieta, Chicama Valley, Peru Bayovar-1, Sechura Desert, Peru Pampa de los fósiles, Cu-pisnique Desert, Peru Budi, Chile	[Sr] range (µmol • g -1 ) 27-51 21-185 21-101 17-50 17-60 14-22	[Sr] at analysis points (µmol • g -1 ) 36.6 (A), 35.0 (B), 39.7 (C), 36.2* (D), 27.2 (E), 50.7 (F), 40.4 (G) 42.1 (A), 48.5 (B), 62.5 (C), 58.5 (D), 53.1 (E) 60.0 (A), 79.5 (B), 93.3* (C), 90.5 (D), 93.9 (E), 74.9 (F), 64.5 (G), 49.0 (H) 18.4, 48.7* 54.1* 14.9, 15.7, 16.8, 21.5, 22.2*
	111 GP-292	Micropogonias altipinnis Galeichthys peruvianus	14/06/2000 26/03/2012	Puerto Lopez, Ecuador Chorillos, Peru	7-12 15-52	7.1, 12.2* 29.1, 51.1*

Table 2 :

 2 Unit cell parameters of aragonite, strontianite, calcite, and vaterite, using data fromCaspi et al. (2005);Hayakawa et al. (2008);

  .

		Aragonite	Strontianite	Calcite	Vaterite
	Ca coordination System Space group a (Å)	9 Orthorhombic Pmcn 4.96183 (1)	9 Orthorhombic Pmcn 5.107	6 Rhombohedral R 3c 4.989	8 Hexagonal P6 3 /mmc 4.130
	b (Å)	7.96914 (2)	8.414	4.989	4.130
	c (Å) α (°) β (°) γ (°) Unit cell volume (Å 3 )	5.74285 (2) 90 90 90 227.081	6.029 90 90 90 259.07	17.062 90 90 120 424.67	8.490 90 90 120 125.41
	4.2. Chemical environment of strontium from the micro-to mil-
	limetre scale

An infraclass of bony fish (superclass osteichthyes) with fin rays (class actinopterygii) exhibiting a more evolved jaw structure. Most living fishes are teleosts(Miller and

A family of ostariophysian catfish inhabiting warm or tropical waters.

A region with a uniform crystallographic phase, different from neighbouring regions.

Extended X-ray Absorption Fine Structure. A technique for probing the local environment up to several Ångströms around a target element. See Section 3.4.

Organisation below microstructure; nanostructure.

Scanning Electron Microscopy, a microscopy technique using electrons instead of photons to provide higher magnification and increased depth of field. See Appendix C.

"Minor" concentration range being defined as between 0.1 and 1 % by weight.

A characteristic (e.g. Sr concentration) which can be measured as a substitute for direct in situ measurement of environmental characteristics (e.g. temperature, salinity).

Ontogenesis: the development of an organism from conception to maturity.

An approach to collect a full powder diffractogram from a small sample (including from a single crystal) by continuously rotating about two axes.

The relation between X-ray energy, lattice spacing, and incident angle such that diffraction occurs. See Section 3.7.

The total width of a measured peak (e.g. spectral, diffraction) at half of its maximum intensity.

Where both energy and number of photons are measured.

Attenuation length: The distance at which a photon beam has been reduced to 1/e of its original intensity.

Standard temperature and pressure (STP): T=273 K, P=100 kPa.

A kernel density plot is produced by placing a kernel (a smooth, peaked function) on the X-axis where each point of data lies. The total area of all kernels is summed to produce the kernel density(Silverman, 1998).

Preprint submitted to Chemical GeologyFriday 28 th August, 2015

Preprint submitted to Geochimica et Cosmochimica Acta

Acknowledgements

This work has been developed as part of the IPANEMA/ Muséum national d'Histoire naturelle agreement on collaborative research. The IPANEMA platform is a joint unit of CNRS and the French Ministry of Culture and Communication, and benefits from a CPER grant (MENESR, Région Île-de-France) (Bertrand et al., 2011). Beamtime was funded by Synchrotron SOLEIL under projects 20120498 and 20121125. The authors thank D. Thiaudière, S. Réguer and F. Alvès at the DiffAbs beamline for support during the experiments, and P. Gueriau (MNHN UMR 7207) for his help during data collection. They thank P. Béarez (MNHN UMR 7209) and T. Dillehay (Vanderbilt University) for providing the samples for study, M. Lemoine (MNHN UMR 7209) for support in sample preparation. They thank S. Cohen (IPANEMA) for helpful discussion regarding data analysis and providing the SpectralImage 'R' package. We thank the referees of this paper for their helpful and insightful comments.

Acknowledgements

This work has been developed as part of the IPANEMA / Muséum national d'histoire naturelle agreement on collaborative research. The IPANEMA platform is jointly developed by CNRS, the French Ministry of Culture and Communication and MNHN, and benefits from a CPER grant (MENESR, Région Ile-de-France) (Bertrand et al., 2011). This work was supported in part by the French Agence Nationale de Recherche under the LabEx ANR-10-LABX-0003-BCDiv within the "Investissements d'avenir" program reference n o ANR-11-IDEX-0004-02. The authors acknowledge SOLEIL for provision of synchrotron radiation facilities under projects no. 20120498 and 20121125 and would like to thank D. Thiaudière and F. Alvès for assistance at the DiffAbs beamline. The authors wish to acknowledge the support of E. Curis (Université René Descartes, Paris, France) for helpful discussion on EXAFS interpretation, and S. Cohen (IPANEMA) for advice in analysis of multispectral data and modelling. The authors thank P. Béarez (MNHN UMR 7209, Paris, France) and T. Dillehay (Vanderbilt University, Nashville, Tennessee, USA) for providing the otoliths samples studied. M. Lemoine (MNHN UMR 7209), O. Tombret (MNHN UMR 7209) and H. Boucher (LOCEAN, IRD, Bondy, France) provided valuable support during sample preparation and/or experiments.

Appendices

A C O N T R I B U T I O N S

Submissions to peer-reviewed journals in press Cook, P. K., Languille, M.-A., Dufour, E., Mocuta, C., Fortuna, F., Tombret, O., and Bertrand, L. "Biogenic and diagenetic

Abstract

Fish otoliths are biogeochemical archives of environmental conditions and are a valuable tool for examining life traits. These sclerochronological life history records, accessed through geochemical analyses, are widely used in fisheries sciences, and are seeing growing use as palaeoenvironmental archives or for reconstruction of past human activities. The fidelity of such reconstructions relies on the preservation of the biogenic geochemistry, which may be altered through diagenetic processes. In this work, a methodology is provided that enables measurement of elemental concentrations with a high precision with simultaneous quality control of the data to ensure high reliability and a multi-technique sample marking strategy to ensure precise alignment. Thin sections of four archaeological and two modern otoliths from the Pacific coast of South America were examined by synchrotron µXRF. High-definition elemental maps of sample sections up to 3.2 × 1.0 mm 2 (V×H) were produced with an on-sample spot size of 6.0 × 16 µm 2 . Thirteen elements were detected, some of which may be useful indicators of diagenetic alteration. Strontium was accurately quantified and one sample presented local concentrations above the previously described range in literature. The quantity of collected data allows the use of statistical approaches to examine the intra-and inter-sample Sr distribution. A highly reliable profile, produced by integrating multiple points, additionally permits identification of defects and potential alteration along its length through changes in median absolute deviation. Contamination at the edge of some of the specimens was evidenced from preparation and other post-mortem alterations, even in modern samples, however diagenetic alteration in the elemental signals was not evidenced significantly further than a few hundred micrometres deep in the samples. Mediumenergy SR-µXRF therefore provides a fast and sensitive elemental probe without a visible effect on the sample, and the integration volume which limits the attainable lateral resolution in this energy range is discussed. This paper discusses the methodological limitations and opens new perspectives in the analysis of biomineral carbonate palaeoproxies.

Table S1: A summary of the characteristics of various techniques described by Campana et al. (1997) and Geffen et al. (2013) compared to that used in the present work. Method identifiers are those used by the respective publications. Where different conditions were used for a given method, the shortest analysis time per point and smallest spot size were taken. Transect measurement time and area scan rate were calculated assuming a step size equal to spot size and without considering deadtime between analysis points. 0.00 0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 X (mm) 0.00 0.50 X (mm) 0.00 0.50 X (mm) 0.00 0.50 X (mm) 0.00 0.50 X (mm) 0.00 0.50 X (mm) 0.00 0.50 C2 Table S4: EXAFS fitting results. Sr-M corresponds to a Ca shell in all samples except strontianite, where it is Sr. Continued from previous page.

Supplementary Information

SEM Images of otoliths

Section

Term Definiton

Littoral Near to shore.

Ontogenesis

The development of an organism from conception to maturity.

Ostariophysi A superorder of teleost fish, one characteristic of which is a "Weberian apparatus" connecting the inner ear to the swim bladder.

Palaeoenvironment The environment in the geological past.

Palaeoenvironmental proxy

Proxies for environment, climate, ecology, etc. in the geological past. See environmental proxy.

Pitch and roll

On the DiffAbs beamline, orthogonal angular axes of the monochromator used to align the beam for downstream optics.

Shape factor

A unitless value which account for overall crystallite shape (spherical, cubic, etc.); typically ca. 0.9 is used for unknown shapes.

Teleost

An infraclass of bony fish (superclass osteichthyes) with fin rays (class actinopterygii) exhibiting a more evolved jaw structure. Most living fishes are teleosts [START_REF] Miller | [END_REF]. Illumination can be in transmitted or reflected mode, with configurations for bright-and dark-field imaging, as well as polarising filters. Ultraviolet (UV) luminescence can be performed using a Xe lamp and one of two excitation/detection spectrum configurations. The "FL2" configuration was uses an excitation of 450-490 nm with a 505 nm dichroic mirror and a 520 nm high-pass emission filter. The "FL1" cube uses an excitation of 330-380 nm with a 400 nm dichroic mirror and a 435 nm high-pass emission filter. The "FL2" configuration was used for all UV luminescence microscopy presented in this work. Photomicrographs of images in the present work were recorded as mosaics at 5X and stitched together using the Grid/collection stitching plugin [START_REF] Preibisch | Globally optimal stitching of tiled 3d microscopic image acquisitions[END_REF] for FIJI [START_REF] Schindelin | Fiji: an open-source platform for biologicalimage analysis[END_REF] and Appendix E).

Ultrastructure

c.2 scanning electron microscopy

Unless otherwise noted, observations were performed on the Zeiss Supra 55VP Field Emission Scanning Electron Microscope (IPANEMA SEM laboratory). A low accelerating voltage (typically 0.5-1 kV) was used to minimise sample charging and potential effects on the sample. Most images presented in the present work are secondary electron images. The system is also equipped with an InLens backscattered electron detector. Images with high magnification over large areas were recorded as a mosaic and stitched together using Adobe Photoshop CS5 (Appendix E).

SEM images supplied by E. Dufour were recorded on a Philips 505 SEM (Université Paris Sud) or Tescan Vega (Université Montpellier II) with a solid state secondary electron detector.

c.3 benchtop x-ray diffraction

Measurements were performed on the Bruker D2 Phaser (SOLEIL Chemistry laboratory). Cu Kα X-rays were produced with a Cu target sealed tube using 30 kV accelerating voltage and 10 mA current. A knife edge was placed 1 mm above the sample. Polycarbonate sample

A brief summary of the notable software and plugins used with notes on use cases and application in this work.

Name (Reference) [License*, URL]

Scientific application in the present work Absorbix [START_REF] Michalowicz | Max: Multiplatform applications for xafs[END_REF] R package for generating colour palettes selected for high readability and representativeness in plots and maps.

FIJI [START_REF] Schindelin | Fiji: an open-source platform for biologicalimage analysis[END_REF] [GPL, http://fiji.

sc]

Repackaged version of ImageJ with a standard suite of plugins. Used for image stitching.

Grid/collection stitching [START_REF] Preibisch | Globally optimal stitching of tiled 3d microscopic image acquisitions[END_REF] [Art., http://fly.mpi-cbg.de/preibisch] FIJI/ImageJ plugin for automated stiching of mosaic photomicrographs.

colophon

This document was typeset using the typographical look-and-feel classicthesis developed by André Miede. The style was inspired by Robert Bringhurst's seminal book on typography "The Elements of Typographic Style". classicthesis is available for both L A T E X and L Y X:

http://code.google.com/p/classicthesis/ Final version as of October 14, 2015 (classicthesis).