Sturgeon and Paddlefish (Acipenseridae) Saggital Otoliths Are Composed of the Calcium Carbonate Polymorphs Vaterite and Calcite

Journal of Fish Biology (2016) doi:10.1111/jfb.13085, available online at wileyonlinelibrary.com

Sturgeon and paddlefish (Acipenseridae) saggital otoliths are composed of the calcium carbonate polymorphs vaterite and calcite

B. M. Pracheil*†, B. C. Chakoumakos‡, M. Feygenson§, G. W. Whitledge‖, R. P. Koenigs¶ and R. M. Bruch¶

*Environmental Science Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, U.S.A., ‡Quantum Condensed Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, U.S.A., §Chemical and Engineering Materials Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, U.S.A., ‖Center for Fisheries, Aquaculture and Aquatic Sciences, Southern Illinois University, Carbondale, IL 62901, U.S.A. and ¶Wisconsin Department of Natural Resources, Oshkosh, WI 54903, U.S.A.

This study sought to resolve whether sturgeon (Acipenseridae) sagittae (otoliths) contain a non-vaterite fraction and to quantify how large a non-vaterite fraction is using neutron diffraction analysis. This study found that all otoliths examined had a calcite fraction that ranged from 18 ± 6to36± 3% by mass. This calcite fraction is most probably due to biological variation during otolith formation rather than an artefact of polymorph transformation during preparation. © 2016 The Fisheries Society of the British Isles

Key words: Acipenser fulvescens.

Otoliths, the calcium carbonate (CaCO3) ear bones of Osteichthyes, are truly a wonder of the Animal Kingdom. These tri-part structures essential for hearing and balance in fishes are composed of some form of3 CaCO and can be used to determine fish environmental history as well as fish age through enumeration of daily and annular rings (Pracheil et al., 2014). The three most common forms, or polymorphs, of CaCO3 are vaterite, which is the least energetically stable, intermediately stable aragonite and the most stable, calcite. All have all been reported from fish otoliths (Gauldie, 1993; Oliveira et al., 1996; Melancon et al., 2005). The polymorph or polymorphs of CaCO3 contained in the otolith of a particular fish species has been reported to reflect some phylogenetic patterning, with vaterite being the primary constituent of otoliths in the most primitive fishes and aragonite being the primary constituent of otoliths inmore modern fishes (Carlström, 1963; Gauldie, 1993). Despite its thermodynamic favourability and its dominance in the otoliths of land animals (Carlström, 1963), otoliths of only a few fishes have been reported to contain the most stable CaCO3 polymorph, calcite. Piranhas (Characiformes), hake (Gadi- formes), orange roughy (Beryciformes) and snapper (Perciformes), for example, have

†Author to whom correspondence should be addressed. Tel.: +1 865 241 5622; email: [email protected] 1

© 2016 The Fisheries Society of the British Isles 2 B. M. PRACHEIL ET AL. been reported to contain otoliths made of all three polymorphs of CaCO3 (Gauldie, 1993; Oliveira et al., 1996). Previous reports have described sturgeon otoliths as being entirely composed of vaterite, although there was mention of small calcite and aragonite fractions that were potentially an artifact of preservation (Carlström, 1963; Gauldie, 1993). The mechanism that might lead to a preservation-induced polymorph transition, however, is not clear. The coexistence of multiple polymorphs in a single otolith has been reported from other fishes, but polymorph composition is controlled by proteins involved in otolith formation (Söllner et al., 2003; Tohse et al., 2009; Ren et al., 2013) rather than transformation of polymorphs post-nucleation. When taken together, this suggests the possibility of sturgeon otoliths actually containing fractions of other polymorphs. There have been some reports of visible differences among CaCO3 polymorphs observed using light microscopy (Gauldie, 1986; Bowen et al., 1999; Tomás & Geffen, 2003; Tomás et al., 2004); reliable visual determination among polymorphs, however, has not been quantitatively demonstrated. Rather, methods for polymorph determination must rely on scattering techniques for determining the crystallographic structure of the CaCO3, e.g. powder diffraction or Raman spectroscopy. Earlier studies of otolith polymorph composition have used X-ray diffraction (Carlström, 1963; Gauldie, 1993; Oliveira et al., 1996) or Raman spectroscopy (Melancon et al., 2005, 2008; Tzeng et al., 2007; Jolivet et al., 2008), but with these methods the whole otolith must be ground to a powder to obtain the average composition. In contrast, neutron diffraction is a method that can allow for non-destructive identification and quantification of CaCO3 polymorphs in an intact otolith; i.e. because of the large penetration depth of neutrons in these materials a whole otolith can be easily sampled. This study uses neutron diffraction analysis to resolve whether sturgeon otoliths contain a non-vaterite fraction and quantify the per cent composition of a non-vaterite fraction if it does exist. Lake sturgeon Acipenser fulvescens Rafinesque 1817 sagittal otoliths (here- after, otoliths; n = 12; Fig. 1) were removed from fish harvested during the annual spear-fishing season on the Lake Winnebago system, Wisconsin, U.S.A. (44∘ 00′ N; 88∘ 30′ W). Harvest of A. fulvescens from the Lake Winnebago system has been actively managed for >100 years and represents one of the only demonstrated sus- tainable sturgeon fisheries in the world (Bruch, 1999). The paddlefish Polyodon spathula (Walbaum 1792) otolith (n = 1) was obtained from the Illinois Department of Natural Resources from a fish collected in the Illinois portion of the Ohio River. While a sample size of one is generally not ideal, limitations on instrument time and sample numbers prevented the examination of more P. spathula otoliths. It was still desirable, however, to characterize this otolith to compare the polymorph composition of A. fulvescens otoliths with that of a closely related species. Also, the novelty of neutron diffraction for characterizing otolith polymorph composition coupled with its sensitivity provided a unique opportunity to create an accurate initial crystallographic characterization of a fish species that had not previously been characterized. Neutrons were chosen because they have a greater penetration depth than X-rays in crystallographic studies. X-rays interact with the electron cloud surrounding each atom and the scattering intensity is proportional to the atomic number, whereas neutrons interact directly with the nucleus of the atom and the scattering intensity is isotope specific and does not vary periodically. For the elements Ca, C and O, their scattering powers follow the order Ca > C > O for X-rays and O > C > Ca for neutrons. Therefore, neutrons offer a slightly better contrast to determine the carbonate group positions as

(a) (b)

1 mm

Fig. 1. Typical otolith from Acipenser fulvescens: (a) an entire otolith with mm scale and (b) close-up of a vaterite portion of the otolith. compared with that of X-rays. For this study, two neutron diffractometers were used: the nanoscale-ordered materials diffractometer (NOMAD) at the Oak Ridge National Laboratory (ORNL) spallation neutron source [SNS (https://neutrons.ornl.gov/sns/); n = 6 homogenized A. fulvescens otoliths] and the wide-angle neutron diffractometer (WAND) at the ORNL’s high flux isotope reactor (https://neutrons.ornl.gov/hfir/; n = 6 whole A. fulvescens otoliths; n = 1 whole P. spathula otolith). The combination of wide detector coverage of NOMAD and high intensity neutron beams available at SNS allowed for measurement of small samples (<100 mg) (Neuefeind et al., 2012), with volumes typically used for X-ray diffraction. The diffraction data were normalized by the scattering from a solid vanadium rod and the background from the empty cap- illary was subtracted. The WAND, in contrast, is a high-intensity, medium-resolution, fixed-wavelength diffractometer. For the 3CaCO polymorphs, NOMAD has sufficient resolution to allow full crystal structure refinements in addition to quantitative phase analysis and the WAND enables quantitative analysis. Because of the high penetration depth of neutrons, the entire volume of the sample was probed in a single diffraction experiment. The preliminary neutron diffraction data collected at NOMAD for shovelnose sturgeon Scaphirhynchus platorynchus (Rafinesque 1820) otoliths (B. C. Chakoumakos, unpubl. data) suggested presence of a calcite fraction, which had not been previously reported in studies of acipenserid otoliths. In developing a protocol for sample preparation and data analysis of otoliths using neutron diffraction, it was considered that the carbonate crystallites in whole otoliths might exhibit a preferred crystallographic orientation, i.e. a singular direc- tion of crystalline growth. In such a case, the radial averaging of data from detectors can result in increased or decreased intensities of particular Bragg peaks. It is well known that preferred orientation can make phase identification and quantitative analysis more difficult and reducing or modelling the preferred orientation generally yields more accurate quantitative analysis. Preferred orientation was suspected, but the extent was not known. Another concern was whether the metastable vaterite polymorph could transform to calcite during handling and specimen preparation. Both concerns were tested using two methods. First, in addition to whole otolith neutron diffraction, six A. fulvescens saggital otoliths were ground into powder using an agate mortar and pestle. This method homogenized the otoliths so that any preferred crystal orientation was greatly reduced. Approximately 50 mg of the resulting powder was then loaded

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13085 4 B. M. PRACHEIL ET AL. into three 3 mm silica glass capillaries and subjected to neutron diffraction using the NOMAD instrument. Additionally, a series of timed grinding experiments was then conducted to ensure that the calcite fraction observed was not an artefact of preparation. For the grinding tests, portions of a single otolith were subjected to 5, 10 and 25 min of grinding and the intensities of the diffraction peaks were compared for the different grinding times. The powder samples analysed in this experiment were measured inside 3 mm silica capillaries for 1 h. For the second, the WAND was used to reduce the effects of preferred orientation by spinning the otolith while it was in the neutron beam. The WAND was chosen for these measurements because the instrument configuration allowed for a device used to spin otoliths to be easily installed. Ideally,a crystal measured in such way can be considered a powder, because a greater extent of all possible crystallographic orientations are equally probed during the measurement, considering that the temporal frequency of spinning is much shorter than an averaged neutron measurement time. Otoliths were suspended in the neutron beam with a piece of fluorocarbon fishing line glued to the otolith with superglue and spun byamotor in the neutron beam during analysis. Neither the glue nor the fishing line affected the diffraction data, since their volumes were miniscule compared with the volume of the otolith (<0·001%). Although this reduced the preferred orientation, the calcite phase still exhibited a significant preferred orientation, implying that the calcite crystallites were radially oriented from the nucleus to the rim of the otolith. Crystalline structural models were fitted using Rietveld refinements of the neutron powder diffraction data using GSAS software (Rietveld, 1969; Larson & Von Dreele, 2000; Toby, 2001). Both vaterite and calcite phases were included using standard structural data for calcite and the vaterite structural model proposed by Wang & Becker (2009). For the calcite phase a March–Dollase preferred orientation parameter was included; however, inclusion of this parameter into the model for the vaterite phase did not improve the quality of the fit in any significant way. 5The P6 22 space group symmetry model of Wang & Becker (2009) was refined to fit the neutron diffraction data of the powdered otolith sample collected with the NOMAD instrument and then this refined structural model was held fixed to do quantitative phase analysis byfitting the neutron diffraction data of whole otoliths collected with the WAND instrument. No change in the relative phase fractions was observed with increased grinding, thus demonstrating that calcite was present in the otoliths and not the result of data misin- terpretation due to preferred orientation in the samples (Fig. 2). The quantitative phase analysis of neutron diffraction data of this sample revealed 75·9% (by mass) vaterite and 24·1% calcite. Quantitative phase analysis for fractions of vaterite and calcite from six A. fulvescens and one P. spathula from whole otolith samples, conducted using the WAND, demonstrated a substantial calcite fraction in A. fulvescens and P. spathula otoliths (Table I). Acipenser fulvescens otoliths ranged from 65 ± 3% (mean ± s.e., by mass) to 82 ± 6% composition of vaterite and 18 ± 6% to 35 ± 3% composition of calcite (Table I). All otoliths measured had vaterite + calcite %mass fractions that summed to 100% and all otoliths had a measurable calcite fraction. Similarly, the P. spathula otolith contained 66 ± 3% mass composition of vaterite and 34 ± 3% composition of calcite. Although otoliths containing calcite have been reported for other fish species, as far as is known, this is the first report of a large calcite fraction from acipenserid otoliths and the first report of per cent polymorph composition by mass. Previous reports have described acipenserid otoliths as consisting entirely of vaterite (Carlström, 1963;

1·4

1·2

y 0·8 it s

0·6 Inten

0·4

0·2

0 2 2·2 2·4 2·6 2·8 3 3·2 3·4 D-spacing (Å)

Fig. 2. Results from ground Acipenser fulvescens otoliths showing peak intensity from neutron diffraction for the same sample after ( )5,( )15and( ) 25 min of grinding. Normalizing data to the sample with the highest intensity peaks (5 min of grinding) shows that differences in intensities of peaks in samples of different grinding times is simply an artefact of different amounts of material in capillaries. , vaterite peak values; , calcite peak value.

Gauldie, 1993). Results from the present study, however, demonstrate that otoliths from freshwater acipenserids are mostly vaterite containing a substantial calcite fraction, with the six A. fulvescens otoliths and the one P. spathula otolith having similar values. Interestingly, Carlström (1963) noted the presence of what looked like calcite and aragonite fractions in the otoliths of the Russian sturgeon Acipenser gueldenstaedtii Brandt & Razeberg 1833, although this was not the case in other species of acipenserids he examined. He suspected that these fractions were the result of the transformation of vaterite to calcite and aragonite through formalin preservation. While he did not observe an aragonite fraction in these otoliths, it is probable that calcite fractions observed in the A. gueldenstaedtii otoliths were real rather than preservation artefact and not discernable due to method sensitivity. Neutron diffraction

Table I. Mean ± s.d. percentages by mass (%M)ofsixAcipenser fulvescens and one Polyodon spathula otolith vaterite and calcite measured using a wide-angle neutron diffractometer

Vaterite (%M) Calcite (%M) A. fulvescens 78·4 ± 1·321·6 ± 0·5 82·0 ± 5·717·9 ± 6·0 68·2 ± 3·331·8 ± 4·1 65·0 ± 2·635·0 ± 2·9 72·9 ± 4·227·0 ± 4·7 78·4 ± 1·321·6 ± 0·5 P. spathula 65·6 ± 3·434·3 ± 3·1

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13085 6 B. M. PRACHEIL ET AL. provides much greater sensitivity to polymorph differences because of its much greater penetration depth. While X-rays mostly characterize the surface, neutrons are able to characterize the entire volume of the otolith thus allowing for determination of per cent composition of polymorphs in the material being characterized. In addition to grinding experiments that showed vaterite’s stability during mechani- cal grinding, the transformation to calcite does not occur until >375∘ C (B. C. Chak- oumakos, unpubl. data) further suggesting that although this polymorph is metastable, its thermodynamic stability is great enough to resist transformation to calcite under routine methods. Perhaps another explanation is that calcite has not been reported in previous studies because all other acipenserid species where the polymorph composition has been described were anadromous. Gauldie (1993), for example, reported on the polymorph composition of the anadromous shortnose sturgeon Acipenser brevirostrum Lesueur 1818 as only containing a vaterite fraction. It is possible that the chemistry of sea water, which influences the chemistry of the endolymph fluid that surrounds the otolith (Campana, 1999), inhibits formation of a calcite fraction. Sea water contains Mg concentrations several times higher than that of fresh water (Turner et al., 1981). Increased Mg concentrations have been shown to strongly inhibit calcite formation (Folk, 1974; Berner, 1975), which could explain a much smaller or non-existent calcite fraction in anadromous acipenserid otoliths. The A. gueldenstaedtii mentioned in Carlström (1963) are anadromous, however, and it was suggested that those otoliths had areas resembling a calcite fraction. Examination of the otoliths of anadromous acipenserids using neutron diffraction could resolve this discrepancy. Protein expression has been shown in several cases to control the type of CaCO3 polymorph nucleated in fish otoliths. Earlier studies suggest that most fish species contain only a single polymorph of CaCO3 (Carlström, 1963; Gauldie, 1993), but it may be possible that under certain conditions, changes in protein expression manifest- ing in changes in otolith polymorph composition occur (Söllner et al., 2003; Tohse et al., 2009; Ren et al., 2013; Weigele et al., 2016). It may be that other species of fish reported as only having single-polymorph otoliths actually have genetic makeup that allows for other polymorphs to crystallize during otolith formation. Söllner et al. (2003) experimentally demonstrated that as activity for the gene linked to aragonite nucleation in zebrafish Danio rerio (Hamilton 1822) otoliths was progressively reduced, the proteins involved in aragonite nucleation were altered leading to a progression towards a completely calcite otolith. This was a particularly interesting finding because D. rerio have fully aragonite otoliths and the change from normal gene expression led to a change in otolith polymorph composition. These findings in combination with the appearance of all three polymorphs of CaCO3 at various points throughout the evolutionary lineage of fishes suggest the possibility that more, or potentially even all, fish may have retained the genetic information to make otoliths with fractions of any of the three common polymorphs. Previous studies have pointed to environmental stress as the source for more than one polymorph in otoliths of an individual fish (Morat et al., 2008) and it could be that environmental stressors cause a change in protein expression leading to a change in polymorph composition. Similarly, it has also been suggested that hatchery fishes and other fishes under physiological stress from across the evolutionary tree maybe more likely to have vaterite incorporated into their otherwise aragonitic otoliths. These reports have included such distant taxa as coho salmon Oncorhynchus kisutch (Wal- baum 1792) (Sweeting et al., 2004) and red drum Sciaenops ocellatus (L. 1766) (David

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13085 ACIPENSERIDAE OTOLITHS ARE VATERITE AND CALCITE 7 et al., 1994). In fact, examination of large samples of otoliths in earlier studies than the present study has shown that there can be variability in CaCO3 polymorph composition among individuals of the same species. This variability may be attributable to individual changes in protein expression thus a greater variation in polymorph composition among species than commonly thought. For example, Gauldie (1993) examined 4000 orange roughy Hoplostethus atlanticus Collett 1889 otoliths (majority or total fraction aragonite) and found 0·4% of those to have partially vateritic saggitae, 0·1% to have totally vateritic saggitae, 0·1% to have partially calcitic saggitae and 0·05% to have a mixture of vaterite and calcite in their saggitae. Because the genetic pathways that led to nucleation of different polymorphs are unlikely to be repeatedly lost and re-evolved in fishes, examining the otoliths of more species using sensitive techniques such asneu- tron diffraction would probably yield reports of many more species with vaterite and calcite otolith fractions. In turn, an enhanced understanding of polymorph distribution among species may help to elucidate molecular pathways involved in otolith formation and a greater understanding of the evolution of otoliths and hearing. While the genetic factors and phenotypic responses responsible for nucleation of otoliths containing different polymorphs has been characterized (Söllner et al., 2003; Tohse et al., 2009; Ren et al., 2013), the crystalline structure of vaterite still remains the subject of debate (Demichelis et al., 2012, 2013; Falini et al., 2014). Overcoming these knowledge gaps may lead to new industrial and biomedical advances as vaterite already has a number of applications in the biomedical and personal care industries (Trushina et al., 2014). Fish otoliths may present a unique opportunity for understanding of the basic material properties of vaterite, potentially leading to a greater understanding of its diversity of morphologies (Kabalah-Amitai et al., 2013) and material properties (Falini et al., 2014). Moreover, fish otoliths present one of the few opportunities to understand links between this vaterite diversity and genetic controls due to its presence among multiple and diverse fish taxa. Using neutron diffraction to obtain quantitative information about the crystalline structure of fish otoliths is an important step towards understanding these materials and their evolutionary rise on a fundamental level. Determination of the CaCO3 polymorph composition of otoliths has important impli- cations for studies of otolith microchemistry, a technique that quantifies the elemental fingerprints in otoliths incorporated from water chemistry to reconstruct individual environmental history. Previous studies have reported differences in elemental concentrations between or among polymorphs using laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS; Melancon et al., 2005; Tzeng et al., 2007; Veinott et al., 2009). Because these studies, however, were unable to take newly discovered differences in polymorph densities into account during data post-processing (Wang & Becker, 2009; Demichelis et al., 2012, 2013), conclusions regarding differences in elemental concentrations among polymorphs must be re-examined. Crystalline density varies by polymorph where vaterite is the least dense form of CaCO3: marginally less dense than calcite and substantially less dense than aragonite (Demichelis et al., 2013). While there may be nothing about the polymorphs themselves that change the actual element: Ca ratios (mmol:mol), different polymorphs have different numbers of moles of Ca per identically sized piece of otolith. By far, the most commonly used technique for quantifying elemental concentrations in otoliths is LA-ICP-MS (Pracheil et al., 2014). During the data reduction phase of LA-ICP-MS analysis, data reduction software [i.e. GLITTER (Griffin et al., 2008), GeoPro (CETAC, 2011), AMS Mutchler et al., 2008)] normalizes concentrations of other elements to a constant value of Ca,

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13085 8 B. M. PRACHEIL ET AL. which was determined from aragonite otoliths. Because the Ca normalization value, however, is held constant by software for all data even though the actual Ca value is varying by polymorph, the elemental concentrations derived from the data reduction software will differ from the actual concentrations in the otolith. In other words, unless the Ca normalization value is changed for data collected from each polymorph, the element:Ca ratios will be incorrect. This issue is illustrated by Melancon et al. (2005) where Ca values of 1 × 106 were used as the constant, normalization value of Ca for both aragonite and vaterite fractions even though the actual values of Ca would differ due to differences in polymorph crystal densities. As a result, the element:Ca ratios are calculated with the same constant value for vaterite and aragonite even though the actual value of Ca would not be constant between polymorphs, thus leading to inaccurate results. Whether or not the differences are significant will probably depend on which polymorphs are present in an otolith and the proportions of the different polymorph fractions. Additional studies will need to be conducted to determine whether these differences in polymorph densities lead to significant differences in elemental concentrations determined in data reduction if the Ca normalization value is held constant.

The research at ORNL’s high flux isotope reactor and spallation neutron source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. This manuscript has been authored by employees of UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the U.S. Department of Energy. Accordingly, the United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.

References Berner, R. A. (1975). The role of magnesium in the crystal growth of calcite and aragonite from sea water. Geochimica et Cosmochimica Acta 39(4), 489–504. Bowen, C. A., Bronte, C. R., Argyle, R. L., Adams, J. V. & Johnson, J. E. (1999). Vateritic sagitta in wild and stocked lake trout: applicability to stock origin. Transactions of the American Fisheries Society 128, 929–938. Bruch, R. M. (1999). Management of lake sturgeon on the Winnebago System – long term impacts of harvest and regulations on population structure. Journal of Applied Ichthy- ology 15, 142–152. Campana, S. E. (1999). Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Marine Ecology Progress Series 188, 263–297. Carlström, D. (1963). A crystallographic study of vertebrate otoliths. Biological Bulletin 125, 441–463. David, A. W., Grimes, C. B. & Isely, J. J. (1994). Vaterite sagittal otoliths in hatchery-reared juvenile red drums. The Progressive Fish-Culturist 56, 301–303. Demichelis, R., Raiteri, P., Gale, J. D. & Dovesi, R. (2012). A new structural model for disorder in vaterite from first-principles calculations. CrystEngComm 14, 44–47. Demichelis, R., Raiteri, P., Gale, J. D. & Dovesi, R. (2013). The multiple structures of vaterite. Crystal Growth & Design 13, 2247–2251. Falini, G., Fermani, S., Reggi, M., Njegic-Džakula,´ B. & Kraljb, D. (2014). Evidence of structural variability among synthetic and biogenic vaterite. Chemical Communications 50, 15370–15373. Folk, R. L. (1974). The natural history of crystalline calcium carbonate: effect of magnesium content and salinity. Journal of Sedimentary Research 44(1), 40–53. Gauldie, R. W. (1986). Vaterite otoliths from Chinook salmon (Oncorhynchus tshawytscha). New Zealand Journal of Marine and Freshwater Research 20, 209–217.

Gauldie, R. W. (1993). Polymorphic crystalline structure of fish otoliths. Journal of Morphology 218, 1–28. Gauldie, R. W., Dunlop, D. & Tse, J. (1986). The remarkable lungfish otolith. New Zealand Journal of Marine and Freshwater Research 20, 81–92. Griffin, W. L., Powell, W. J., Pearson, N. J. & O’reilly, S. Y. (2008). GLITTER: data reduction software for laser ablation ICP-MS. In Laser Ablation ICP-MS in the Earth Sciences: Current Practices and Outstanding Issues (Sylvester, P.,ed), pp. 204–207. Mineralogical Association of Canada Short Course Series 40. Quebec: Mineralogical Association of Canada. Kabalah-Amitai, L., Mayzel, B., Kauffmann, Y., Fitch, A. N., Bloch, L., Gilbert, P. U., & Pokroy, B. (2013). Vaterite crystals contain two interspersed crystal structures. Science, 340(6131), 454–457. Jolivet, A., Bardeau, J.-F., Fablet, R., Paulet, Y.-M. & de Pontual, H. (2008). Understanding otolith biomineralization processes: new insights into microscale spatial distribution of organic and mineral fractions from Raman microspectrometry. Analytical and Bioana- lytical Chemistry 392, 551–560. Melancon, S., Fryer, B. J., Ludsin, S. A., Gagnon, J. E. & Yang, Z. (2005). Effects of crystal structure on the uptake of metals by lake trout (Salvelinus namaycush) otoliths. Canadian Journal of Fisheries and Aquatic Sciences 62, 2609–2619. Melancon, S., Fryer, B. J., Gagnon, J. E. & Ludsin, S. A. (2008). Mineralogical approaches to the study of biomineralization in fish otoliths. Mineralogical Magazine 72, 627–637. Morat, F., Betoulle, S., Robert, M., Thailly, A. F., Biagianti-Risbourg, S. & Lecomte-Finiger, R. (2008). What can otolith examination tell us about the level of perturbations of salmonid fish from the Kerguelen Islands? Ecology of Freshwater Fish 17, 617–627. Mutchler, S. R., Fedele, L. & Bodnar, R. J. (2008). Analysis Management System (AMS) for reduction of laser ablation ICPMS data. In Laser Ablation ICP-MS in the Earth Sciences: Current Practices and outstanding Issues (Sylvester, P., ed), pp. 318–327. Mineralogical Association of Canada Short Course Series 40. Quebec: Mineralogical Association of Canada. Neuefeind, J. C., Feygenson, M., Carruth, J., Hoffmann, R. & Chipley, K. (2012). The nanoscale ordered materials diffractometer NOMAD at the spallation neutron source SNS. Nuclear Instruments and Methods in Physics Research Section B 287, 68–75. Oliveira, A. M., Farina, M., Ludka, I. P. & Kachar, B. (1996). Vaterite, calcite and aragonite in the otoliths of three species of piranha. Naturwissenschaften 83, 133–135. Pracheil, B. M., Hogan, J. D., Lyons, J. & McIntyre, P. B. (2014). Using hard-part microchemistry to advance conservation and management of North American freshwater fishes. Fisheries 39, 451–465. Ren, D., Feng, Q. & Bourrat, X. (2013). The co-effect of organic matrix from carp otolith and microenvironment on calcium carbonate mineralization. Materials Science and Engi- neering C: Materials for Biological Applications 33, 3440–3449. Rietveld, H. M. (1969). A profile refinement method for nuclear and magnetic structures. Jour- nal of Applied Crystallography 2, 65–71. Söllner, C., Burghammer, M., Busch-Nentwich, E., Berger, J., Schwarz, H., Riekel, C. & Nicol- son, T. (2003). Control of crystal size and lattice formation by starmaker in otolith biomineralization. Science 302, 282–286. Sweeting, R. M., Beamish, R. J. & Neville, C. M. (2004). Crystalline otoliths in teleosts: com- parisons between hatchery and wild Coho salmon (Oncorhynchus kisutch)intheStrait of Georgia. Reviews in Fish Biology and Fisheries 14, 361–369. Toby, B. H. (2001). EXPGUI, a graphical user interface for GSAS. Journal of Applied Crystal- lography 34, 210–213. Tohse, H., Saruwatari, K., Kogure, T., Nagasawa, H. & Takagi, Y. (2009). Control of polymor- phism and morphology of calcium carbonate crystals by a matrix protein aggregate in fish otoliths. Crystal Growth & Design 9, 4897–4901. Tomás, J. & Geffen, A. J. (2003). Morphometry and composition of aragonite and vaterite otoliths of deformed laboratory reared juvenile herring from two populations. Journal of Fish Biology 63, 1383–1401.

Tomás, J., Geffen, A. J., Allen, I. S. & Berges, J. (2004). Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein? Comparative Biochemistry and Physiology Part A 139, 301–308. Trushina, D. B., Bukreeva, T. V., Kovalchuk, M. V. & Antipina, M. N. (2014). CaCO3 vaterite microparticles for biomedical and personal care applications. Materials Science and Engineering C: Materials for Biological Applications 45, 644–658. Turner, D. R., Whitfield, M. & Dickson, A. G. (1981). The equilibrium speciation of dissolved components in freshwater and seawater at 25∘ C and 1 atm pressure. Geochimica et Cos- mochimica Acta 45, 855–881. Tzeng, W. N., Chang, C. W., Wang, C. H., Shiao, J. C., Iizuka, Y., Yang, Y. J., You, C. F. & Ložys, L. (2007). Misidentification of the migratory history of anguillid eels by Sr/Ca ratios of vaterite otoliths. Marine Ecology Progress Series 348, 285–295. Veinott, G. I., Porter, T. R. & Nasdala, L. (2009). Using Mg as a proxy for crystal structure and Sr as an indicator of marine growth in vaterite and aragonite otoliths of aquaculture rainbow trout. Transactions of the American Fisheries Society 138, 1157–1165. Wang, J. & Becker, U. (2009). Structure and carbonate orientation of vaterite (CaCO3). Ameri- can Mineralogist 94, 380–386. Weigele, J., Franz-Odendaal, T. A. & Hilbig, R. (2016). Not all inner ears are the same: otolith matrix proteins in the inner ear of sub-adult cichlid fish, Oreochromis mossambicus, reveal insights into the biomineralization process. The Anatomical Record 299, 234–245.

Electronic References CETAC (2011). GeoPro Software User’s Guide. Omaha, NE: CETAC Technologies. Available at https://www.yumpu.com/en/document/view/45720708/geopro-software-users-guide- cetac-technologies/3/. Larson, A. C. & Von Dreele, R. B. (2000). General Structure Analysis System (GSAS).Los Alamos National Laboratory Report LAUR 86-748. Los Alamos, NM: University of California. Available at https://subversion.xor.aps.anl.gov/EXPGUI/gsas/all/GSAS% 20Manual.pdf/.