DOI: 10.5061/dryad.7h44j0zs4
title: Data from: Evaluating tooth strontium and barium as indicators of weaning age in Pacific walruses

description: A dataset of calcium-normalized 88Sr and 137Ba concentrations from laser
 ablation transects across the cementum layer of
 107 (female: n = 84, male: n = 23) Pacific
 walrus (Odobenus rosmarus divergens) teeth. Dataset includes spreadsheets
 containing elapsed time of laser ablation transect in seconds
 (ElapsedTime_s; laser transect speed = 5μm/s), calcium-normalized
 strontium concentrations (Sr_ppm_m88; values below limit of detection
 replaced with 1/2*limit of detection, see methods), and calcium-normalized
 barium concentrations (Ba_ppm_m137; values below limit of detection
 replaced with 0.5*limit of detection). Photos (in .tif format) of
 tooth cementum used to estimate the positions of cementum growth layer
 groups are included for each animal. For teeth where more than one laser
 ablation scar is visible in the photo, an arrow is included to indicate
 the laser ablation scar that corresponds with the included trace element
 data. Finally, a Word document containing metadata (Catalog Number/ID,
 Sex, Median Age Est., Coll. Year, Est. Birth Year), weaning age
 estimates produced by both a visual and mathematical method (Sr Vis. Est.,
 Sr Math. Est., Ba Vis. Est., Ba Math. Est.), and a descriptive
 grouping of the patterns of accumulation of Sr and Ba (Sr Pat., Ba Pat.)
 are included. See methods and accompanying paper in Methods in Ecology and
 Evolution for more details.
description: <i>Trace element analysis and data
 processing</i> Walrus teeth used for this study
 (female: n = 84, male: n = 23) were on loan from the University of Alaska
 Museum in Fairbanks, Alaska, the National Museum of Natural History,
 Smithsonian Institution, in Washington DC, and the Alaska Department of
 Fish & Game. These specimens each represented an individual
 animal, and were collected at various locations throughout the Pacific
 walrus range in the Bering and Chukchi seas. Most teeth originated from
 Alaska Native subsistence harvests, though a small number came from
 research expeditions. Dates of collection ranged from 1932 to 2016. In
 preparation for analysis, teeth were sectioned longitudinally using a
 slow-speed, water-cooled saw equipped with a diamond blade, creating a
 cross section of the center of the tooth with a thickness of ~1.5mm. This
 cross section was then polished using a rotary polishing wheel with a 3000
 grit smoothing disc, rinsed with ultrapure water, and allowed to dry.
 Specimens were rinsed and dried again immediately prior to trace element
 analysis.             Trace element analyses were
 conducted in the Advanced Instrumentation Lab at the University of Alaska
 Fairbanks (UAF), Fairbanks, Alaska. Concentrations of
 <sup>88</sup>Sr and <sup>137</sup>Ba were measured
 using an Agilent 7500ce Inductively Coupled Plasma Mass Spectrometer
 (ICP-MS; fitted with an Agilent 7500cs lens stack to improve sensitivity)
 coupled with a New Wave UP213 laser. Instrumental precision for the ICP-MS
 is reported at ± 5%. <sup>43</sup>Ca was used as an internal
 standard for these analyses, and all results are reported in parts per
 million (ppm). Measured element concentrations were compared to a United
 States Geological Survey microanalytical phosphate standard (MAPS-4), as
 well as a National Institute of Standards and Technology Standard
 Reference Material (SRM 610). Accuracy and precision were estimated by
 comparing element concentrations measured during ablation of the reference
 materials (n = 363) with reported concentrations. Sr and Ba measurements
 were both accurate to within 1% of reported values, with a precision (± 1
 standard deviation) of ± 4% and ± 5%, respectively. Laser transects were
 ablated at a beam width of 25μm, at 55% power, with a pulse frequency of
 10Hz, and a transect speed of 5μm/s. Dwell times were 0.02 seconds for
 <sup>43</sup>Ca, 0.01 seconds for <sup>88</sup>Sr,
 and 0.15 seconds for <sup>137</sup>Ba. Ablation was conducted
 at locations that maximized distance from the root of the tooth, where
 cementum GLGs converge and become distorted, while avoiding areas of tooth
 wear near the crown, where not all cementum layers are present for
 sampling. Each transect was ablated from the cementum-dentin interface
 (first year of life) to the outer edge of the tooth (final year of life),
 thereby measuring lifetime changes in element concentrations for each
 walrus (Fay, 1982).             Trace element data were
 extracted and processed in Igor Pro version 6.37 using the Iolite software
 package version 3.0. Statistical analyses were conducted using R version
 3.6.3 (R Core Team, 2020) with RStudio version 1.2.5033 (RStudio Team,
 2015). Limits of detection were calculated for each analytical run using
 the standard method applied by Iolite (Longerich, Jackson, &
 Günther, 1996). Typical limits of detection were 0.18ppm for Sr and
 0.34ppm for Ba. Element concentrations falling below the limit of
 detection were replaced with a value of one half the limit of detection
 (U.S. Environmental Protection Agency, 2000). Data points that were more
 than 4 standard deviations above or below the mean were considered
 outliers (Tukey, 1977). These were typically single data points believed
 to represent measurement errors generated during data collection and
 processing, rather than changes in element concentrations within the
 tooth, thus they were removed from subsequent analyses.
             All specimens used in this study were obtained from
 museum collections and/or Alaska Native subsistence harvests, thus this
 work is Institutional Animal Care and Use Committee (IACUC) exempt.
 Specimens from subsistence harvests were transferred to UAF for analysis
 under a Letter of Authorization from the United States Fish and Wildlife
 Service (USFWS) to Dr. L. Horstmann. <i>Weaning
 age assignment</i> After analysis on the ICP-MS, teeth
 were photographed under a Leica M165 C optical microscope coupled with a
 Leica DFC295 camera using reflected light. Level of magnification used
 when taking photographs varied depending on the size of the tooth, and was
 selected to maximize visibility of the GLGs. The first five GLGs in the
 tooth cementum were counted (Fay, 1982; Garlich-Miller, Stewart, Stewart,
 & Hiltz, 1993) and their positions marked on the images to denote
 the positions of the first five years of life on the laser ablation
 transect. Growth layer groups consist of paired bands of cementum, which
 accrete onto the tooth annually (Fay, 1982). The terminology used to
 describe growth layer groups has not been standardized and differences in
 the appearance of growth layers under reflected and transmitted light may
 cause confusion. In this study, the term “light layer” refers to the
 opaque, hypercalcified layer that accretes during periods of faster
 growth. This layer represents the period from approximately mid-April to
 mid-December (Clark, Horstmann, & Misarti, 2020). Under reflected
 light, this layer appears white, but it is dark under transmitted light.
 The term “dark layer” refers to the more translucent, hypocalcified layer
 that is built during periods of slower growth. The dark layer represents
 the period from around mid-December to mid-April (Clark et al., 2020).
 This layer appears dark under reflected light, because it allows light to
 pass through, thus it appears white when using transmitted light.
 Individually, each light or dark layer is referred to as a growth layer,
 whereas a pair of light and dark layers, representing one year of growth,
 is referred to as a growth layer group (Laws, 1952). Growth layers were
 identified and marked collaboratively by three observers (C.T.C., L.H.,
 and N.M.), and the positions of these growth layers were revisited on at
 least two additional days to confirm their positions on the laser ablation
 transect. Estimates of overall age (age at death) were also generated for
 each animal using the methods described in Clark et al. (2020), and median
 age estimates were used to calculate approximate birth year for all
 animals examined in this study (Table S1). Analysis of Sr and Ba data for
 age-at-weaning estimation was restricted to the first five years of life.
 Prior to analysis, data were smoothed using a Savitzky-Golay filter from
 the R package <i>prospectr</i> (Stevens &
 Ramirez-Lopez, 2014) with a window of 15 data points. This approach was
 chosen because it reduces noise in the data, allowing underlying patterns
 to be seen more clearly, but results in very little data loss and uses a
 centered method, thus does not shift the data left or right like some
 other smoothing methods. Weaning is a process, rather than a single event,
 beginning with the first intake of non-milk food items and ending when
 consumption of mother’s milk has ceased completely. For the purposes of
 this study, a walrus was considered to have weaned at the point where the
 early life signal in its cementum Sr or Ba time series, associated with
 consumption of milk, transitioned to relatively stable values that
 persisted throughout the animal’s adult life. The weaning age estimates
 generated here thus represent the end of the weaning process and the
 transition to an entirely non-milk diet. Data were first analyzed
 visually, with a single observer (C.T.C.) identifying patterns in Sr and
 Ba indicative of weaning (e.g., abrupt decreases/increases in
 concentration, changes in slope, etc.) based on expected patterns from the
 published literature (Humphrey, Dean, et al., 2008; Humphrey, Dirks, et
 al., 2008; Austin et al., 2013; Humphrey, 2014; Tsutaya & Yoneda,
 2015; Smith et al., 2017). Because Pacific walrus tooth cementum typically
 begins growing in the second or third month after birth (Fay, 1982), a
 signal associated with the onset of nursing (low and stable Sr
 concentrations/rapidly increasing Ba concentrations) was not expected.
 Once a suspected weaning signal was selected, a plot of the data was
 overlaid on an image of the individual tooth from which they were
 collected, with the x-axis aligned to the laser ablation scar (Fig. 1). In
 this way, the location of the estimated weaning signal could be directly
 assigned to the growth layer in which it occurred. After visual analysis,
 weaning estimates were made using a mathematical approach, in which the
 data were subjected to an iterative method of change point detection using
 the R package <i>segmented</i> (V.R.M. Muggeo, 2008). This
 tool detects underlying changes in slope within a time series and, for the
 purposes of this analysis, was restricted to assigning a single change
 point within the first five years of an animal’s life. Because the
 mathematical approach was susceptible to both positive and negative
 changes in slope, and was restricted to assigning only a single change
 point to the elemental time series, periods of increasing or stable
 element concentrations at the beginning of an animal’s life (relatively
 rare, and possibly associated with the onset of and/or a period of
 sustained nursing) were omitted from the Sr and Ba data when applying the
 mathematical weaning estimation approach. While these variations in the
 patterns of element accumulation in the first year of life may contain
 important information, they were excluded for their tendency to interfere
 with the ability of the segmented regressions to assign a change point
 associated with the end of weaning and the attainment of an entirely
 non-milk diet.             Weaning age data were
 analyzed qualitatively, by comparing estimates of age-at-weaning generated
 by the visual and mathematical estimation methods for Sr and Ba to walrus
 weaning age estimates from the published literature. The performance of
 these two different estimation methods was compared by examining the
 average differences in the predictions generated by the two approaches.
 Chi-squared tests were used to examine differences in the weaning age
 predictions generated for female and male walruses. Significance was
 assessed using an alpha of 0.05. Weaning age estimates were plotted by
 approximate year of birth for each individual, and visually examined to
 determine whether any patterns in estimated age-at-weaning existed across
 the ~100 year span represented by the walrus teeth in this study. Regional
 differences in weaning age estimates were not assessed, as only location
 of harvest/collection was available for the animals in this study. The
 Pacific walrus population is large and panmictic, with little evidence for
 internal structure (Beatty et al., 2020). Individual walruses move widely
 over the species’ range (Jay, Udevitz, Kwok, Fischbach, & Douglas,
 2010; Jay, Fischbach, & Kochnev, 2012; Beatty et al., 2016), thus
 location of collection is unlikely to provide useful information about the
 regions in which an individual walrus spent its life or to reflect
 association with a distinct group within the larger population.