Total mercury concentrations in Steller sea lion bone: Variability among locations and elements.

Mercury is a global contaminant that bioaccumulates in a tissue-specific manner in long-lived predators such as Steller sea lions (SSL). Bone is a well-preserved material amenable for studying millennial scale trends; however, little is known about the distribution and variability of total mercury concentrations ([THg]) within individual bones and among bone elements in SSL. We assessed SSL bone [THg] variability with respect to physiologic age, bone type, longitudinally within a bone, and among bone elements. Pup bones (mean ± SD; 31.4 ± 13.58 ppb) had greater [THg] than adults (7.9 ± 1.91 ppb). There were greater and more variable [THg] within individual long bones near epiphyses compared to mid-diaphysis. Pup spongy bone in ribs (62.7 ± 44.79 ppb) had greater [THg] than long bones (23.5 ± 8.83 ppb) and phalanges (19.6 ± 10.78 ppb). These differences are likely due to variability in bone composition, growth, and turnover rate. This study informs standardized sampling procedures for [THg] in bone to improve interpretations of mercury variability over time and space.

Ancient DNA indicates a century of overhunting did not reduce genetic diversity in Pacific Walruses (Odobenus rosmarus divergens).

Pacific Walruses (Odobenus rosmarus divergens [Illiger 1815]) are gregarious marine mammals considered to be sentinels of the Arctic because of their dependence on sea ice for feeding, molting, and parturition. Like many other marine mammal species, their population sizes were decimated by historical overhunting in the nineteenth and twentieth centuries. Although they have since been protected from nearly all commercial hunting pressure, they now face rapidly accelerating habitat loss as global warming reduces the extent of summer sea ice in the Arctic. To investigate how genetic variation was impacted by overhunting, we obtained mitochondrial DNA sequences from historic Pacific Walrus samples in Alaska that predate the period of overhunting, as well as from extant populations. We found that genetic variation was unchanged over this period, suggesting Pacific Walruses are resilient to genetic attrition in response to reduced population size, and that this may be related to their high vagility and lack of population structure. Although Pacific Walruses will almost certainly continue to decline in number as the planet warms and summer sea ice is further reduced, they may be less susceptible to the ratcheting effects of inbreeding that typically accompany shrinking populations.

Steroid hormones in Pacific walrus bones collected over three millennia indicate physiological responses to changes in estimated population size and the environment.

The Pacific walrus (Odobenus rosmarus divergens) is an iconic Arctic marine mammal and an important resource to many Alaska Natives. A decrease in sea ice habitat and unknown population numbers has led to concern of the long-term future health of the walrus population. There is currently no clear understanding of how walrus physiology might be affected by a changing Arctic ecosystem. In this study, steroid hormone concentrations (progesterone, testosterone, cortisol and estradiol) were analysed in walrus bones collected during archaeological [3585-200 calendar years before present (BP)], historical [1880-2006 common era (CE)] and modern (2014-2016 CE) time periods, representing ~ 3651 years, to track changes in reproductive activity and cortisol concentrations (biomarker of stress) over time. Our results show that modern walrus samples have similar cortisol concentrations (median = 43.97 ± standard deviation 904.38 ng/g lipid) to archaeological walruses (38.94 ± 296.17 ng/g lipid, P = 0.75). Cortisol concentrations were weakly correlated with a 15-year average September Chukchi Sea ice cover (P = 0.002, 0.02, r 2 = 0.09, 0.04, for females and males, respectively), indicating a possible physiological resiliency to sea ice recession in the Arctic. All steroid hormones had significant negative correlations with mean walrus population estimates from 1960 to 2016 (P < 0.001). Progesterone in females and testosterone in males exhibited significant correlations with average September Chukchi Sea ice cover for years 1880-2016 (P < 0.001 for both, r2 = 0.34, 0.22, respectively). Modern walruses had significantly lower (P = < 0.001) reproductive hormone concentrations compared with historic walruses during times of rapid population increase, indicative of a population possibly at carrying capacity. This is the first study to apply bone as a tool to monitor long-term changes in hormones that may be associated with changes in walrus population size and sea ice cover.

Walrus teeth as biomonitors of trace elements in Arctic marine ecosystems.

Effective biomonitoring requires an understanding of the factors driving concentrations of the substances or compounds of interest in the tissues of studied organisms. Biomonitoring of trace elements, and heavy metals in particular, has been the focus of much research; however, the complex roles many trace elements play in animal and plant tissues can make it difficult to disentangle environmental signals from physiology. This study examined the concentrations of 15 trace elements in the teeth of 122 Pacific walruses (Odobenus rosmarus divergens) to investigate the potential for walrus teeth as biomonitors of trace elements in Arctic ecosystems. Elemental concentrations were measured across cementum growth layer groups (GLGs), thereby reconstructing a lifetime history of element concentrations for each walrus. The locations of GLGs were used to divide trace element time series into individual years, allowing each GLG to be associated with an animal age and a calendar year. The elements studied exhibited a great deal of complexity, reflecting the numerous factors responsible for generating tooth trace element concentrations. Generalized linear mixed models were used to investigate the importance of age and sex in explaining observed variation in trace element concentrations. Some elements exhibited clear physiological signals (particularly zinc, strontium, barium, and lead), and all elements except arsenic varied by age and/or sex. Pearson's correlations revealed that elements were more strongly correlated among calendar years than among individual walruses, and correlations of trace elements within individual walruses were generally inconsistent or weak. Plots of average elemental concentrations through time from 1945 to 2014 further supported the correlation analyses, with many elements exhibiting similar patterns across the ~70-year period. Together, these results indicate the importance of physiology in modulating tooth trace element concentrations in walrus tooth cementum, but suggest that many trace elements reflect a record of environmental exposure and dietary intake/uptake.

Evaluating tooth strontium and barium as indicators of weaning age in Pacific walruses.

Lactation length and weaning age provide important information about maternal investment, which can reflect the health and nutritional status of the mother, as well as broader reproductive strategies in mammals. Calcium-normalized strontium (Sr) and barium (Ba) concentrations in the growth layers of mammalian teeth differ for nursing animals and those consuming non-milk foods, thus can be used to estimate age-at-weaning. To date, this approach has been used only for terrestrial animals, and almost exclusively for primates.The goal of this study was to determine whether Sr and Ba concentrations in the cementum of Pacific walrus Odobenus rosmarus divergens teeth can be used to estimate weaning age. Teeth from 107 walruses were analysed using laser ablation inductively coupled plasma mass spectrometry, and calcium-normalized 88Sr and 137Ba concentrations were quantified.For most walruses, both Sr and Ba concentrations exhibited rapid changes in early life. Ba concentrations matched closely with expected patterns in the published literature, rapidly declining from high to low concentrations (typically from ~10 ppm to ~5 ppm). In contrast, Sr exhibited a pattern opposite to that presented in studies of terrestrial mammals, appearing nearly identical to Ba (typically declining from ~400 ppm to ~200 ppm). To explain these findings, we present conceptual models of the factors generating weaning signals in Sr and Ba for terrestrial mammals, as well as a new, hypothetical model for walruses. Both a visual and mathematical approach to weaning age estimation indicated a median weaning age of walruses at the end of the second year of life (in the second dark layer of the tooth cementum), with many walruses estimated to have weaned in their third year of life, and a smaller group weaning in their fourth or fifth year. This is later than expected, given a published estimate of walrus weaning at 18-24 months.These results do not conclusively support the use of tooth Sr and Ba for estimating weaning age in walruses, and further research is warranted to better understand the drivers of the observed patterns of Ba and Sr accumulation in walrus teeth.

Determining sex of adult Pacific walruses from mandible measurements.

Pacific walruses (Odobenus rosmarus divergens) play a vital role in Arctic marine ecosystems and the subsistence lifestyle of Alaska Native communities. Museum collections contain numerous archaeological and historic walrus specimens that have proven useful in a variety of studies; however, for many cases, the sex of these specimens is unknown. Sexes of adult (> 5 years determined by tooth aging) Atlantic walruses (Odobenus rosmarus rosmarus) have been accurately determined in previous studies using mandible measurements. We tested the validity of this approach for Pacific walruses, and used full fusion of the mandibular symphysis to define adults. Using high precision digital calipers (± 0.01 mm), four measurements were taken either on the left or right side of 91 walrus mandibles: 80 modern mandibles (70 known-sex specimens; 10 unknown-sex specimens) and 11 archaeological mandibles of unknown sex. We used linear discriminant function analysis (LDFA) to determine what measurements best distinguished Pacific walrus males from females. Minimum mandible thickness had the most predictive power, whereas mandible length, height, and depth, were less predictive. Posterior probabilities indicated that LDFA classified the known-sex Pacific walruses with 100% accuracy, and unknown sex with ≥ 90% probability. The ability to define the sex of unknown individuals accurately could greatly increase the sample size of future projects dealing with skeletal remains, and will improve future research efforts.

Zinc concentrations in teeth of female walruses reflect the onset of reproductive maturity.

Age at maturity is an important parameter in many demographic models and, for some species, can be difficult to obtain using traditional methods. Incremental growth structures act as biological archives, recording information throughout an organism's life and possibly allowing for the reconstruction of life history events. Concentrations of zinc (Zn) in animal tissues are known to be linked to life history, physiology and reproduction and may be retained in incremental growth structures. This study reconstructed lifetime Zn concentrations in teeth (n = 93) of female Pacific walruses (Odobenus rosmarus divergens) collected from 1932-2016. Zn displayed a characteristic pattern of accumulation, with a change point marking the beginning of a lifelong, linear increase in Zn concentrations. We hypothesized that this change point marks the onset of reproductive maturity. The age at which the change point occurred (agecp) was estimated by counting tooth cementum growth layers. These estimates closely matched literature values of timing of first ovulation in female walruses. Total number of ovulations (estimated from ovary corpora counts from paired tooth/ovary specimens) was closely related to reproductive lifespan (total lifespan - agecp; R2 = 0.70). Further, agecp tracked changes in Pacific walrus population size as a proportion of carrying capacity, decreasing when the population was depleted by commercial hunting and peaking when carrying capacity was exceeded. This novel approach will aid walrus management, and is likely applicable to other species, offering a potentially powerful tool for research, management and conservation of wildlife populations.

Author Correction: Lipid normalization and stable isotope discrimination in Pacific walrus tissues.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Lipid normalization and stable isotope discrimination in Pacific walrus tissues.

Analysis of stable carbon and nitrogen isotope values (δ13C and δ15N) of animal tissues can provide important information about diet, physiology, and movements. Interpretation of δ13C and δ15N values, however, is influenced by factors such as sample lipid content, tissue-specific isotope discrimination, and tissue turnover rates, which are typically species- and tissue-specific. In this study, we generated lipid normalization models for δ13C and investigated the effects of chemical lipid extractions on δ13C and δ15N in Pacific walrus (Odobenus rosmarus divergens) muscle, liver, and skin. We also evaluated tissue-specific isotope discrimination in walrus muscle, liver, skin, and bone collagen. Mean δ13Clipid-free of skin and bone collagen were similar, as were mean δ15N of muscle and liver. All other tissues differed significantly for both isotopes. Differences in δ13Clipid-free and δ15N among tissues agreed with published estimates of marine mammal tissue-specific isotope discrimination factors, with the exception of skin. The results of this work will allow researchers to gain a clearer understanding of walrus diet and the structure of Arctic food webs, while also making it possible to directly compare the results of contemporary walrus isotope research with those of historic and paleoecological studies.

A novel method to measure steroid hormone concentrations in walrus bone from archaeological, historical, and modern time periods using liquid chromatography/tandem mass spectrometry.

RATIONALE: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was validated and utilized to measure and analyze four steroid hormones related to stress and reproduction in individual samples from a novel tissue, Pacific walrus (Odobenus rosmarus divergens, herein walrus) bone. This method determines steroid hormone concentrations in the remote walrus population over millennia from archaeological (>200 bp), historical (200-20 bp), and modern (2014-2016) time periods. METHODS: Lipids were extracted from walrus bone collected from these periods using methanol before LC/MS/MS analysis. Isotopically labeled internal standards for each target hormone were added to every sample. Analytical and physiological validations were performed. Additionally, a tissue comparison was done among paired walrus bone, serum, and blubber samples. A rapid resolution liquid chromatography system coupled to a QqQ mass spectrometer was used to analyze all samples after derivatization for progesterone, testosterone, cortisol, and estradiol concentrations. Multiple reaction monitoring was used for MS analysis and data were acquired in positive electrospray ionization mode. RESULTS: Progesterone, testosterone, cortisol, and estradiol were linear along their respective standard calibration curves based on their R2 values (all > 0.99). Accuracy ranged from 93-111% for all hormones. The recovery of extraction, recovery of hormones without matrix effect, was 92-101%. The overall process efficiency of our method for measuring hormones in walrus bone was 93-112%. Progesterone and testosterone concentrations were not affected by reproductive status among adult females and males, respectively. However, estradiol was different among pregnant and non-pregnant adult females. Overall, steroid hormones reflect a long-term reservoir in cortical bone. This method was also successfully applied to walrus bone as old as 3585 bp. CONCLUSIONS: LC/MS/MS analysis of bone tissue (0.2-0.3 g) provides stress and reproductive data from elusive walruses that were alive thousands of years ago. Based on physiological validations, tissue comparison, and published literature, steroid hormone concentrations measured in walrus cortical bone could represent an accumulated average around a 10-20-year time span. By investigating how stress and reproductive physiology may have changed over the past ~3000 years based on bone steroid hormone concentrations, this method will help answer how physiologically resilient walruses are to climate change in the Arctic.

Compound-specific amino acid δ15 N values in archaeological shell: Assessing diagenetic integrity and potential for isotopic baseline reconstruction.

RATIONALE: Reconstructing stable isotope (SI) ratios at the base of paleo-food webs is often challenging. For coastal systems, the SI ratios of organic matter in archeological shell represents a possible solution, providing a direct record of primary consumer SI ratios in the littoral zone. However, shell is often porous, with organic compounds susceptible to diagenetic alteration or contamination. If molecular isotopic information is well preserved, compound-specific amino acid isotope analysis (CSI-AA) has the potential to provide direct proxies for baseline SI ratios, bypassing many contamination issues, and to allow assessment of the diagenetic state. METHODS: We collected shell from both archeological middens and nearby littoral zones in coastal Alaska, and used a simple organic extraction approach based on decalcification with sequential weak HCl additions to liberate organic material. We measured CSI-AA patterns, molar AA distributions, and the CSI-AA degradation parameter (ΣV), in the context of bulk SI ratios in fossil shell, modern shell, and soft tissue from five common taxa (urchin, limpet, mussel, periwinkle, chiton). RESULTS: CSI-AA patterns in both soft tissue and shell were consistent with primary consumers, and were indistinguishable in most modern and fossil shell pairs, showing that amino acid δ15 N values can be well preserved in archeological shell. AA molar distributions were also similar, although most fossil shell was enriched in Asx and Gly. Comparison between CSI-AA results from modern specimens confirmed that the source AA group (tracking isotopic baselines) are transferred without substantial modification into the shell record. In contrast, the Trophic AA group had elevated δ15 N values in shell versus soft tissue for all taxa examined, suggesting that a correction factor will be required for any CSI-AA proxies using these AAs. CONCLUSIONS: Overall, this new data indicates that the CSI-AA analysis of fossil shell represents a promising new approach to determining isotopic baselines in coastal paleo-ecosystems.

Maize (Zea mays) consumption in the southern andes (30°-31° S. Lat): Stable isotope evidence (2000 BCE-1540 CE).

OBJECTIVES: The timing and dietary role of maize agriculture is central to archaeological discussions in the Andean region. In the semi-arid region of northern Chile (SARNC), archaeological models propose that maize was adopted during the Early Ceramic period in tandem with pottery and sedentism. Through stable isotope (SI) analyses, of bone collagen and apatite, this study assesses the timing of maize introduction, diachronic changes (2,000 BCE to 1,540 CE.), and synchronic dietary variability in the prehistoric SARNC. MATERIALS AND METHODS: Fifty-two prehistoric individuals from SARNC were analyzed for δ13 Cap , δ13 Ccol, and δ15 N. Descriptive statistics were used to characterize the results by period and location (inland and coast). Between-periods (ANOVA or Kruskal-Wallis tests), and synchronic comparisons (inland vs. coast; Student's t-tests), were conducted. A SIAR model was run to further evaluate dietary changes. Dietary interpretations are based on food web data. RESULTS: Coastal groups show significant changes in the diet during the Middle (900-1,000CE; enrichment in δ13 C), and Late Intermediate periods (100-1450CE; when the Δ13 Cap-col is above 5.2‰). In the inland, significant changes in SI occurred in the Late Intermediate period (δ13 C enrichment). In the Late period, the inland diet became enriched for δ15 N. Synchronic comparisons showed coastal individuals to have higher δ15 N. DISCUSSION: The popularization of maize in the SARNC was not associated with the appearance of pottery and/or sedentism, and its role as a dietary staple was a late phenomenon (c.a. 1,000CE). The results obtained in this study show that the adoption and consumption of maize varied dramatically in the Southern Andes.

Antiquity and geographic distribution of cranial modification among the prehistoric groups of Fuego-Patagonia, Chile.

OBJECTIVES: Nineteenth and twentieth century documents testify that four ethnic groups, generally classified as terrestrial hunters or canoe nomads, inhabited Fuego-Patagonia. Archaeologically, however, their presence and temporal depth remains unknown. This study analyzes the antiquity and geographic distribution of cranial modification, a highly visible symbol of social identity, in Fuego-Patagonia, Chile, to assess whether it expressed ethnic affiliation. MATERIALS AND METHODS: A total of 60 adult skulls from Southern Patagonia (n = 32; 53.3%) and Tierra del Fuego (n = 28; 46.7%) were examined for age-at-death, sex and cranial modification with standard methods. Individuals were further categorized as terrestrial (n = 26; 43.3%), marine (n = 21; 35%) or indetermined hunter-gatherers (n = 13; 21.7%) based on the archaeological site's characteristics, geographic location, and isotopic information. RESULTS: Thirty percent (n = 18) of the skulls in this study were modified, and most of the modified skulls (n = 15) presented a tabular-erect shape. No statistically significant differences were identified between Fuegians and Patagonians, males or females, or between the different types of adaptation and geographic locations. DISCUSSION: Thus, this Late Holocene, widely distributed practice, was not a reflection of ethnicity, but a material expression of information circulation and the complex social relations that these small-size groups had with one another. These results suggest that the emergence of modern ethnic identities in the region is a historic process that resulted from the interaction of local groups with European and Criollos.