Palaeoproteomic identification of breast milk protein residues from the archaeological skeletal remains of a neonatal dog.

Accurate postmortem estimation of breastfeeding status for archaeological or forensic neonatal remains is difficult. Confident identification of milk-specific proteins associated with these remains would provide direct evidence of breast milk consumption. We used liquid chromatography coupled to tandem mass spectrometry (MS) to confidently identify beta-lactoglobulin-1 (LGB1) and whey acidic protein (WAP), major whey proteins associated with a neonatal dog (Canis lupus familiaris) skeleton (430-960 cal AD), from an archaeological site in Hokkaido, Japan. The age at death of the individual was estimated to be approximately two weeks after birth. Protein residues extracted from rib and vertebra fragments were analyzed and identified by matching tandem MS spectra against the dog reference proteome. A total of 200 dog protein groups were detected and at least one peptide from canine LGB1 and two peptides from canine WAP were confidently identified. These milk proteins most probably originated from the mother's breast milk, ingested by the neonate just before it died. We suggest the milk diffused outside the digestive apparatus during decomposition, and, by being absorbed into the bones, it partially preserved. The result of this study suggests that proteomic analysis can be used for postmortem reconstruction of the breastfeeding status at the time of death of neonatal mammalian, by analyzing their skeletal archaeological remains. This method is also applicable to forensic and wildlife studies.

"Mind the gap"-Assessing methods for aligning age determination and growth rate in multi-molar sequences of dietary isotopic data.

OBJECTIVES: Creating multi-tooth sequences of micro-sampled stable isotope (SI) analytical data can help track 20+ years of individual dietary history. Inferences about individual and population level behavioral patterns require cross-calibration of the timing of dietary changes recorded by each tooth. Dentin sections from contemporaneous tissues (eg, in M1 and M2) reflect dietary signature for the time of growth. Contemporary sections should produce similar values, allowing alignment of temporally overlapping portions of teeth into multi-tooth sequences. Published methods for determining the ages of incremental sections do not provide guidance for adjustment when poor alignment between individual tooth sequences is encountered. The primary objective is to address this problem; examine cause(s), assess the effects of the standard growth-model on available age-assessment techniques, and provide a viable solution. METHODS: Investigating difficulty in aligning a 3-molar sequence at Shamanka II, an Early Neolithic (7000-5700 BP) Kitoi hunter-gatherer cemetery in Cis-Baikal, Siberia, we employed 10 age assessment models and 13 variants of 2 published growth rate methods on 3 individuals of different age and sex. RESULTS: At Shamanka II, dentin initiation and/or growth rates were different from the mostly European, reference populations used to create published age-estimation/growth rate models. Initiation ages for M2 and M3 were delayed. Root formation rates were on the rapid end of known development parameters. CONCLUSIONS: Age-assessment methods customized to dentin initiation ages and growth parameters of Siberian populations produced a hybrid growth rate model for dentin section ages and improved alignment for multi-tooth SI sequences over published models.

The first horse herders and the impact of early Bronze Age steppe expansions into Asia.

The Yamnaya expansions from the western steppe into Europe and Asia during the Early Bronze Age (~3000 BCE) are believed to have brought with them Indo-European languages and possibly horse husbandry. We analyzed 74 ancient whole-genome sequences from across Inner Asia and Anatolia and show that the Botai people associated with the earliest horse husbandry derived from a hunter-gatherer population deeply diverged from the Yamnaya. Our results also suggest distinct migrations bringing West Eurasian ancestry into South Asia before and after, but not at the time of, Yamnaya culture. We find no evidence of steppe ancestry in Bronze Age Anatolia from when Indo-European languages are attested there. Thus, in contrast to Europe, Early Bronze Age Yamnaya-related migrations had limited direct genetic impact in Asia.

Barley (Hordeum vulgare) in the Okhotsk culture (5th-10th century AD) of northern Japan and the role of cultivated plants in hunter-gatherer economies.

This paper discusses archaeobotanical remains of naked barley recovered from the Okhotsk cultural layers of the Hamanaka 2 archaeological site on Rebun Island, northern Japan. Calibrated ages (68% confidence interval) of the directly dated barley remains suggest that the crop was used at the site ca. 440-890 cal yr AD. Together with the finds from the Oumu site (north-eastern Hokkaido Island), the recovered seed assemblage marks the oldest well-documented evidence for the use of barley in the Hokkaido Region. The archaeobotanical data together with the results of a detailed pollen analysis of contemporaneous sediment layers from the bottom of nearby Lake Kushu point to low-level food production, including cultivation of barley and possible management of wild plants that complemented a wide range of foods derived from hunting, fishing, and gathering. This qualifies the people of the Okhotsk culture as one element of the long-term and spatially broader Holocene hunter-gatherer cultural complex (including also Jomon, Epi-Jomon, Satsumon, and Ainu cultures) of the Japanese archipelago, which may be placed somewhere between the traditionally accepted boundaries between foraging and agriculture. To our knowledge, the archaeobotanical assemblages from the Hokkaido Okhotsk culture sites highlight the north-eastern limit of prehistoric barley dispersal. Seed morphological characteristics identify two different barley phenotypes in the Hokkaido Region. One compact type (naked barley) associated with the Okhotsk culture and a less compact type (hulled barley) associated with Early-Middle Satsumon culture sites. This supports earlier suggestions that the "Satsumon type" barley was likely propagated by the expansion of the Yayoi culture via south-western Japan, while the "Okhotsk type" spread from the continental Russian Far East region, across the Sea of Japan. After the two phenotypes were independently introduced to Hokkaido, the boundary between both barley domains possibly existed ca. 600-1000 cal yr AD across the island region. Despite a large body of studies and numerous theoretical and conceptual debates, the question of how to differentiate between hunter-gatherer and farming economies persists reflecting the wide range of dynamic subsistence strategies used by humans through the Holocene. Our current study contributes to the ongoing discussion of this important issue.

A 9,000 Year History of Seal Hunting on Lake Baikal, Siberia: The Zooarchaeology of Sagan-Zaba II.

Sagan-Zaba II, a habitation site on the shore of Siberia's Lake Baikal, contains a record of seal hunting that spans much of the Holocene, making it one of the longest histories of seal use in North Asia. Zooarchaeological analyses of the 16,000 Baikal seal remains from this well-dated site clearly show that sealing began here at least 9000 calendar years ago. The use of these animals at Sagan-Zaba appears to have peaked in the Middle Holocene, when foragers used the site as a spring hunting and processing location for yearling and juvenile seals taken on the lake ice. After 4800 years ago, seal use declined at the site, while the relative importance of ungulate hunting and fishing increased. Pastoralists began occupying Sagan-Zaba at some point during the Late Holocene, and these groups too utilized the lake's seals. Domesticated animals are increasingly common after about 2000 years ago, a pattern seen elsewhere in the region, but spring and some summer hunting of seals was still occurring. This use of seals by prehistoric herders mirrors patterns of seal use among the region's historic and modern groups. Overall, the data presented in the paper demonstrate that Lake Baikal witnessed thousands of years of human use of aquatic resources.

Skeletal growth in early and late Neolithic foragers from the Cis-Baikal region of Eastern Siberia.

Skeletal growth is explored between Early Neolithic (EN) (8000 to 6800 BP) and Late Neolithic (LN) (6000 to 5200 BP) foragers from the Cis-Baikal region of Eastern Siberia. Previous studies suggest that increased systemic stress and smaller adult body size characterize the EN compared to LN. On this basis, greater evidence for stunting and wasting is expected in the EN compared to LN. Skeletal growth parameters assessed here include femoral and tibial lengths, estimated stature and body mass, femoral midshaft cortical thickness, total bone thickness, and medullary width. Forward selection was used to fit polynomial lines to each skeletal growth parameter relative to dental age in the pooled samples, and standardized residuals were compared between groups using t tests. Standardized residuals of body mass and femoral length were significantly lower in the EN compared to LN sample, particularly from late infancy through early adolescence. However, no significant differences in the standardized residuals for cortical thickness, medullary width, total bone thickness, tibial length, or stature were found between the groups. Age ranges for stunting in femoral length and wasting in body mass are consistent with environmental perturbations experienced at the cessation of breast feeding and general resource insecurity in the EN compared to LN sample. Differences in relative femoral but not tibial length may be associated with age-specific variation in growth-acceleration for the distal and proximal limb segments. Similarity in cortical bone growth between the two samples may reflect the combined influences of systemic and mechanical factors on this parameter.

Lower limb activity in the Cis-Baikal: entheseal changes among middle Holocene Siberian foragers.

Lower limb entheseal changes are evaluated in order to reconstruct activity levels and more fully understand cultural and behavioral variation among the middle Holocene (ca. 9,000-3,000 years BP) foragers of Siberia's Cis-Baikal region. The four cemetery samples examined span a period of diachronic change characterized by an 800- to 1,000-year discontinuity in the use of formal cemeteries in the region. Two of the cemetery samples represent the early Neolithic Kitoi culture, dating from 8,000 to 7,000/6800 cal. BP; the other two represent the late Neolithic-early Bronze Age Isakovo-Serovo-Glazkovo (ISG) cultural complex, dating from 6,000/5,800 to 4,000 cal. BP. Findings suggest a dynamic pattern of cultural variability in the Cis-Baikal, with spatial distribution (i.e., site location within particular microregions) appearing to be just as important a factor as cultural/temporal affiliation in explaining intersample differences in entheseal morphology. In addition, intrasample comparisons reveal increasing sexual disparity with advancing age at death, emphasizing the influence of sex-related activities on lower limb entheseal changes. Finally, results from the separate fibrous and fibrocartilaginous datasets appear to be largely congruous, implying that activity patterns in the Cis-Baikal may have similar effects on the morphology of both types of entheses.

Infant and child diet in Neolithic hunter-fisher-gatherers from Cis-Baikal, Siberia: intra-long bone stable nitrogen and carbon isotope ratios.

Analysis of stable nitrogen and carbon isotopes (δ(15) N and δ(13) C) from subadults and adults allows for assessment of age-related dietary changes, including breastfeeding and weaning, and adoption of an adult diet. In one of the first studies of hunter-fisher-gatherer subadults from Eurasia, three Neolithic (8,800-5,200 calBP) mortuary sites from southwestern Siberia are analyzed to evaluate hypothesized differences in weaning age between Early versus Late Neolithic groups. An intra-individual sampling methodology is used to compare bone formed at different ages. Collagen samples (n = 143) from three different growth areas of long bones-the proximal metaphysis, diaphysis, and distal metaphysis-were obtained from 49 subadults aged birth to 10 years. In infants (birth to 3 years, n = 23) contrasting the δ(15) N values of the metaphysis, which contains newer bone, to the δ(15) N values of the diaphysis, which contains older bone, permits a more precise determination of breastfeeding-weaning status. In Early and Late Neolithic groups breast milk was the major protein source until the age of 2-3 years. However, there are differences in the age of weaning completion and duration: Early Neolithic groups weaned their infants at a later age and over a shorter amount of time. Differences may have affected infant morbidity and mortality, and female fecundity and inter-birth intervals. Stable isotope values in older subadults (4-10 years, n = 26) do not differ from adults suggesting the absence of age-based food allocation.

Upper limb musculoskeletal stress markers among middle Holocene foragers of Siberia's Cis-Baikal region.

This evaluation of musculoskeletal stress markers (MSMs) in the Cis-Baikal focuses on upper limb activity reconstruction among the region's middle Holocene foragers, particularly as it pertains to adaptation and cultural change. The five cemetery populations investigated represent two discrete groups separated by an 800-1,000 year hiatus: the Early Neolithic (8000-7000/6800 cal. BP) Kitoi culture and the Late Neolithic/Bronze Age (6000/5800-4000 cal. BP) Isakovo-Serovo-Glaskovo (ISG) cultural complex. Twenty-four upper limb MSMs are investigated not only to gain a better understanding of activity throughout the middle Holocene, but also to independently assess the relative distinctiveness of Kitoi and ISG adaptive regimes. Results reveal higher heterogeneity in overall activity levels among Early Neolithic populations-with Kitoi males exhibiting more pronounced upper limb MSMs than both contemporary females and ISG males-but relative constancy during the Late Neolithic/Bronze Age, regardless of sex or possible status. On the other hand, activity patterns seem to have varied more during the latter period, with the supinator being ranked high among the ISG, but not the Kitoi, and forearm flexors and extensors being ranked generally low only among ISG females. Upper limb rank patterning does not distinguish Early Neolithic males, suggesting that their higher MSM scores reflect differences in the degree (intensity and/or duration), rather than the type, of activity employed. Finally, for both Kitoi and ISG peoples, activity patterns-especially the consistently high-ranked costoclavicular ligament and deltoid and pectoralis major muscles-appear to be consistent with watercraft use.

Osteoarthritis in Siberia's Cis-Baikal: Skeletal indicators of hunter-gatherer adaptation and cultural change.

This examination of osteoarthritis in Siberia's Cis-Baikal region focuses on the reconstruction of mid-Holocene mobility and activity patterns with particular interest in an alleged fifth millennium BC biocultural hiatus. Five cemetery populations--two representing the pre-hiatus Kitoi culture (6800-4900 BC) and three the post-hiatus Serovo-Glaskovo (4200-1000 BC)-are considered. The objective is to investigate osteoarthritic prevalence and distribution (patterning) within and among these populations in order to reconstruct mobility and activity patterns among the Cis-Baikal foragers, and to test for possible disparities that may reflect differing adaptive strategies. The data reveal that levels of activity remained relatively constant throughout the mid-Holocene but that mobility and specific activity patterns did not. Although results are consistent with the current understanding of distinct Kitoi and Serovo-Glaskovo subsistence regimes, specifically the lower residential mobility and narrower resource base of the former, they also draw attention to adaptive characteristics shared by all occupants of the Cis-Baikal.