Preservation of samples

We analyzed 80 bone collagen and 108 dental enamel samples from 137 individuals from 60 archaeological sites (Tables 1–4; Fig. 1). Samples were separated into four chronological periods based on relative and absolute dating (Early [Neolithic - Bronze Age], Early Iron, Xiongnu, and Mongol; see Supplementary Table S4 for AMS dates). As there is only a single individual from the Neolithic period (c. 4400–3000 B.C.E.), this sample was combined with Bronze Age individuals dating to prior to 800 B.C.E. (n = 23; collagen n = 14, enamel n = 16) to create a single period labelled as ‘Early’. The Iron Age samples were split into two chronological periods, corresponding to the pre-imperial Early Iron Age (c. 800–200 B.C.E.) and the Xiongnu (c. 200 B.C.E. – 250 C.E.). The Early Iron Age samples include 16 individuals (collagen n = 7, enamel n = 16) from one site. From the subsequent Xiongnu, we analyzed 59 individuals (collagen n = 23, enamel n = 54) from 28 sites. Individuals from the later Mongol Empire (c. 1200–1375 C.E.) are grouped together and consist of 28 individuals (collagen n = 28, enamel n = 21) from 19 sites.

Table 1 Average bone collagen values for individuals in this study by time period, individual values presented in Supplementary Table 1 (results include additional individuals from previously published articles20,29,30,44). Full size table

Table 2 Average tooth enamel bioapatite values by time period. Full size table

Table 3 Average human and faunal bone collagen δ13C and δ15N values between the steppe (>250 mL annual precipitation) and dry (<250 mL annual precipitation) regions. Full size table

Table 4 Average human dental enamel δ13C and δ18O values between the steppe (>250 mL annual precipitation) and dry (<250 mL annual precipitation) regions. Full size table

Figure 1 Maps of sites used in this study. These maps were created for this study and were produced using QGIS 3.089 https://qgis.org/en/site and using the Natural Early Data maps from https://www.naturalearthdta.com/downloads/ by Shevan Wilkin and Michelle O’Reilly (Graphic Designer for the Max Planck Institute for the Science of Human History, Jena, Germany). Full size image

All of the human bone collagen samples included in this study had atomic C:N ratios between 3.1 and 3.5 and were thus within the accepted range for good collagen preservation35 (Supplementary Table S1). The collagen yields of these samples ranged between 6 and 30%, with none falling below 1%, a further check of data quality35. Furthermore, the majority of collagen samples have greater than 11% N and greater than 30% C, within acceptable ranges36. Each bone collagen sample was run in duplicate, and averages are presented in Supplementary Table S1 along with their standard deviation.

Bone collagen carbon and nitrogen stable isotope results

δ13C and δ15N results from human bone collagen are grouped into four chronological periods, as detailed above, for comparative analysis. The pre-Bronze and Bronze Age average values are the closest to those of the average faunal values, although the faunal values have a higher standard deviation. The widest range of carbon and nitrogen isotope values were found in the Xiongnu and Mongol Period populations. For all of the individual δ13C and δ15N values from each time period see Supplementary Table 1.

Dental enamel carbon stable isotope results

The data from δ13C values of human enamel bioapatite are divided into the same chronological periods as the bone collagen data. The Xiongnu population had the largest range of stable carbon isotope values, followed by the Mongol period and Early Iron Age (Table 2). The pre-Bronze/Bronze period had the lowest range of stable carbon isotope values when compared to the later populations. For all of the individual δ13C and δ18O values from each time period see Supplementary Table 2 (Samples with both collagen and enamel Supplementary Table 3).

Environmental differences

As stable carbon and nitrogen isotope values may vary in different environments (i.e. temperature and aridity), to adequately assess human δ13C and δ15N values from normal steppe (>200 mL of annual precipitation) and dry (<200 mL of annual precipitation) regions, we also determined the average values for each environmental type (Table 3). In these tables we have separated the previously published faunal stable carbon and nitrogen isotope values into the “steppe” or “dry” regions according to modern annual rainfall37,38.

Statistical tests

Boxplots of our results can be found in Fig. 2A–C, and statistical comparisons between the time periods can be found in Supplementary Table 5 (δ13C bone collagen), Supplementary Table 6 (δ13C enamel bioapatite), and Supplementary Table 7 (δ15N bone collagen). For the bone collagen data, both the Xiongnu and the Mongol average δ13C values were significantly higher than those of Early individuals (p < 0.05). The same trend was seen for tooth enamel δ13C, with Early Iron, Xiongnu, and Mongol samples having δ13C significantly higher than that of the Early group (for the overall p < 0.05, and the specific pairwise comparisons are available in Supplementary Table 6). There was no significant difference between average dental enamel values for the Early Iron, Xiongnu, and Mongol periods δ13C (p > 0.05). Bronze Age δ15N values were also significantly higher (p < 0.05) than those of the Early Iron, Xiongnu, and Mongol periods (Supplementary Table 7).

Figure 2 Boxplots showing the range of carbon values for all individuals from each period. Outliers are shown as individual data points. (A) Comparison of the bone collagen carbon values for humans and fauna. Faunal data derives from previously published data20,29,30,44, and all human data is from this study. (B) Difference in human enamel values between Early, Early Iron, Xiongnu, and Mongol periods. The Early Iron, Xiongnu, and Mongol period average values are significantly higher than the Early period average. (C) Boxplots showing the range of oxygen values from enamel samples. There are no significant differences between any of the time periods. Full size image

Isotopic temporal trends in Mongolia and environmental impacts

Higher δ13C values in individuals from the Early Iron Age, Xiongnu, and Mongol periods could be the product of the increased direct consumption of C 4 crops or wild plants or animals consuming C 4 plants. It should also be understood that both Mongolians and foreign travellers would have been moving within and outside of the imperial borders, and dietary intake likely varied greatly in different regions. In areas with environmentally-linked variation in wild C 4 and C 3 plant distributions, such as Mongolia, it is important to rule out a climatically driven change (see Supporting Information Text 1). Modern plant samples from Mongolia have yielded δ13C values ranging from −28.3 to −23.4‰ for C 3 photosynthetic pathways and an average δ13C of −14.7‰ for plants following the C 4 photosynthetic pathway39. Notably, wild C 4 plants make up a much smaller proportion of Mongolian and other Central Asian environments than C 3 plants40,41. Overall, contemporary studies suggest that leaf δ13C values decrease with increasing mean annual precipitation42, both as a product of reduced C 4 plants in wetter landscapes and aridity-driven changes in δ13C among C 3 plants (see Supplemental Text S1).

While C 4 plants make up a relatively limited portion of the biotic community today, we established local isotopic baselines for Mongolia in the past using archaeological fauna in order to determine if shifts in δ13C values through time are the product of environmental variations or social and economic choices. Isotopic studies of modern and archaeological herd animals have shown differences in δ13C values between more and less arid regions43,44,45, and that there is variation in the availability of C 3 and C 4 plants across the country46,47. While there were no fauna associated with the human remains collected for this study, we were able to use previously published faunal stable isotope data from the Minusinsk Basin of Siberia (just north of Mongolia)(MNSK, AD, AM; n = 21)20,29, the Gobi (BGC; n = 14)30,48, Gobi-Altai (SBR; n = 5)30, and north central Mongolia (EG; n = 13)30 areas to show that regional herbivores generally consumed C 3 plants, with some having higher stable carbon isotope values, indicative of C 4 plant consumption, in the hyper-arid desert regions30,48.

Statistical tests further support this assessment, with humans having higher δ13C values than the available fauna in all periods, with the greatest difference occurring in the Xiongnu and Mongol periods (p ≤ 0.005)(Figs. 2A and 3A). For terrestrial faunal remains (Fig. 3A), there is a significant correlation between δ15N and δ13C bone collagen values (R2 = 0.64, p-value <0.001) which is a product of higher levels of aridity leading to a higher availability of C 4 plants in the natural vegetation cover. However, no such correlation is observed in humans (Fig. 3A,B), either between δ15N and δ13C bone collagen values (R2 = 0.01, p-value = 0.15) or between δ15N bone collagen and δ13C enamel values (R2 = 0.05, p-value = 0.13). Given this, alongside the consistent elevation of human δ13C values over the available fauna δ13C values, this indicates that higher δ13C values in human bone collagen and enamel is a product of direct consumption of non-wild C 4 plants.

Figure 3 Carbon and nitrogen values from bone collagen with ellipses showing ranges at 95% confidence. (A) Individuals included in this study as well as humans and faunal values from previously published data31 (B) Humans included in this study showing the variation between those in the “Dry” and “Steppe” zones. “Dry” sites have less than 250 mm of annual precipitation, “Steppe” sites have over 250 mm of precipitation per year. (C) δ15N from bone collagen versus δ13C values from dental enamel demonstrating the shift from primarily C 3 reliant diets in the Early period to a wider range of carbon and nitrogen values, indicating an increase in the diversity of diets in the later three periods. (D) δ18O from dental enamel versus δ15N from bone collagen showing the values in “Dry” and “Steppe” areas. Full size image

Mean bone collagen δ13C values for faunal remains from steppe regions are typically C 3 (−19.3 ± 1.3‰), and the stable carbon isotopic offset between bone collagen of herbivores and carnivores is c. 1‰49. Thus, human bone collagen steppe samples dating to the Early period (prior to 800 B.C.E.) do not show δ13C values indicative of a millet dietary contribution (−18.3 ± 0.1‰). This same offset applies to human bone collagen samples from dry regions from all periods since these are elevated by up to 1‰ (Early −17.2 ± 0.7‰; Early Iron = −16.2 ± 0.9‰; Xiongnu −16.1 ± 1.1‰; Mongol −16.1 ± 1.4‰) when compared to the bone collagen mean for faunal samples from dry regions (−17.2 ± 1.5‰). However, for later time periods in steppe regions average human bone collagen δ13C values are elevated by c. 3‰ (Early Iron −16.3 ± 0.7‰; Xiongnu −16.1 ± 1.2‰; Mongol −16.8 ± 2.2‰) when compared to faunal values. Thus, the higher (c. 2‰) human bone collagen δ13C values observed for later periods in steppe regions when compared to the early period is indicative of a temporal increase in millet-based food consumption. Furthermore, given that bone collagen reflects primarily consumed protein, and that millet has a poor protein content, the dietary caloric contribution from millet was likely much higher than its protein contribution50. This is corroborated by human enamel δ13C values given that this isotopic proxy reflects the carbon dietary mix50. Steppe human enamel samples for the later periods show mean δ13C values higher by c. 3.5‰ when compared to the Early period. For dry areas, mean human enamel samples δ13C values are higher by c. 1‰ when compared to the Early period, which indicates a temporal increase in millet-based food consumption although considerably smaller than that observed in the steppe regions as shown also in the model estimates for millet caloric contributions (Fig. 4).

Figure 4 Sites in and around Mongolia with archaeological or archaeobotanical evidence for C 3 (wheat and barley) and C 4 (broomcorn and foxtail millet) grain cultivation during the Iron Age. This map was newly created for this study and produced using QGIS 3.089 https://qgis.org/en/site and using the Natural Early Data maps from https://www.naturalearthdta.com/downloads/ by Shevan Wilkin, Bryan K. Miller, and Michelle O’Reilly (Graphic Designer for the Max Planck Institute for the Science of Human History, Jena, Germany). Full size image

Further evidence for C 4 plant consumption is offered by the distribution of isotopic values. For faunal remains there is a positive significant correlation (R2 = 0.67, p-value <0.05, correlation coefficient =1.126) between δ15N and δ13C bone collagen values which is expected given that an increase in aridity leads to a higher availability of C 4 plants in the vegetation cover. A similar correlation, albeit with isotopic offsets, would be expected if humans relied exclusively on animal products. However, no clear environmentally-driven correlation is observed for the human groups. There is no significant correlation for the Early (R2 = 0.65, p-value <0.08, correlation coefficient = 0.65) and Xiongnu (R2 = 0.0, p-value <0.46, correlation coefficient = 0.11) periods, and although the correlation is significant for the Mongol period (R2 = 0.13, p-value <0.02, correlation coefficient = 0.39), it only explains 39% of the variability. For similar δ15N bone collagen values across the human individuals, there are wide ranges in δ13C collagen values. Whereas during the Xiongnu period one can observe a significant negative correlation (R2 = 0.0, p-value <0.46, correlation coefficient = 0.11), which implies the contribution from a food source with higher δ13C values but lower δ15N values when compared to animal food sources. These isotopic relationships are indicative of varying individual intake of a food with elevated δ13C values, such as millet, and having relatively uniform δ15N and δ13C values across regions with varying levels of aridity.

Bayesian spatial modelling of C 4 plant caloric consumption

To further confirm that the increased δ13C values in human bone collagen and tooth enamel through time is a product of the consumption of crops rather than changing availabilities of baseline C 4 /C 3 plant ratios or the availability of samples in different local environments, we developed a Bayesian model to produce a C 4 dietscape, representing estimates of spatial distribution of C 4 plants based on per capita caloric consumption (See SI for detailed discussion). Stable carbon isotope data of dental enamel was used, and individuals were separated into two periods, Early (Neolithic - Bronze Age) or Late (Xiongnu, and Mongol). The results for the two models show that during the Early period C 4 caloric contributions were very low across Mongolia, likely including consumption of local plants and livestock consuming natural vegetation, with mean estimates varying between c. 2.5 and 5.0% of calories (interpolation 1-sigma uncertainty up to 0.5% calories) (Fig. 5A,B). During the later periods, the variability in millet-based food consumption increases considerably as shown by the range in the mean estimate (between 3 and 26% of per capita millet calories) and in the 1-sigma interpolation uncertainty for each location (between c. 3 and 6% of per capita millet calories) (Fig. 5C,D). The C 4 plant dietscape for the late period also shows that millet consumption is concentrated in central northern Mongolia (reaching the highest mean value [26% per capita millet calories]), an area where environmental increase of carbon values would not be expected naturally (Figs. 4 and 5).