Stable C & N isotopes in 2100 Year-B.P. human bone collagen indicate rare dietary dominance of C4 plants in NE-Italy

Abstract

C₄ plants (e.g. maize, millet), part of our current diet, are only endemic of reduced areas in South-Europe due to their need of warm climates. Since the first vestiges of agriculture in Europe remains of C₄ plants were recorded but their overall proportion in the human diet remains unknown. Therefore, isotopic (δ¹³C and δ¹⁵N) composition of bone collagen from the skeletal remains (human and animals) of a Celtic population, Cenomani Gauls, from Verona (3^rd to 1^st century BC) in the NE Italy provide a new perspective on this matter. The δ¹³C collagen values of 90 human skeletal individuals range between −20.2‰ and −9.7‰ (V-PDB) with a mean value of −15.3‰. As present day C₄ plants have δ¹³C values around −11‰, which is equivalent to −9.5‰ for samples of preindustrial age, the less negative δ¹³C values in these individuals indicate a diet dominated by C₄ plants. This palaeodietary study indicates that some European populations predominantly consumed cultivated C₄ plants 2100 year B.P. This is supported by the paleobotanical records and ancient Roman sources (e.g. Pliny the Elder), which indicate that millet was a staple food in South-Europe.

Introduction

The term C₄ plants refers to the type of photosynthetic pathway (Hatch-Slack or C₄ cycle) used by these plants, which has less isotopic discrimination than the C₃ cycle (C₃ plants). In the C₄ cycle the first stable compound namely oxaloacetic acid is formed by 4 carbon atoms. This group includes warm climate grasses like maize, millets, sorghum and sugar cane, among others¹.

Paleobotanical studies have shown the presence of domesticated C₄ plants (in particular broomcorn or Panicum Miliaceum L. and foxtail millet or Setaria Italica L. Beauv.) in Central and Eastern Europe as early as 5000-4000 BC for Panicum Miliaceum and Late Bronze Age for Setaria Italica²; while in Asia (China) and in the Caucasian region (Georgia) this is documented as early as ca. 10000 years B.P.^3,4,5. Based on the botanical evidence, it seems that millet was first introduced into Central and Eastern Europe from the Steppe regions during the Neolithic period^6,7,8. Broomcorn millet seems to have arrived in the Mediterranean zone from the north or the north-east⁹; in northern Italy, millet appears in early Bronze Age settlements (1700-1500 BC)². The earliest records of foxtail millet (Setaria Italica L. Beauv.) come from Peigang and Cishan (North China) in the 6^th millennium BC³. In Europe the first carbonized seeds of foxtail millet appear in the 2^nd millennium BC, from Bronze Age settlements in central Europe¹⁰ and France¹¹. It is also reported from the late Bronze age Kastanas site (Macedonia, Greece)¹². In the Near East the earliest evidence for foxtail millet cultivation dates back to the Iron Age (c. 600 BC) in Tille Höyük, south-east Turkey⁷. However, these data only indicate the presence of millet, but do not reveal their role in the human diet.

C₄ plants are typical of warm climates and their abundance is highly correlated with climatic factors, such as temperature, precipitation and the degree of aridity. In general, they are not present in environments where night temperatures are lower than 8 °C^13,14. Nowadays in Europe C₄ biomass is very scarce; however these plants are naturally present in small proportions especially in the SW-Europe (e.g. Spain, Portugal, SW-France and Italy)¹⁵. Before the onset of agriculture, herbivores collagen isotopic δ¹³C values from S-Italy (32.6 to 13.3 ka B.P.) suggest that C₄ biomass was practically absent (calculated vegetal biomass is around −25‰ vs V-PDB)¹⁶. Even during the warmer periods of the Late Miocene, fossil isotopic data indicate the absence of C₄ biomass in central and southern Europe^17,18. Man has introduced certain species, such as millet, with a planting strategy in the warm season (i.e. summer), possibly as a response to Holocene arid periods¹¹.

The stable isotope composition (δ¹³C and δ¹⁵N) is a very effective tool to study the trophic web in ecosystems^{19,20,21,22,23}. This methodology is crucial for studying past ecosystems and paleodiets, because direct observation is impossible^24,25,26,27. In addition this technique allows quantitative analysis, which even in current ecosystems is difficult to obtain with the classical methods of observation. Isotopic carbon traceability suffers less change in the steps of the food chain than isotopic nitrogen one. Therefore, the footprint of primary producers (e.g. plants) is clearer²⁸.

The importance of carbon isotopes as a racer for the presence of C₄ plants in the diet, is that atmospheric CO₂ is fixed into organic matter by the enzyme PEP carboxylase (C₄ plants) which discriminates much less against ¹³C than the enzyme RuBisCO (C₃ plants)²⁹. In present day C₃ plants biomass show typical δ¹³C values around −26‰ (V-PDB), while C₄ plants are around −11‰ (V-PDB)^30,31, −24.5‰ and −9.5‰ (V-PDB), respectively in preindustrial age, because δ¹³C values of CO₂ were less negative by 1.5‰ compared to the present values (−8‰ V-PDB). This 15‰ difference between C₃ and C₄ plants allows an accurate calculation of the percentage of C₄ plants in the diet. The δ¹³C_collagen values of humans and animals are proportional to the isotopic signal from the base of the food chain (plants), but enriched by 0.8 and 1.3‰ for herbivores and carnivores, respectively³². The δ¹⁵N of the collagen is also related with the food chain and can be used to evaluate the trophic system, because in a particular ecosystem there is a significant enrichment in ¹⁵N between 3 and 5‰ for each trophic level^25,26,27.On the other hand δ¹⁵N cannot be used to distinguish between C₃ and C₄ plants consumption.

There is little knowledge about the nutritional habits of protohistorical populations such as Pre-Roman-Celtic ones. Therefore, we performed an isotopic study to unveil the dietary habits of the Celtic population Cenomani Gauls, from the necropolis of Seminario Vescovile in Verona (Italy) dated between the 3^rd to 1^st century BC³³. This necropolis counts with a minimum of 174 skeletons in a good state of preservation, and the majority of them are non-adults (see Supplementary Information for detailed descriptions of the archaeological context). Surprisingly, we recorded relatively high, i.e. less negatives, δ¹³C_collagen values in the human samples, and even in some animals found in the necropolis. In our initial considerations, we suspected the presence of protein in the diet from marine sources, or even anomalous fresh water sources (enriched ¹³C in DIC-Dissolved Inorganic Carbon, “dead carbon” from marine carbonates), or alternatively an extensive consumption of C₄ plants. The first hypothesis seemed from the beginning improbable, taking into account the geographic location of the necropolis, which was far from the sea (about 120 km). Additionally, the less negative δ¹³C collagen, −10‰ (V-PDB), is higher than what would be expected for a signal from marine primary production²². A second alternative explanation is a freshwater tropic web with an anomalous enriched ¹³C source of carbon (DIC) from dissolution or thermic decomposition of carbonates. This geochemical scenario is present in some areas of Italy³⁴ in which natural water becomes enriched in CO₂. This archaeological site was near to the Adige River that runs through Verona. Therefore, water samples from the Adige river and its tributaries were collected and analysed. In addition, ¹⁴C dating was done on some human remains to detect the presence of “dead carbon”.

The main objective of this study is to demonstrate that these less negative δ¹³C values are related to the consumption of C₄ plants and refute the improbable hypothesis of a significant marine or anomalous fresh water dietary intake (enriched in ¹³C). At the same time, we set out to quantify the proportion of C₄ plants in the diet and propose an interpretation about the alimentary habits of Celtic populations of Italy which up to now have been elusive.

Results

Isotopic values of the human and animal sample from Verona

The human δ¹³C values range from −20.2‰ to −9.7‰ (V-PDB) with a mean value of −15.3‰ (±2.2‰) (V-PDB) while δ¹⁵N values are between +6.9‰ and +12.9‰ (AIR), showing a mean value of +9.4‰ (±1.3‰) (AIR) (Fig. 1; Table 1; Supplementary Table S2). The atomic C/N ratio of the samples falls inside the range of 2.9–3.6, which corresponds to typical values of well-preserved samples, as suggested by DeNiro³⁵. Isotopic data from animal bones (Tables 1 and 2) suggests that domesticated herbivores had a diet mainly based on C₃ plants, although one of these (VRAR-1 with −17.2‰ V-PDB) showed a less negative δ¹³C value that could suggest that this animal partly fed on C₄ plants. Dogs (n = 2) usually eat men’s leftovers, showing relatively high δ¹³C values (≈ −13‰ V-PDB). One of them also has a relatively low δ¹⁵N value for an omnivore. This implies a diet poor in animal proteins, supporting the hypothesis of an important influence in their diet of human food scabs, rich in (C₄) plants.

Table 1 Summary table with Number of samples (N), Minimum (Min), Maximum (Max), Mean (M) and standard deviation (SD) of the isotopic values (‰) of human and animal remains.

Table 2 Isotopic values (δ¹⁵N and δ¹³C) of the animal samples with the estimated percentage of C₄ plants in their diet. %PP-C₄: minimum percentages of diet based on C₄ primary production.

Figure 1

Scatter plot of the data for δ¹⁵N and δ¹³C analyses of human and animal samples from Verona.

The δ¹³C data of human bone collagen range between −20.2 and −9.7‰ (V-PDB), and reveals the significance of C₄ plant in the diet, with less negative values indicating an almost exclusive diet of C₄ plants and the more negative values representing a diet lacking C₄ plants. The range of δ¹⁵N values (from 6.9 to 12.9‰ AIR) compared with those of animals suggests a mixed terrestrial diet (meat or dairy products and vegetal foodstuffs).

Isotopic values of DIC of Adige’s river and Radiocarbon data

The DIC δ¹³C data (Table 3 and Supplementary Figure S1b) of the Adige river ranges between −4.5‰ and −5.0‰ (V-PDB); this, after isotopic fractionation processes of photosynthesis in algae, would result in values ranging from −23.5‰ and −24‰ (V-PDB) and consequently it would imply δ¹³C values in fish tissues very close to these values (≈ −22.5‰ to −23‰ V-PDB). These data would result in a value for human collagen resulting from an exclusively fish diet (an extreme that would be relatively rare), between −20‰ to −22‰ (V-PDB), which is not compatible with the least negative values found in this work. The rest of δ¹³C values of DIC of other tributaries, wells, etc. are even more negative so they also cannot justify the higher δ¹³C values found in collagen of human remains³⁶.

Table 3 DIC (dissolved inorganic carbon) isotopic composition of the water of the zone.

On the other hand, the age obtained from ¹⁴C dating of bone collagen from four individuals (those with less negative δ¹³C values) is consistent with that extrapolated (Table 4) from the preliminary archaeological analysis of the grave goods³⁶. This indicates the absence of “dead” carbon (e.g. dissolution of marine carbonate rocks), which would lead to apparently much older ages. Such dissolved inorganic carbon (DIC) have δ¹³C values close to +0‰ or positive values, which would also lead to an aquatic trophic chain with less negative values of δ¹³C, this being the only alternative to the consumption of C₄ plants. The absence of “dead” carbon in the humans collagen rules out this hypothesis, and thus consequently supports the presence of C₄ plants in the diet of these individuals.

Table 4 ¹⁴C dating of the less negative δ¹³C human collagen samples.

Discussion

The isotopic results show the important role that C₄ plants had in the diet of this Celtic population from Verona (Fig. 1). Generally, δ¹³C values of −20‰ (V-PDB) or less negatives would indicate some C₄ plant contributions. Thus the vast majority of studied individuals (over 90%) seem to include C₄ plants in a direct way (or indirectly through the consumption of herbivores that fed on them) in their diet. Additionally, adult females (mean value of 48.8 ± 15.1%) show significantly (Mann-Whitney U test = 198.0; p = 0.008) higher consumption of C₄ plants than adult males (mean of 37.4 ± 16.8%) (Fig. 2). We also found a significant statistical difference in δ¹⁵N values between sex in the adult group (t = 3.15 p ≤ 0.01): men display higher δ¹⁵N values compared to women (Fig. 3). Thus, there is a clear differentiation in the diet according to sex with a higher intake of animal protein (meat and meat products) for men, while the diet of women included more cereals and vegetable proteins. It is important to note, that some adults related with high intakes of animal protein (highest values in δ¹⁵N) have less negative values in δ¹³C (≈−12‰ vs V-PDB, see for example individuals VRSV-12 and VRSV-24 in the Supplementary Table S2), indicating that domestic herbivorous have also consumed large amount of C₄ plants, although the few herbivorous studied show a consumption of C₃ plants. However, two dogs show δ¹³C values which are very similar to those of humans (Fig. 1). Especially the younger dog (VRSV-94) presents a typical value of the middle-low animal protein intake of omnivores: δ¹⁵N = +7.1‰ (AIR). While the δ¹³C value of −13.7‰ (V-PDB) is very close to those of the humans with a C₄ diet (Table 2). As mentioned before, a possible explanation is that dogs were fed with the impoverished-protein waste from the meals of their owners.

Figure 2

C₄ plants minimum percentages (%) in the diet of the individuals from Verona.

Some individuals show C₄ plants proportion above 60% in their alimentation, and especially female adults reach significantly higher percentages of C₄ plants consumption compared to adult males.

Figure 3

δ¹⁵N values of adult females and males. Adult males have higher δ¹⁵N values compared to adult females.

DIC values (Table 3) from local meteoric water show relatively negative δ¹³C values (from −4.5 to −13.3‰ vs V-PDB; i.e. an aquatic diet should be approximately between −23.5 and −32.3‰ vs V-PDB) and ¹⁴C values of the human samples with less negative δ¹³C_collagen values does not indicate the presence of “dead carbon”. Consequently, this excludes a freshwater diet to explain the measured less negative isotopic values of this particular Celtic population and thus supports the hypothesis of a large consumption of C₄ plants in their diet. Furthermore, δ¹⁵N values in humans (+9.4‰ AIR) plot in about a one trophic level above terrestrial herbivorous (+4.7‰ AIR)³⁶, rule out a marine diet, in agreement with the relative large distance from the coast.

Considering that there are not many known cultivated C₄ plant species in this time period (3^rd-1^st BC), the hypothesis of millet consumption seems reasonable based on the archaeological record and ancient written source. In fact, the investigated necropolis is located in an area which is still today characterized by a fertile plane (called Pianura Padana or Po valley): it offers ideal climatic and environmental conditions for the cultivation of C₄ plants during the warm seasons (spring-summer). Pliny the Younger (Epistulae, IV, 6,1) praised the Po Valley region and wrote “in regione Transpadana summa abundantia”³⁷. Since the Middle and Late Bronze age (1550-1170 years BC) this plane was characterized by intensive agriculture, pastoralism and the demand of large amounts of wood for building sites. This lead to a heavy deforestation of the whole Po plain and converting it into an artificial steppe devoted to cereal crops³⁸. Furthermore, paleoclimate records of lake sediments and speleothems show trends towards a drier climate, or alternating wet and dry climates, between 2500 and 2000 BP^39,40. This might have induced a change in cropping strategies and the consequent introduction of C₄ plant cultivation (probably millet).

Unfortunately, during the archaeological excavation of Verona, archaeologists have not found seeds (or charcoal) and the ceramic grave goods (vessels and others) are still under study and initially seem not to indicate macroscopic remains of organic waste. Some previous archaeobotanical studies of Neolithic, Bronze and Iron Age sites of the NE of Italy, and particularly of the area of Verona, seem to confirm the presence of Panicum Miliaceum and of Setaria italica among the carpological remains^41,42. Another comparative paleobotanical study relating to some Iron Age sites located between the Northern and Southern Alps (Eastern Switzerland, Austria and Northern Italy), also describes the presence of millet (Panicum Miliaceum L.) and foxtail millet (Setaria Italica L. Beauv.) remains⁴³. This is also confirmed by the results of the study about carpological remains found at the site of Oppeano (Verona), dated to the second Iron Age (about 6^th to 3^rd century BC) and located in the same geographical context of the Seminario Vescovile necropolis. The majority of the determined cereal remains correspond to millet (Panicum miliaceum L.) and foxtail millet (Echinochloa crus-galli L. Beauv. and Setaria italica L. Beauv)⁴⁴. Thus, the Iron Age seems to be characterized by a greater crops specialization that fits with the assumption of the pursuit of a cereal type that best fits different climates (biomass increase with more efficiency in water use). In this case of the sandy Pianura Padana (Verona), the crops of millet were preferred, because it is a plant that adapts very well to poor substrates characterized by water scarcity during summer⁴⁴.

Finally, other isotopic results from bone collagen analysis of some individuals proceeding from the Bronze Age necropolis of Olmo di Nogara, Verona (1600-1200 cal. BC) and of Arano di Cellore, Verona (2040-1890 cal. BC) support the hypothesis of C₄ plants consumption, and in particular of millet in some areas of the Verona province^45,46(Fig. 4). In conclusion, to these archaeological data we add the testimonies of some ancient authors such as Pliny the Elder (Naturalis Historiae XVIII, 83-84) and Columella (De Re Rustica, 2, 9, 14–16) who report the use of millet flour for the production of bread and a sort of porridge cooked in water and salt and often accompanied with vegetables and cheese and very rarely with meat. Pliny (XVIII.XXIV) exactly noted: “millet is used to prepare a very white puls (i.e. similar to present-day polenta). Panic, when ground and freed from bran, and millet as well, makes a porridge which, especially with milk, is not to be despised even in time of plenty”. Columella (2.9.19), agreed with Pliny and wrote: “bread is made of millet, and it may be eaten without distaste before it cools”.

Figure 4

Stable carbon and nitrogen isotope data of human bone collagen from Verona (VRSV) compared with the individual’s values from Olmo di Nogara (ODN)⁴⁵ and Arano di Cellore (AC)⁴⁶.

We have also plotted the faunal ranges from the three necropolises. While Verona and Olmo di Nogara (Middle Bronze Age) have very similar isotopic values, confirming a largely C₄ plants based diet, Arano di Cellore, dated to Early Bronze Age shows a diet based on C₃ cereal-type plants.

The isotopic data obtained in this study reveal that the proportion of C₄ plants was substantial (above ≈40%) in the daily diet of this Celtic population. Traces of C₄ plant consumption are reported in other studies^45,46 from more ancient necropolis of the zone. While Olmo de Nogara (ODN) dated to Middle Bronze Age share very similar isotopic values with Verona (Fig. 4), confirming a preponderant C₄ plants based diet, Arano di Cellore (AC), dated to Early Bronze Age, shows a diet based on the consumption of C₃ cereal-type plants. Hence, the data from AC and ODN firmly place the shift in C₄ crop use in the region at a transition period between the late phases of the Early Bronze Age and the beginning of the Middle Bronze Age⁴⁶, consequently our data show that successively, in pre-Roman times, the diet of most habitants of the zone was almost exclusively based on C₄ plants. This, indirectly indicates that this Celtic population had mastered agriculture techniques to such an extent that they could live off the harvest of their cultivated crops the year round.

Methods

A sample of 90 human ribs, consisting of both sexes and different ages (Supplementary Information Table S1), and 7 animal bones, was selected for analysis of bone collagen. The animal bone sample is composed of 5 herbivorous species (2 horses, 1 goat/sheep and 2 cows) and 2 omnivorous species (2 dogs). The extraction of collagen is performed using the protocol described by Bocherens et al.^47,48 and following the routine procedures of the Stable Isotope Biogeochemistry Laboratory of the Andalusian Institute of Earth Sciences (CSIC, Granada, Spain). We also determined the current DIC values of the Adige’s river (and of some of its tributaries) and dated with ¹⁴C the collagen samples of the less negative human values (a source of “dead” inorganic carbon would show abnormally high ages).

The δ¹³C mean values of present day C₃ and C₄ plants are respectively −26‰ and −11‰ (V-PDB)^49,50, but before the industrial revolution the isotopic composition of atmospheric CO₂ was 1.5‰ less negative⁵¹. Hence, to make a more precise estimate we considered that these two carbon sources would respectively correspond to values of −24.5‰ and −9.5‰ (V-PDB) (see Supplementary Information for detailed descriptions about the different methodologies applied).