Chapter 3: Stable Isotope Analysis
3.2 Dietary Variation in the Abundance of Carbon
3.2.1 Identification of Terrestrial Plant Consumption C 3 and C 4 plants
plants
During photosynthesis, fractionations that actively discriminate against the heavier 13C isotope occur at both the diffusion of atmospheric CO2 into the leaf and during subsequent chemical enzymatic reactions, resulting in the plant being isotopically depleted in its 13C/12C ratio (O’Leary 1981). The extent of isotopic fractionation varies depending on the photosynthetic pathway utilised by the plant (O’Leary 1981).
The majority of plants, including all trees, shrubs and those native to temperate climates, follow the C3 (Calvin-Benson) pathway, which heavily discriminates against the 13C isotope, resulting in a depleted mean δ13C of -26‰ (range -22 to -38‰) (O’Leary 1988). In contrast, some grasses adapted to xeric and tropical environments use the C4 (Hatch-Slack) pathway, which discriminates against 13C to a lesser extent (Smith and Epstein 1971). As a result, C4 plant species are less depleted than C3 plants, with an average δ13C of -12.5‰ (range -9 to -16‰) (O’Leary 1988). Laboratory (DeNiro and Epstein 1978; Teeri and Schoeller 1979) and field analysis (Vogel and van der Merwe 1977) has demonstrated these consistent and non- overlapping differences in the δ13C of C3 and C4 plants are retained throughout the food web, with the isotopic composition of an animal’s tissues reflecting the mean dietary δ13C ingested in life, thereby allowing the assessment of their relative importance in a consumer’s diet. This method has been used archaeologically to identify the incorporation of C4 cultigens, such as maize, millet, sugar cane and sorghum, into previously C3 dominated food chains. This analysis has been successfully applied to the identification of changes in subsistence strategies in North America attributed to the introduction of maize horticulture (for review see Larsen, 1997). As C4 plant species are confined to tropical and arid regions, the application of this method in temperate Europe is limited. The only C4 plant found in Europe in the Roman period is millet, noted in Northern Europe from 3000BCE (Zvelebil and Dolukhanov 1991). The
importance of this crop within the Roman Empire appears limited and within Roman-Britain is considered negligible (Cummings 2008). However, a previous isotopic study of the Roman population of London has identified some individuals exhibiting a C4 signal (Redfern 2011 pers comm.). As this study was limited to only a few individuals, the importance of C4 plants in Roman London requires further investigation.
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(i) Non-Dietary Variations in δ
13C Values of Terrestrial Plants
Although, photosynthesis is the main determinant in the variation of the natural abundance of carbon ratios within the biosphere, deviations in source carbon alongside climatic,environmental and genetic factors can influence the extent of fractionation at any stage of the process (Chisholm 1989). These factors introduce non-dietary variations in the endpoint values detected in tissues (see below). While it is possible to correct for some of these factors, a culmination of several may be impossible to distinguish (Chisholm 1989). These non-dietary variations highlight the importance of the utilisation of local fauna and flora in the
reconstruction of palaeodietary food webs to avoid introducing errors into analyses.
(a) Variations in Source Carbon
The δ13C value of terrestrial plants is ultimately derived from its carbon source.Therefore, if fluctuations in the isotopic composition of a carbon reservoir occur, they will be incorporated into primary producers and passed through the food chain. The global average δ13C for atmospheric CO2 is approximately -8.0‰, but, slight deviations associated with anthropogenic factors have been noted (Friedli et al. 1986; Marino and McElroy 1991). Since the industrial revolution, the burning of 13C-depleted fossil fuels (with an average δ13C value of -27.28‰) has led to the depletion of atmospheric CO2, making it approximately 1.5‰ more negative than pre-1800 averages (Leavitt and Long 1986). Whilst studies conducted on populations prior to the burning of fossil fuels will not be affected by these shifts in δ13C, any food web
reconstructions that utilise modern samples of possible food sources must correct for this offset to avoid introducing errors into analyses (van Klinken et al. 2000).
Spatial variations in atmospheric CO2 have also been identified in connection with forest ecosystems. In dense closed canopy forest environments, where the free-mixing of atmospheric CO2 is restricted, a vertical cline of 2-5‰ in the δ13C of forest vegetation is observed, with δ13C values increasing with height. This phenomenon, termed the “canopy effect”, is the result of a change in photosynthesis activity in response to decreased light intensity, and the assimilation of recycled CO2 from the decomposition of forest litter and soil respiration (Tieszen 1991; van der Merwe and Medina 1991; Heaton 1999; Drucker et al. 2008). This canopy effect occurs in both tropical and temperate forests (Drucker et al. 2008). Therefore, plants grown on forest floors, and the animals that eat them, will have more negative δ13C values (by up to 5‰) than those in open terrestrial ecosystems (Ambrose 1993).
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(b) Climatic and Environmental Variations
On a global scale, climatic factors, namely temperature, humidity and irradiance, can significantly affect the δ13C values of terrestrial plant species, creating spatial patterning in 13
C/12C ratios (van Klinken et al. 1994; van Klinken et al. 2000). A negative correlation is observed between temperature and δ13C, with an average depletion of 0.3‰ per °C increase, as above optimum temperatures reduce the ability of carboxylation enzymes to take up CO2 (Chisholm 1989; Tieszen 1991). Similarly, a negative correlation also exists with humidity, in the order of -0.1‰ per increase in the percentage of relative humidity. In conditions of low humidity stomatal closure occurs in an attempt to prevent water loss, leading to decreased discrimination and δ13C enrichment (Tieszen 1991; Heaton 1999). Increases in the levels of light intensity will also lead to enrichment of isotopic values (up to 3‰) as carboxylation efficiency increases with higher levels of irradiance (Smith et al. 1976; O’Leary 1981). Low light intensity is one of the main contributory factors for the depletion of δ13C values of understory species in forests (see above).
A general regional patterning of δ13C has been observed across Western Europe, producing a northwest-southeast cline of 2 to 4‰ (van Klinken et al. 2000). This regional variation
introduces systematic non-dietary differences in the δ13C values observed in the countries encompassed by the Roman Empire, with a 1.6‰ difference in bone collagen δ13C values observed between Britain and Italy (van Klinken et al. 2000). Therefore, it is necessary to ensure that comparisons of stable isotope data within the Roman Empire acknowledge and correct for these differences.
In addition to large scale climatic variations, localised differences within the microhabitats of plants can also lead to significant deviations from recognised mean δ13C values. Within the soils that plants are grown, variations in type and drainage can lead to differences of up to 2‰. In addition, poor soil nutrition is thought to lead to depletion in δ13C values due to its effects on carboxylation (Chisholm 1989; Heaton 1999). However, as plants grown in poor soil will have low yields, their contribution to dietary δ13C is thought to be minimal.
Further variations in δ13C are observed with water availability, as plants subject to water stress can be enriched by 3-4‰ compared to unstressed plants. This enrichment is attributed to stomatal closure in an attempt to avoid water loss, resulting in decreased discrimination against 13C (O’Leary 1981). In addition, altitude has a strong and consistent effect on plant 13
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O2 partial pressure increases enzymatic carboxylation efficiency (Smith et al. 1976; Livingstone and Clayton 1980; Ambrose 1993; Heaton 1999).
(c) Genetic variations
Within C3 and C4 classifications, genetic differences occur between plant species and plant forms that can lead to average differences of up to 3‰ in the δ13C values observed. Within the same geographic region, short-lived shrub species exhibit δ13C values up to 5‰ higher than evergreen species as a result of differences in the rate of photosynthesis (Heaton 1999). In addition to inter-species variations, intra-species genetic diversity introduces small
disparities in a plant’s physiological response to its environment, altering its δ13C value. Studies of individual plants of the same species at the same site have demonstrated that deviations of ±0.8 to ±1.5‰ are not uncommon in areas covering only a few hectares (Heaton 1999). While it is difficult to establish the extent of intra-species variation in the absence of environmental factors, differences of up to 3‰ might be observed.
Further significant variations in δ13C are observed within individual plants themselves. Studies of isotopic variation between whole plant δ13C values and those of their summative parts have identified shifts from bulk values of -1.3‰ for grains, legumes and fruit (Deines 1980). A difference of +3.8‰ has been recorded between seeds and leaves of the same plant (Deines 1980). These differences are thought to be due to variations in the concentration of
isotopically distinct biochemical fractions (proteins, carbohydrates and lipids) within different plant tissues (O’Leary 1981). For example, lipids are depleted in δ13C by 5‰ compared to whole plants, making any tissue with large concentrations of lipids isotopically lighter than bulk values (Chisholm 1989). This is potentially important for palaeodietary studies as preferential selection of specific plant parts (such as seeds and grain) may not be accurately represented by average δ13C values derived from whole plant values or those derived from leaves (Tieszen 1991).