FROEHLE ET AL. 2012 Multivariate Carbon and Nitrogen

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 147:352369 (2012)
Multivariate Carbon and Nitrogen Stable Isotope Model for the Reconstruction of Prehistoric Human Diet
A.W. Froehle,1* C.M. Kellner,2 and M.J. Schoeninger3,4
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Department of Community Health, Wright State University, Dayton, OH 45435 Department of Anthropology, Northern Arizona University, Flagstaff, AZ 86011 3 Department of Anthropology, University of California, San Diego, La Jolla, CA 92093 4 Center for Academic Training and Research in Anthropogeny (CARTA), La Jolla, CA 92093 KEY WORDS cluster analysis; discriminant function analysis; apatite; collagen; bioarchaeology variance, providing a multivariate model for diet reconstruction. Both carbon variables dominate the rst function, while d15N most strongly inuences the second. Independent support for the functions ability to accurately classify individuals according to diet comes from a small sample of experimental rats, which cluster as expected from their diets. The new model also provides a statistical basis for distinguishing between food sources with similar isotopic signatures, as in a previously analyzed archaeological population from Saipan (see Ambrose et al.: AJPA 104(1997) 343-361). Our model suggests that the Saipan islanders 13C-enriched signal derives mainly from sugarcane, not seaweed. Further development and application of this model can similarly improve dietary reconstructions in archaeological, paleontological, and primatological contexts. Am J Phys Anthropol 147:352369, 2012. V 2011 Wiley Periodicals, Inc.
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ABSTRACT Using a sample of published archaeological data, we expand on an earlier bivariate carbon model for diet reconstruction by adding bone collagen nitrogen stable isotope values (d15N), which provide information on trophic level and consumption of terrestrial vs. marine protein. The bivariate carbon model (d13Capatite vs. d13Ccollagen) provides detailed information on the isotopic signatures of whole diet and dietary protein, but is limited in its ability to distinguish between C4 and marine protein. Here, using cluster analysis and discriminant function analysis, we generate a multivariate diet reconstruction model that incorporates d13Capatite, d13Ccollagen, and d15N holistically. Inclusion of the d15N data proves useful in resolving protein-related limitations of the bivariate carbon model, and splits the sample into ve distinct dietary clusters. Two signicant discriminant functions account for 98.8% of the sample
Recent meta-analyses (Kellner and Schoeninger, 2007; Froehle et al., 2010) have developed a new, regressionbased model for prehistoric diet reconstruction using published experimental animal bone stable isotope data (Hare et al., 1991; Ambrose and Norr, 1993; Tieszen and Fagre, 1993; Howland et al., 2003; Jim et al., 2004; Warinner and Tuross, 2009). When carbon stable isotope ratios (13C:12C, represented as d13C) of bone collagen (d13Ccollagen) and bone apatite (d13Capatite) are plotted against one another, these experimental data sort into two discrete groups according to protein source (C3 vs. C4/marine). The resulting d13Ccollagen vs. d13Capatite regression lines for the two protein-specic groups differ in their vertical intercepts (on the d13Ccollagen axis), but not for slope (Froehle et al., 2010). Applied to diet reconstruction, the position of an individual relative to one or the other line along the d13Ccollagen axis provides isotopic information on sources of dietary protein, while placement along the d13Capatite axis indicates the ratio of C3 to C4 foods in whole diet (d13Cdiet), as Ambrose and Norr (1993) originally proposed. The regression model offers more detailed and accurate dietary estimates than either d13Ccollagen or d13Capatite does alone, and avoids much of the redundancy inherent in the apatite-collagen spacing model (the arithmetic difference between d13Capatite and d13Ccollagen values, denoted as D13Capatite-collagen: see Krueger and Sullivan, 1984; Lee-Thorp et al., 1989; Ambrose et al., 1997). Although the bivariate regression model has advantages over previous methods, a plot of archaeological human data against the experimentally derived regresC V 2011
sion lines reveals two limitations concerning protein sources (Fig. 1a). First, the model distinguishes poorly between C4 and marine sources of protein, confounding determination of protein sources in populations living in coastal areas that also harbor C4 wild vegetation or agricultural crops. Second, many individuals fall between the protein-specic lines, the meaning of which, with regard to protein sources, remains somewhat unclear. Previous work showed that swine eating an experimental diet with mixed protein ($80% C3, $20% C4) fell, as expected, slightly above the C3 protein regression line derived from rodents consuming 100% C3 protein (Froehle et al., 2010). Given the small rodent sample size and scatter within data, however, the swine were statistically indistinguishable from the 100% C3 protein rodent population. It is therefore difcult to say whether the position of the archaeological humans close to, but not on, one of the protein-specic lines reects normal variation in populations consuming monoisotopic protein, or whether it indicates mixed protein consumption.
*Correspondence to: Andrew Froehle, Department of Community Health, Wright State University, Dayton, OH. E-mail: andrew.froehle@wright.edu Received 23 June 2011; accepted 29 October 2011 DOI 10.1002/ajpa.21651 Published online 30 December 2011 in Wiley Online Library (wileyonlinelibrary.com).
WILEY PERIODICALS, INC.
MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION
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Because we are aware of only one study that has reported experimental animal d15N values (Ambrose, 2000), the present analysis uses published data from archaeological human populations with reliable, nonisotopic evidence of diet. The experimental animal data (Ambrose and Norr, 1993; Ambrose, 2000) serve as a post hoc check of our interpretation of the human data. Finally, we provide an example application of the new model to an archaeological dataset, for which previous work left a dietary question unanswered (Ambrose et al., 1997).
STABLE ISOTOPE BIOGEOCHEMISTRY

It is well established that carbon and nitrogen stable isotope ratios in the tissues of animals, including humans, reect the isotope ratios in the foods they eat (DeNiro and Epstein, 1978, 1981; Schoeninger and DeNiro, 1984; Lee-Thorp et al., 1989; Ambrose and Norr, 1993; Tieszen and Fagre, 1993; Howland et al., 2003; Jim et al., 2004). Carbon-isotope ratios mainly reect the photosynthetic pathway of the plants an animal eats, or in carnivores, the plants that prey animals ate. The two most common photosynthetic pathways are C3 and C4, so named because the resulting sugars contain three and four carbon atoms, respectively. Compared to reference standards, C3 plants are relatively depleted in the 13C isotope compared to C4 plants, which are relatively 13Cenriched. Both d13Capatite and d13Ccollagen reect overall d13Cdiet, but dietary sub-components appear to inuence the tissues carbon-isotope ratios differently (Sullivan and Krueger, 1981; Lee-Thorp et al., 1989; see Schwarcz, 2000 for a review). Data from experimental feeding studies (Ambrose and Norr, 1993; Tieszen and Fagre, 1993; Howland et al., 2003; Jim et al., 2004) and meta-analyses of those data (Kellner and Schoeninger, 2007) suggest that d13Capatite correlates most closely with d13Cdiet, equally reecting all subcomponents in proportion to their abundance in the diet. In contrast, d13Ccollagen also reects d13Cdiet, but is skewed toward the isotopic signature of protein (Kellner and Schoeninger, 2007; Warinner and Tuross, 2009; Froehle et al., 2010). Nitrogen from the atmosphere enters the human food web mainly through dietary plants and animals. Many leguminous plants have symbiotic relationships with bacteria that x nitrogen directly from the atmosphere, and are thus not enriched in nitrogen (d15N 0% by denition). Other legumes do not participate in this kind of symbiosis (Muzuka, 1999; Codron et al., 2005), and are correspondingly enriched in 15N relative to the atmosphere: this probably explains the lack of low d15N values in some human populations known to ingest beans and other leguminous plants (Schwarcz and Schoeninger, 1991). Most nonleguminous plants obtain nitrogen from compounds in the soil and have higher values of d15N than the atmosphere (Rennie et al., 1976; Wada and Hattori, 1976; Shearer and Kohl, 1978; Amarger et al., 1979; Delwiche et al., 1979; Virginia and Delwiche, 1982). Animal nitrogen is enriched in 15N relative to the plants and animals they consume, such that animals have d15N values $3% higher than their diets (Minagawa and Wada, 1984; Schoeninger and DeNiro, 1984; Ambrose, 2000). In marine ecosystems, vertebrates exhibit d15N values 6-8% higher than animals at similar trophic levels in most terrestrial environments (Schoeninger and DeNiro, 1984). Thus, d15N in human bone collagen provides information on trophic position American Journal of Physical Anthropology
Fig. 1. Means (with 95% condence intervals of the mean) for d13Ccollagen and d13Capatite in (a) archaeological human populations and (b) cluster centroids, plotted against experimental animal regression lines and their 95% prediction intervals (experimental data compiled in Froehle et al., 2010; data from Ambrose and Norr, 1993; Tieszen and Fagre, 1993; Howland et al., 2003; Jim et al., 2004; Warinner and Tuross, 2009). The long-dashed lines represent the 95% prediction interval of the mostly-C3-protein regression, while the short-dashed lines delimit the 95% prediction interval of the C4/marine-protein regression. Human data sources are in Table A1. The experimental data from which the regression lines derive have been adjusted to reect pre-industrial atmospheric carbon levels by adding 1.5% (Marino and McElroy, 1991). Cluster diets: (1) 100% C3 diet/protein; (2) 30:70 C3:C4 diet, >50% C4 protein; (3) 50:50 C3:C4 diet, marine protein; (4) 70:30 C3:C4 diet, !65% C3 protein; (5) 30:70 C3:C4 diet, !65% C3 protein.
In order to resolve these protein-related ambiguities, here we expand the model beyond the sole use of carbon variables by adding nitrogen stable isotope data (d15N). Values of d15N in bone collagen closely reect protein sources, varying with consumption of different proportions of plant vs. animal protein, as well as with terrestrial vs. marine protein sources (Schoeninger et al., 1983; Minagawa and Wada, 1984; Schoeninger and DeNiro, 1984). This study also introduces multivariate statistical techniques to the study of stable isotopes. We employ cluster analysis to investigate relationships between diet and the three isotope variables holistically, and develop a discriminant function model for the simultaneous use of the three variables in dietary reconstruction.
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A.W. FROEHLE ET AL. using the average (UPGMA) and Wards method, two common measures in archaeological analyses (Baxter, 1994). K-means cluster analysis differs from hierarchical analysis in that it does not combine discrete groups into a unied, nested hierarchy (Everitt et al., 2001), but instead produces a user-specied number of centroids (geometric cluster centers, in this case numbering from one through six) around which group members aggregate. We used two main criteria to determine which number of clusters best reected natural groupings in the data. First, we sought the greatest level of consistency between hierarchical and k-means analyses in assignment of cases to clusters. Second, we evaluated the dietary validity of the k-means cluster centroids, which are multivariate-average cluster centers with cluster-specic average d13Ccollagen, d13Capatite, and d15N values. We only accepted centroids that we could interpret clearly in dietary terms, deriving our judgments in part from the placement of the centroids along the d13Ccollagen and d13Capatite axes relative to regression lines derived from experimental animal data (Froehle et al., 2010). We used previous evidence of diet for the populations in the dataset to check for consistency between cluster membership and cluster diet as implied by the centroids, and to further rene our centroid diet interpretations. After determining the most realistic number of clusters, we used linear discriminant function analysis to generate equations for predicting group membership from d13Ccollagen, d13Capatite and d15N. Discriminant function analysis uses known group membership from a training sample (in this case, our database) to produce linear equations (functions) describing how groups vary in terms of the independent variables (Everitt and Dunn, 2001). Our data met the assumptions of multivariate normality within groups and equality of covariance matrices between groups (Garrett, 1989; Everitt and Dunn, 2001). To estimate the resulting functions misclassication rate, we employed a post-hoc leave-one-out test, evaluating the results for consistency with classications dened by the cluster analysis. We further checked the functions validity against the experimental rat data of Ambrose and Norr (1993) and Ambrose (2000). To demonstrate how archaeological studies of diet might use our new model, we applied the discriminant functions to a population from the island of Saipan, part of the Marianas Islands in the northwestern Pacic. Archaeological and isotopic evidence for diet have remained somewhat inconclusive (Ambrose et al., 1997): though Saipans ecosystem is C3-dominated, stable isotope data indicated moderate consumption of 13C-enriched foods. Two candidates, C4 sugarcane and seaweed, were identied as potential sources of this signal, but the previous analysis offered no clear choice between the two (Ambrose et al., 1997). Here, we show how our discriminant function model can distinguish between the signals of the two foods in the bones of the Saipan islanders, a method that may be applicable to similar problems in other populations.
(Schoeninger, 1985; Phillips and Koch, 2002) and the degree of reliance on marine resources (Schoeninger et al., 1983). This latter aspect of nitrogen-isotope data is critical to the present analysis, since the relationship between d13Capatite and d13Ccollagenvalues in marine resources converges with that of C4 protein sources (Kellner and Schoeninger, 2007). By adding d15N values in this analysis, we expect to be capable of distinguishing between populations consuming C4 vs. marine protein.
METHODS Archaeological data

The database includes only adults with individual measurements for each of the relevant variables (d13Ccolla13 15 gen, d Capatite and d N), resulting in a sample size of N 5 158 (see Table A1). The data come from eight different populations for which substantial nonisotopic archaeological evidence for diet exists: Ontario preagriculturists and agriculturists; San Nicolas Island sher-foragers (Harrison and Katzenberg, 2003); American Bottom oodplain and upland agriculturists; Illinois River agriculturists (Hedman et al., 2002); Cahokia Mound 72 agriculturists (Ambrose et al., 2003); and Tierra del Fuego sher-foragers (Yesner et al., 2003). All studies from which data were drawn used established sample preparation and analysis methods. Although most studies sampled mainly ribs, other bones were occasionally includedthis should not be a problem for our meta-analysis given that only wellpreserved samples were included (DeNiro and Schoeninger, 1983; Ubelaker, 1995).
Multivariate statistical analysis

We began by using cluster analysis to determine whether individuals associated with one another according to their isotope values in the manner expected from previous evidence for diet. By comparing measures of interindividual proximity in multivariate space (Everitt et al., 2001), cluster analysis assigns data to clusters that maximize both intragroup homogeneity and intergroup heterogeneity. We constrained our analyses by assuming that the sample would divide into a minimum of one diet group, and a maximum of six groups, testing every integer from one through six. The minimum represents the case where no dietary differences exist in the sample, while the maximum represents every possible combination of diet and protein end-members (C3 vs. C4 for the nonprotein portion of diet; C3, C4, and marine for protein). All three isotope variables use the same scale and occupy roughly the same magnitude of range across that scale: thus, for all analyses we used raw, untransformed isotope data, and used squared Euclidian distance as the interindividual proximity measure. We conducted all analyses using SPSS 16.0 for Windows. To determine the optimal number of natural clusters in this dataset, we used both agglomerative hierarchical and k-means clustering (Baxter, 1994). Agglomerative hierarchical cluster analysis proceeds via the stepwise merging of individuals into larger and larger groups, using decreasingly stringent criteria for within-group homogeneity. This analysis produces a dendrogram in which branch length expresses the relative degree of similarity between individuals and groups (Everitt et al., 2001). Because the specic branching pattern of any dendrogram partly reects the intergroup proximity method used, we compared results from the unweighted pair-group method American Journal of Physical Anthropology
RESULTS Cluster analysis

The data split best into ve diet groups, on the basis of the degree of overlap between the clustering methods, and for consistency with previous archaeological and isotopic interpretations of diet in the sample. The one-, two-, and three-group clustering regimes were thrown out: high degrees of within-cluster heterogeneity limited

TABLE 1. K-means cluster analysis centroids d13Capatite % (PDB) Cluster Cluster Cluster Cluster Cluster 1 2 3 4 5 (n (n (n (n (n 5 5 5 5 5 17) 74) 37) 17) 13) 214.8 25.0 27.8 211.0 25.9 d13Ccollagen % (PDB) 220.3 211.3 210.5 218.6 215.3 d15N % (AIR) 12.0 10.1 17.7 11.4 8.8 Diet description C3:C4 diet; proteina 100:0; C3 protein 30:70; [50% C4 protein 50:50: marine protein 70:30; !65% C3 protein 30:70; !65% C3 protein
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Euclidian distance between centroids Cluster 1 13.47 13.29 4.17 10.75 Cluster 2 Cluster 3 Cluster 4
3.21 9.60 4.28
10.79 10.32
6.69
a Centroid carbon isotope values interpreted for percent C3 and C4 in whole diet using experimental animals analyzed in Froehle et al. (2010). For this comparison, experimental animal carbon data were adjusted to reect preindustrial atmospheric carbon levels by adding 1.5% (Marino and McElroy, 1991). Collagen carbon and nitrogen isotope values were interpreted for protein sources following DeNiro (1987) and Froehle et al. (2010).
their utility in precise dietary reconstruction, and were inconsistent with the substantial variability in diet within and between populations in the database. The six-group regime was thrown out because it produced a lower rate of overlap between hierarchical and k-means case assignments than did the four- and ve-group clustering regimes. Assuming ve groups, the UPGMA hierarchical and k-means methods assigned 151 of 158 (96%) cases to clusters in the same way. When we assumed four groups, UPGMA and k-means also classied 151 of 158 cases the same way, while Wards and k-means assigned a slightly higher number of cases, 154 of 158 (97%), to the same clusters. Among all such comparisons, the latter had the highest rate of consistency between clustering methods. We nevertheless chose to adopt the slightly less-consistent ve-group clustering regime because we found that it revealed more dietary discrimination than did the four-groups cluster assignments. To interpret diet for each group, we plotted the k-means cluster centroids isotope values (Table 1) against the experimental animal carbon-isotope regression lines (Fig. 1b): four of the centroids fell on or near one of the two protein-specic diet regression lines, while the fth cluster fell between the lines. It is important to note that the sample from which the C3 protein regression line is derived includes animals eating 95100% (N 5 12), 7585% (N 5 11), and 65% (N 5 1) C3 protein: as such, centroids falling on this line can be interpreted as consuming at least 65% C3 protein (although more likely !75% C3 protein). Dietary similarities and differences between clusters were also evaluated using Euclidian distances between centroids (Table 1) and the dendrograms splitting pattern (Fig. 2), both of which matched and conrmed that the two analyses organized the clusters similarly. The placement of centroids on the experimental animal plot indicates that Cluster 1 likely represents a fully C3 diet, and that Cluster 4 encompasses a 70% C3 diet with mostly C3 protein. The similarity of these two clusters relative to the rest of the sample is reected in their low-level split in the dendrogram, and their small Euclidian distance (4.17; smallest of any two centroids). The dendrograms highest-order split occurred between the high-C3 Clusters 1 and 4 on the one hand, and Clusters 2, 3, and 5 on the other. Given their placement along the d13Capatite axis on the experimental animal carbon plot, this is consistent with the latter three groups all having diets consisting of at least 50% C4 foods. Fitting with this split, the two greatest distances between centroids occurred between Clusters 1 and 2 (13.47), and between Clusters 1 and 3 (13.29). Of the three high-C4 groups, Cluster 5 is the easiest to interpret, consisting of a 70% C4 diet with mostly C3 protein. Clusters 2 and 3 defy interpretation using
Fig. 2. Simplied dendrogram showing the results of hierarchical cluster analysis. Average diet descriptions are based on centroid isotope values from the k-means cluster analysis and interpretations of those values using Froehle et al. (2010) and DeNiro (1987), as well as archaeological evidence. Proximity coefcients indicate the degree of similarity at which different groups split/merge. Higher coefcients indicate a higher tolerance for dissimilarity within groups/branches.
only carbon data because although they both clearly had overall diets high in C4 foods (70 and 50% of diet, respectively), protein sources remain indeterminate. It is clear that the vast bulk of the protein consumed by members of Cluster 3 came from 13C-enriched sources, but the carbon data do not indicate whether those were C4 or marine foods. Similarly, the position of Cluster 2 likely indicates a mix of protein from both C3 and 13C-enriched sources: although the centroid falls within the 95% prediction interval of the C4/marine protein line, that interval is quite wide due to the regression being derived from a small sample. Were the sample larger, akin to that of the C3 protein line, we would expect the Cluster 2 centroid to fall just outside the C4/marine prediction interval (see Fig. 1b) and thus between the two lines. We take this to indicate that at least some of the protein consumed by members of Cluster 2 came from C3 sources. As with Cluster 3, however, whether the 13C-enriched American Journal of Physical Anthropology
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A.W. FROEHLE ET AL. between Clusters 2 and 3 due to their nitrogen values. On the basis of these comparisons, it seems reasonable to interpret the diets of these clusters in the following terms: Cluster 2 includes a 70% C4 diet with mixed C3 and C4 protein, while Cluster 3 encompasses a 50% C3 diet with mainly marine protein (interpretations that are also consistent with archaeological evidence for dietsee Discussion).
portion of the protein for Cluster 2 comes from C4 or marine foods is unclear from the carbon-isotope data alone. The inclusion of d15N data in the hierarchical and kmeans cluster analyses helps make sense of protein sources for Clusters 2 and 3. For example, the high-level split of Cluster 3 from Clusters 2 and 5 is suggestive of major dietary differences between these sub-branches of the dendrogram. Clusters 2 and 5 had very closely positioned k-means centroids with a Euclidian distance of just 4.28, similar to the distance between Clusters 1 and 4, and so close that the split was not visible in the fourgroups k-means clustering regime. Clusters 2 and 3 were almost twice as far from each other than were Clusters 1 and 4 (which split much lower in the dendrogram), despite smaller gaps between Clusters 2 and 3 in d13Capatite and d13Ccollagen (Cluster 1 vs. 4 differences: d13Capatite 5 3.8%, d13Ccollagen 5 1.7%; Cluster 2 vs. 3 differences: d13Capatite 5 2.8%, d13Ccollagen 5 0.8%). This pattern suggests that differences in nitrogen, not carbon, are largely responsible for the high-level split between Clusters 2 and 3, and indeed, their centroid d15N values are highly divergent: 10.1% vs. 17.7%, respectively (Clusters 1 and 4 differ by only 1.3% for d15N). Plotting the clusters in three-dimensional space (Fig. 3) emphasizes the difference
Discriminant function analysis

In the discriminant function analysis, we used cluster assignments according to k-means for ve groups as the training sample. We chose k-means over hierarchical classications because the former revises cluster centroids and membership with sequential iterations, while the latter does not (Baxter, 1994). Thus, in cases of disagreement between the two techniques, we expect k-means to better reect natural trends in the data. Clusters were multivariate normal (Mahalanobis distance/chi square plots), but exhibited unequal covariate matrices (Boxs M)violating the latter assumption is not necessarily fatal to discriminant function analysis (Baxter, 1994). Using the leaveone-out assessment, the discriminant functions incorrectly classied just 4 of 158 cases, three of which were statistical outliers in their original studies (see Discussion). Given this low rate of misclassication and the outlier status of the misclassied individuals, we accept the discriminant function analysis results as robust. All three isotope variables exerted a signicant inuence (all P \ 0.01) on the clustering of cases (Table 2). Three signicant linear functions were derived (all P \ 0.01) and the rst two together accounted for 98.8% of variance in the sample (Table 3). Both carbon-isotope variables exercised their heaviest relative inuence on scores derived from Function 1 (indicated by structure coefcientssee Table 3), while nitrogen exerted its strongest effect on scores from Function 2. The third function explained only 1.2% of variance and coincided with none of the independent variables highest structure coefcients, so we dropped it from further use. The remaining functions are:
TABLE 2. ANOVA results (tests of equality of group means) Variable d Capatite d13Ccollagen d15N
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Wilks Lambdaa 0.124 0.840 0.142
F 269.315 415.647 231.264
P-value \0.01 \0.01 \0.01
Fig. 3. Three-variable plot of archaeological sample according to k-means classications, including cluster centroids. Cluster diets: (1) 100% C3 diet/protein; (2) 30:70 C3:C4 diet, >50% C4 protein; (3) 50:50 C3:C4 diet, marine protein; (4) 70:30 C3:C4 diet, !65% C3 protein; (5) 30:70 C3:C4 diet, !65% C3 protein.
a Smaller values indicate greater effect on the discriminant function(s). All three variables contribute signicantly to the discrimination between groups.
TABLE 3. Function Eigenvalues and structure matrix Structure coefcientsb Function 1 2 3 Eigenvalue 13.402 6.939 0.243
a
% of Variance 65.1 33.7 1.2
Cumulative % 65.1 98.8 100.0
Canonical correlation 0.965 0.935 0.442
d Capatite 0.597* 20.561 0.573
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d13Ccollagen 0.898* 20.047 20.438
d15N 0.192 0.891* 0.411
a Eigenvalues indicate the amount of variance in the sample each function accounts for. The higher an Eigenvalue for a particular function, the higher the percentage of variance that function explains, which is also noted in this table. b Structure coefcients indicate the relative level of association between each variable and each function. Where these values are largest for a particular variable, the inuence of that variable on that function is greatest. The largest values for each variable are indicated by * above, and allow the functions to be characterized according to the key inuential variables. In this case, we can name Function 1 Carbon, and Function 2 Nitrogen. Because of its relatively low inuence on the samples variance, and because none of the variables were most highly correlated with it, we dropped Function 3.
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(Fig. 4a,b; see Table A1 for function scores) that facilitate the application of the model to new cases.
DISCUSSION Diet classications and archaeological sites

Overall, the ve-groups cluster assignments and their dietary interpretations conform well to expectations from previous studies results, although in some cases our results differ somewhat from earlier conclusions, which we discuss below. We also address the seven individuals classied differently in the hierarchical vs. k-means clustering analyses, as well as the four cases misclassied by the discriminant function analysis. Clustering disagreements occurred for Cahokia 120, American Bottom ESLSQ 25, Range 14, and IC 114, and Illinois River 65, 66a, and 153. The discriminant function analysis misclassied American Bottom ESLSQ 25 and IC 116, Illinois River 65, and Tierra del Fuego 13. See Table A1 for individual data. Ontario. Within the Ontario sub-sample, all individuals from the post-agricultural period fell in Cluster 2 (70% C4; mixed C3/C4 protein), consistent with heavy reliance on C4 maize (Harrison and Katzenberg, 2003). Eleven Ontario preagriculturists dating to 400 CE or earlier fell into Cluster 1, indicative of a 100% C3 diet as expected given that they antedate the introduction of maize to the region. Seven others aggregated with Cluster 4, which includes a small contribution of C4 foods to whole diet. All seven postdate 500 CE, a date that coincides with evidence for the early presence of maize in the region, possibly as a result of trade preceding full-scale cultivation after 1000 CE (Crawford et al., 1997; Smith and Crawford, 1997; Harrison and Katzenberg, 2003). While we anticipated the above results, the analysis also revealed some divergence from the overall trend. Four individuals predating 500 CE (three from the Donaldson site and one from Serpent Mounds) all fell into Cluster 4, their d13Capatite and d13Ccollagen values well within the range of later samples from the proposed 5001000 CE maize-importation period. Thus, either some people in Ontario consumed maize much earlier than currently accepted, or this early C4 signal may result from consumption of locally available amaranths, as previous authors have suggested (Schwarcz et al., 1985). Finally, an individual dating to 918 CE grouped with Cluster 1, ostensibly due to little consumption of imported maize despite its apparent availability. Across the entire Ontario sample, nitrogen-isotope values fell between $10 to 15%, with no difference in mean d15N between the subgroups assigned to Clusters 1, 2 and 4 (one-way ANOVA: P 5 0.08; means, respectively: 12.5%, 11.7%, 11.9%). Although the differences were not signicant, the tendency toward lower average nitrogen values in Clusters 4 and 5 members coincides with some protein coming from maize after its introduction into the region. Overall, though, the similarity of the nitrogen-isotope data across subgroups ts with consumption of terrestrial game, waterfowl and freshwater sh as the major sources of protein, and the maintenance of that pattern over time despite the introduction of maize (Schwarcz et al., 1985; Harrison and Katzenberg, 2003). Because there were no discrepancies between the hierarchical and k-means analyses for any American Journal of Physical Anthropology
Fig. 4. Archaeological sample discriminant function scores plotted by group, as assigned in the discriminant function analysis, along with function scores at the group centroids. Cluster diets: (1) 100% C3 diet/protein; (2) 30:70 C3:C4 diet, >50% C4 protein; (3) 50:50 C3:C4 diet, marine protein; (4) 70:30 C3:C4 diet, !65% C3 protein; (5) 30:70 C3:C4 diet, !65% C3 protein. (a) Individual data points from the archaeological sample. Using the leave-one-out technique, the discriminant function analysis classied only 4 of 158 cases (labeled on the plot) differently than did the k-means analysis. Three of these cases were statistical outliers in the original studies from which we drew the data. (b) Plot using the same data as above, but without individual data points. Error bars extend to 2r of the means of both sets of function scores (equivalent to functions at centroids) within each cluster. The limits of the boxes represent the extreme minimum and maximum individual case scores for each function within each cluster. This plot is intended to provide a clean working space for the evaluation of new cases (i.e., those not included in the present study) where diet remains indeterminate using other methods.
Carbon : F1 0:322 d13 Capatite 0:727 d13 Ccollagen
0:219 d15 N 9:354
Nitrogen : F2 0:393 d13 Capatite 0:133 d13 Ccollagen 0:622 d15 N 8:703 We calculated function scores for the individual cases in the dataset and the cluster centroids, generating plots
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A.W. FROEHLE ET AL. In the entire study sample, only seven individuals received conicting cluster assignments for the vegroups analysis, the UPGMA hierarchical method placing them in Cluster 2 while k-means relegated them to Cluster 5. All seven came from the broader Cahokia/ American Bottom/Illinois River regions, where the populations appear to have had rather homogeneous diets (at least relative to the overall sample in the present study), and where six of these seven individuals were statistical outliers within their respective populations (Cahokia 120: Ambrose et al., 2003; American Bottom ESLSQ 25, Range 14, IC 114, Illinois River 66a, 153: Hedman et al., 2002; see Table A1 for individual data). It makes statistical sense that different clustering methods might treat these six outliers differently, especially given rather ne isotopic and dietary distinctions between Clusters 2 and 5. The specic characteristics of these individuals, rather than general aws in the clustering regime, probably explain the classicatory discrepancies, and may point to intraregional migration. For example, Cahokia 120 appears to have eaten less C3 protein than other high status individuals at Mound 72, suggesting an origin in the Illinois River or Corbin Mounds (American Bottom) areas (Ambrose et al., 2003). The converse may be true of the three outliers from the American Bottom, all of whom appear to have eaten less C4 protein than their neighbors. These ndings may indicate an origin outside the region (Hedman et al., 2002), or possibly migration from Cahokia to the American Bottom. The Illinois River disagreements are more difcult to interpret due to small sample size and a high degree of variability at this site (Hedman et al., 2002). Finally, it remains unclear what underlies disagreement over the seventh individual, Illinois River 65 (Hedman et al., 2002), who fell into Cluster 2 per the hierarchical analysis, but Cluster 5 for the k-means analysis. Archaeological details of the burial provide no additional explanatory information (see Goldstein, 1980), but given the overall sample size, this one disagreement does not disqualify the clustering results. Three of the four misclassied individuals in the leave-one-out test of the discriminant function analysis also come from the American Bottom/Illinois River region, and are similarly explained as being statistical outliers. Two of these individuals were already noted above (ESLSQ 25, Illinois River 65) since they also received different classications from the hierarchical vs. the k-means clustering methods. A third case (IC 116) was also an outlier for the American Bottom population, although the hierarchical and k-means results did not disagree on this individuals assignment. Again, the statistical uniqueness of these individuals likely explains their misclassication by the discriminant functions. The fourth individual misclassied in the discriminant function analysis came from Tierra del Fuego (13), and is discussed below. Tierra del Fuego. No discrepancies between the hierarchical and k-means analyses occurred for any individuals from Tierra del Fuego. The sample split mainly between Cluster 3 (50% C4 diet; marine protein) and Cluster 1 (100% C3 diet), individuals in the former coming from southern and southeastern regions while those in the latter came mainly from the northern part of Tierra del Fuego (except one from Navarino Island in the south). This split agrees with the original studys suggestion that marine protein played an important dietary role in the south, while C3 guanaco meat served
individuals from Ontario, we accepted the classications as robust. Greater Cahokia region. Data from three areas within the greater Cahokia region (Pauketat, 1998) were included in this study: the American Bottom, the Illinois River Valley, and Cahokia itself. The American Bottom oodplain and upland sites together constitute the largest population in our analysis. As expected from the original study, which reported an extensive reliance on C4 foods in the overall diet (Hedman et al., 2002), 33 of 35 oodplain individuals and 13 of 17 upland individuals fell into Cluster 2. Among those assigned to Cluster 2, d15N values fell within the range of 7.910.7% (mean 5 9.3 6 0.09%), subsuming the intersite variability reported in the original study (Hedman et al., 2002), but consistent with a mixed protein base consisting of wild terrestrial and aquatic game supplemented with wild nuts and cultivated maize. The remaining two oodplain and four upland individuals split between Clusters 1 (100% C3), 4 (70% C3) and 5 (70% C4). While their d15N measurements fell well within the range of the larger population, their d13C values indicated less reliance upon C4 foods, in particular as a source of protein. Three of these individuals also caused disagreement between the hierarchical and k-means analyses, which we discuss below. The Illinois River specimens, from just outside the American Bottom and Cahokia regions proper, split mainly between the high-C4 Clusters 2 (70% C4; mixed protein) and 5 (70% C4) with a single individual falling into Cluster 4 (mostly C3). Despite their seeming randomness, these classications are consistent with archaeological evidence that some of these people incorporated a smaller proportion of C4 foods into their diet and displayed more dietary heterogeneity than their neighbors in the American Bottom (Hedman et al., 2002). On the other hand, the Illinois River population did not appear to differ from the American Bottom in terms of trophic level, demonstrated by their average d15N of 8.9 6 0.23% and the centroid d15N values for Clusters 2 and 5 (10.1% and 8.8%, respectively). We discuss the single Illinois River individual that fell into Cluster 4 below. Cahokia Mound individuals split between Clusters 4 and 5, as expected, along the social status lines reported in the original study (Ambrose et al., 2003). Low status individuals fell into Cluster 5, indicating a heavy reliance on maize as a dietary staple, but not as a source of protein. On the other hand, Cluster 4 incorporated the high status individuals, indicating that they obtained the bulk of their diet, including protein, from C3 sources, with a small overall maize component. Further, these cluster assignments are consistent with a slightly higher trophic level among the high status subgroup, whose mean d15N value was signicantly higher than the low status groups average (10.6 6 0.67% vs. 8.2 6 0.19% respectively; independent samples t-test, P \ 0.01). Interestingly, given the results from the nearly contemporaneous American Bottom (within 125225 years), the cluster assignments suggest that even low status individuals from Cahokia consumed less C4 food, especially as protein, than populations from the surrounding region. At the same time, however, low status Cahokia nitrogen-isotope values indicate a trophic position at the lower end of the range for the entire region, while the high status mean falls near the top of that range (see above). American Journal of Physical Anthropology

TABLE 4. Experimental rat bone stable isotope values and discriminant function scores Diet ID 1A 2B 3C 4D 5E 6F 12.13G
a b c a
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Function scores F1 F2 0.264 1.122 23.742 20.266 20.569 25.197 23.293 Cluster 1 none 2 2 4 5 5
Diet compositionb C3 non-protein/C3 protein (20%) C3 non-protein/C4 protein (5%) C4 non-protein/C3 protein (5%) C3 non-protein/C4 protein (70%) C4 non-protein/C3 protein (70%) C4 non-protein/C3 protein (20%) C4 non-protein/C3 protein (20%)
d13Capatite % (PDB)c 214.2 211.9 21.4 26.2 212.0 23.7 24.1
d13Ccollagen % (PDB)c 219.9 213.2 212.2 28.2 219.2 214.5 215.4
d15Ncollagen % (AIR) 9.7 11.1 9.7 11.4 9.6 6.4 9.4
27.561 21.643 2.158 3.893 26.366 20.977 21.103
Carbon data from Ambrose and Norr (1993); nitrogen data from Ambrose (2000). Diet composition from Ambrose and Norr (1993). Protein as percent of total diet by weight in parentheses. Carbon data adjusted to reect preindustrial atmosphere by adding 1.5% (Marino and McElroy, 1991).
as the primary northern protein source (Yesner et al., 2003). Mean d15N values for the subgroups were also consistent with this interpretation, being signicantly higher in those assigned to Cluster 3 than those falling in with Cluster 1 (17.5 6 0.47% vs. 11.5 6 0.54% respectively; independent samples t-test, P \ 0.01). Our interpretation of whole diet, however, differs from the previous studys, where D13Capatite-collagen spacing values were taken to indicate a small C4 component for the northern subsample. Compared with the experimental animal regression lines (Kellner and Schoeninger, 2007; Froehle et al., 2010) instead of tissue spacing, the northern subset falls at the 100% C3 diet end of the C3 protein line. Therefore, it appears that people from northern Tierra del Fuego also differed from southern populations by eating a diet devoid of 13C-enriched foods, though we are unaware of any independent lines of evidence that support this interpretation. One individual, Tierra del Fuego 13, came from the south and was placed in Cluster 1 by the discriminant function analysis, in contrast to both clustering methods which assigned this individual to Cluster 4. The Cluster 1 assignment would associate this individual with the northern Tierra del Fuegan subgroup, reliant entirely upon C3 resources, while placement in Cluster 4 would indicate about 70% consumption of C3 foods. Tierra del Fuego 13 falls midway between the centroids of Clusters 1 and 4 for 13Capatite, but is enriched in d13Ccollagen and has higher d15N than both cluster centroids. This position suggests a diet midway between Clusters 1 and 4 in terms of C4 foods in overall diet, but also indicates the consumption of higher trophic level and more 13Cenriched protein than in either Cluster 1 or 4. This combination is consistent with a largely C3 diet similar to the northern Tierra del Fuego group, but with some incorporation of marine and perhaps C4 resources in the general fashion of the southern regions (although not sufcient to fall into Cluster 3). Thus, Tierra del Fuego 13 appears to have consumed a diet intermediate to the northern and southern patterns, which in its pairing of mainly C3 resources with some marine foods, is unique in the overall sample studied in the present meta-analysis. This individuals uniqueness may underlie the discrepancy between the discriminant function analysis and the clustering methods classications. San Nicolas Island. Finally, the entire subsample from San Nicolas Island fell into Cluster 3, the marine protein cluster. In fact, all three isotope values for the Cluster 3 centroid match almost exactly the corresponding means for San Nicolas islanders as a group. There were no dis-
agreements between hierarchical, k-means, or discriminant function analyses as to the classication of any individual from San Nicolas Island. This categorization agrees with the emphasis on marine foods described in the original study (Harrison and Katzenberg, 2003). It disagrees, however, in that it suggests a roughly 50% C4 contribution to diet, whereas the previous authors used D13Capatite-collagen values to conclude that the San Nicolas islanders consumed no C4 foods. Adjusted for the industrial effect (Marino and McElroy, 1991), mean d13Capatite and d13Ccollagen values for this sample (29.0% and 211.7%, respectively) are very similar to experimental values for rats consuming a controlled diet of 50:50 C3:C4 nonprotein with tuna sh as the protein source (Jim et al., 2004: d13Capatite 5 28.6%; d13Ccollagen 5 212.2%). Rats from the same experimental study, eating a diet of 100% C3 nonprotein with tuna sh as the protein source, have considerably less-enriched carbon-isotope values, especially for d13Capatite (213.4%). Together, our three-variable assessment and the experimental rat data strongly suggest that the San Nicolas Island population indeed exploited both marine and C4 resources. The source of the C4 signal is unknown, but others have suggested that mortars and pestles common to San Nicolas sites may have been used to process the seeds of native grasses (Meighan and Eberhart, 1953), some of which use the C4 photosynthetic pathway (Thomas, 1995). This hypothesis merits further exploration.
Experimental animal data

As an additional check of our results, we plotted discriminant function values for rats raised on experimental diets (N 5 7 diets; see Table 4) and looked for compatibility with our models expectations. Despite other experimental feeding studies of diet-tissue isotope fractionation, the data used here come from the only such investigation to publish individual d13Ccollagen, d13Capatite and d15N values (Ambrose and Norr, 1993; Ambrose, 2000). In these experimental diets, the protein and nonprotein components were within themselves monoisotopic, but in most cases the protein and nonprotein isotopic signatures diverged from one another. For example, the diet 6F (see Table 4) was 80% nonprotein and 20% protein by weight, with the nonprotein portion coming entirely from C4 sources, and all protein coming from C3 foods (Ambrose and Norr, 1993). Below, this diet is thus referred to as being 80% C4 nonprotein and 20% C3 protein by weight, and other diets are described in the same manner. American Journal of Physical Anthropology
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A.W. FROEHLE ET AL. 70% C3 protein by weight, fell accordingly into Cluster 4 where the centroid indicates an overall 30% C4 diet with !65% of protein coming from C3 sources. In Cluster 5, diet 12/13G fell very near the centroid while diet 6F fell within 2 standard deviations of the centroid, indicating an overall $70% C4 diet with protein coming from !65% C3 sources. This positioning matches well with the compositions of these diets, both of which were 80% C4 nonprotein by weight, with the remainder consisting entirely of C3 protein. Interestingly, despite similar placement along the Function 1/Carbon axis, diet 6F fell lower on the Function 2/Nitrogen axis than did 12/ 13G. The difference corresponds to the lower d15N values observed in the rats on diet 6F vs. 12/13G, and ts given that 6F contained protein from soy and wheat, while 12/ 13Gs protein source was milk casein (Ambrose and Norr, 1993). This nding supports the idea that positioning along the Function 2/Nitrogen axis provides information about trophic level when other aspects of diet are the same. Positioning on the discriminant function plot of diets 2B and 3C was somewhat less straightforward than for the other diets. Diet 2B fell outside of the boundaries of all Clusters, which may be explained by the fact that its specic diet (95% C3 nonprotein and 5% C4 protein by weight) was not represented in our archaeological sample. We might have expected 2B to fall closer to Cluster 1, however, considering that the vast majority of the diet carried a C3 isotopic signature. Diet 3C consisted of 95% C4 nonprotein and 5% C3 protein, and fell just within the boundaries of Cluster 2. This was to be expected in a general sense on the basis of the high C4 content of diet 3C, but with its C3 protein content, we might have predicted it to fall further to the left along the Function 1/ Carbon axis, and nearer to Cluster 5.
For the most part, given the composition of their experimental diets, the rats fell as expected on our discriminant function plot (Fig. 5). For example, the 100% C3 diet 1A fell within the range of the 100% C3 Cluster 1. Diet 5E, which was 30% C4 nonprotein and
Fig. 5. Discriminant function values calculated from published bone isotope values for rats fed experimental diets (Ambrose and Norr, 1993; Ambrose, 2000), and plotted against archaeological human clusters from this study. Rat carbon isotope values were adjusted to match the preindustrial atmosphere (and thus the archaeological data) by adding 1.5% (Marino and McElroy, 1991). Information on the composition of the experimental diets is in Table 4. Cluster diets: (1) 100% C3 diet/protein; (2) 30:70 C3:C4 diet, >50% C4 protein; (3) 50:50 C3:C4 diet, marine protein; (4) 70:30 C3:C4 diet, !65% C3 protein; (5) 30:70 C3:C4 diet, !65% C3 protein.
TABLE 5. Saipan Archaeological and foodweb data (Ambrose et al., 1997) Archaeological remainsa Function Valuesb Site MacHomes MacHomes Nansay Duty Free Site Duty Free Site Duty Free Site Duty Free Site Mean: Burial 2 5 6 1 2 3 4 d Capatite % (PDB) 210.2 27.7 29.7 29.6 28.5 29.1 210.0 29.3
13
d Ccollagen % (PDB) 218.0 218.3 219.0 218.6 218.9 218.7 218.5 218.6
13
d N % (AIR) 8.4 8.4 7.9 7.1 6.1 7.5 9.2 7.8 d13C (%) PDB
15
F1 25.177 24.590 25.852 25.705 25.787 25.529 25.301 25.420
F2 21.864 22.886 22.504 22.988 24.082 22.949 21.511 22.683
Foodweb carbon isotope valuesc Plant/Diet Seaweed Sugarcane C3 end-member

a b
Bone apatited 23.7 24.1 0.5 215.3
Bone collagene 214.9 215.2 212.7 221.1
Gracilaria tsudae Gracilaria tsudae Puried cane sugar
213.9 214.3 29.7 225.5
As reported in Ambrose et al. (1997), reecting preindustrial atmospheric carbon levels. Calculated using the present studys discriminant functions. c Plant/Diet values adjusted to preindustrial atmospheric carbon levels by adding 1.5% to reported modern values (Marino and McElroy, 1991). d Values assume enrichment of d13C in apatite by 10.2% over diet (Froehle et al., 2010), and reect expected values where the food in question constitutes 100% of diet. e Values based on C3 protein linear equation for d13Ccollagen vs. d13Cdiet from experimental data (Froehle et al., 2010), assuming the food in question constitutes 100% of diet.
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rats on diets 2B and 3C were very similar to one another, and intermediate to those of rats consuming either C3 or C4 protein in sufcient amounts (see Table 4). A similar pattern of divergence from population-based expectations in human individuals may indicate protein deciencies, and is worth exploring further. Comparing skeletal remains with evidence for protein malnutrition to apparently healthy individuals within the same population, where diet is otherwise known to be homogeneous, could test the validity of this particular application of the model.
Example application of the model: addressing dietary ambiguity on Saipan

To demonstrate the use of the discriminant function model, we applied it to an unexplained aspect of diet in an archaeological population from Saipan (Marianas Archipelago, western Pacic, 12501350 CE). Ambrose et al. (1997) originally investigated the diet of these people, reconstructing the isotopic composition of the ancient foodweb by analyzing modern foods matched to archaeological data (Table 5). Their analysis revealed that people on Saipan consumed less marine protein and more protein from C3 sources compared with other nearby islands populations. This nding, along with very high, positive D13Capatite-collagen values (mean 5 8.8 6 0.64%) led Ambrose et al. (1997) to conclude that the Saipan population ate a relatively high proportion of 13Cenriched foods, despite a mainly C3 local ecosystem. Two candidates for this enriched signal were sugarcane and seaweed, both of which are consumed in the region today, but neither of which appears in the relevant archaeological record. While the D13Capatite-collagenvalues pointed to the presence of a 13C-enriched dietary component, they could not determine its specic source. Thus, it is unclear whether sugarcane, seaweed, or a mixture of the two accounts for 13C-enrichment in the bones of Saipan islanders. To apply the discriminant function model to the Saipan data, we estimated the proportions of C3 and 13Cenriched resources each individual (excluding Nansay 4, who postdates the main population; and Nansay 7, who was not an adult) consumed, which we accomplished by examining their placement along the axis of the experimental animal carbon-isotope plots (Fig. 6, see Froehle et al., 2010). Our results conrm that, as previously concluded (Ambrose et al., 1997), the Saipan islanders ingested a 6570% C3 overall diet (individual ratios are in Table 6), with !65% of protein coming from terrestrial C3 sources and the remainder coming from lagoon and reef species. We used these percentages and the food web data to generate hypothetical d13Cdiet values that included either sugarcane or seaweed as the 13C-enriched component, making up 3035% of total diet. We estimated hypothetical 13Cdiet values from the individual diet composition ratios, using the Saipan C3-end member (d13CC3) and either sugarcane or seaweed (d13Csugarcane and d13Cseaweed, respectively), as follows:
Sugarcane : d13 Cdietsugarcane %C3 d13 CC3 %C4 d13 Csugarcane
Fig. 6. Collagen and apatite carbon stable isotope values for Saipan islanders from Ambrose et al. (1997) plotted against compiled experimental animal data (see Froehle et al., 2010). Long-dashed lines represent the 95% prediction interval of the mostly-C3-protein regression, while the short-dashed lines dene the 95% prediction interval of the C4/marine-protein regression. Human data sources are in Table A1. The experimental data from which the regression lines derive have been adjusted to reect pre-industrial atmospheric carbon levels by adding 1.5% (Marino and McElroy, 1991). We used the position of the Saipan data relative to the experimental animal regression lines to determine protein sources as well as percent C3 diet. The C4/marine protein line is derived from animals consuming either 100% C4 protein or 100% marine protein. The C3 protein line is derived from animals consuming !65% C3 protein (13 of 25 individual animals in this group ate 5-35% C4 marine protein; see Froehle et al., 2010). On the basis of this comparison, Saipan islanders appear to have consumed mostly C3 protein, up to 35% marine protein, and on average had a diet consisting of $65 to 70% C3 foods. This is consistent with the assessment of Ambrose et al. (1997), and falls closest to this studys Cluster 4, the centroid for which is plotted here along with error bars delimiting two sample standard deviations from the mean. Cluster diets: (1) 100% C3 diet/protein; (2) 30:70 C3:C4 diet, >50% C4 protein; (3) 50:50 C3:C4 diet, marine protein; (4) 70:30 C3:C4 diet, !65% C3 protein; (5) 30:70 C3:C4 diet, !65% C3 protein.
Low protein content (5% of diet by weight) was common to both 2B and 3C, and may explain their somewhat abnormal positioning relative to the other rat diets and this studys clusters. Ambrose (2000) reports that rats from litters eating diets 2B and 3C were proteinlimited, since they achieved adult weights much lower than those on normal-to-high-protein diets. It may therefore be the case that the rats on diets 2B and 3C synthesized amino acids endogenously from nonprotein dietary precursors in order to build bone collagen, rather than incorporating most or all of those amino acids intact from dietary protein (see Schwarcz, 2000). In both cases, the low protein rats fell approximately where expected along the Function 2/Nitrogen axis, but were shifted $4 to 6 units to the right of the centroids for their expected clusters along the Function 1/Carbon axis. This nding makes sense, in that (barring starvation) tissue nitrogen will be derived largely from dietary protein, no matter its percentage contribution to diet. On the other hand, collagen carbon could be skewed toward nonprotein dietary components when protein intake is very low, which may explain why d13Ccollagen values for
Seaweed : d13 Cdietseaweed %C3 d13 CC3 %C4 d13 Cseaweed
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1.77 1.02 1.08 1.25 0.95 1.10 1.41 1.22f EDe
A.W. FROEHLE ET AL.

Percent diet from C3 and C4 sources as derived from experimental animal regression lines from Froehle et al. (2010). See text for methods. Calculated from %C3:%C4 diet using C3 end-member and sugarcane d13C values from Ambrose et al. (1997). See text for methods. Discriminant function values (F1 and F2) are calculated using the present studys discriminant functions and the hypothetical bone stable isotope values for the sugarcane diet. c Calculated from %C3:%C4 diet using C3 end-member and seaweed d13C values from Ambrose et al. (1997). See text for methods. Discriminant function values (F1 and F2) are calculated using the present studys discriminant functions and the hypothetical bone stable isotope values for the seaweed diet. d 15 d N as measured in Ambrose et al. (1997). Sugarcane and seaweed are very low in protein and should not affect bone collagen d15N values. e Euclidian (geometric) distance between discrinmant function values of measured bone stable isotopes (see Table 5) and hypothetical C3/sugarcane or C3/seaweed diets. f Euclidian distance means for sugarcane and seaweed hypothetical diets are sigincantly different from one another, independent samples t-test, two-tailed P\0.01.
TABLE 6. Expected stable isotope and discriminant function values for Saipan hypothetical diets
d N% (AIR)d
Sugarcaneb
d Ccollagen % (PDB)
15
d Capatite % (PDB)
13
d Cdiet % (PDB)
13
221.2 217.9 220.5 220.4 218.9 219.7 220.9 219.9
211.0 27.7 210.3 210.2 28.7 29.5 210.7 29.7
218.8 217.0 218.4 218.4 217.6 218.0 218.7 218.1
8.4 8.4 7.9 7.1 6.1 7.5 9.2 7.8
26.00 23.67 25.64 25.72 24.92 25.17 25.64 25.25
F1
21.67 22.73 22.20 22.74 23.82 22.70 21.26 22.45
F2
0.84 0.94 0.37 0.25 0.91 0.44 0.42 0.60f
EDe
We then modeled expected human bone isotope values reective of each hypothetical diet (Table 6), assuming an enrichment of 10.2% in apatite over d13Cdiet (see Froehle et al., 2010) and using the d13Ccollagen /d13Cdiet equation from Froehle et al. (2010: pooled C3 protein equation, Table 2, p. 2664) to account for enrichment of d13C in bone collagen. We did not adjust d15N values for differential contributions from seaweed vs. sugarcane since both foods are very low in protein (Ambrose et al., 1997) and thus unlikely to differentially affect tissue nitrogen-isotope ratios. Individual hypothetical bone carbon-isotope values and measured nitrogen-isotope values were used to derive corresponding discriminant function scores for each hypothetical diet (Table 6). Euclidian distances were then calculated between each individuals scores for actual bone isotope measurements and their respective hypothetical diet-specic scores for the corresponding seaweed or sugarcane diets. Euclidian distances of the bone data from their respective hypothetical diet points provided information on the proportions of sugarcane and seaweed responsible for the 13C-enriched isotopic signal on Saipan. On average, actual bone values fell signicantly closer (independent samples t-test, two-tailed P \ 0.01) to sugarcane (mean Euclidian distance 5 0.60) than to seaweed (mean Euclidian distance 5 1.22). As a population, the Saipan islanders actual discriminant function scores fell almost entirely within the range expected for a diet
d15N % (AIR)d Seaweedc d Ccollagen % (PDB) d Capatite % (PDB) d Cdiet % (PDB)

13 13 13
222.4 220.0 221.9 221.8 220.8 221.3 222.2 221.5
212.2 29.8 211.7 211.6 210.6 211.1 212.0 211.3
219.4 218.2 219.2 219.1 218.6 218.9 219.3 219.0
8.4 8.4 7.9 7.1 6.1 7.5 9.2 7.8
26.85 25.17 26.63 26.73 26.21 26.31 26.54 26.35
F1
21.28 22.05 21.75 22.27 23.23 22.18 20.85 21.94
F2
Fig. 7. Plot of mean discriminant function values for actual bone vs. the hypothetical sugarcane and seaweed diets. Error bars indicate the 95% condence intervals of the means for each function. The mean Euclidian distance between actual bone and sugarcane of 0.60 was signicantly smaller than the distance of 1.22 between bone and seaweed (independent samples t-test, two-tailed P < 0.01). One-way ANOVA revealed a signicant difference between group mean Carbon/F1 function values (P < 0.01). Post-hoc analysis (Tukey HSD) showed that for Carbon/ F1, seaweed differed signicantly from bone (P 5 0.03) and sugarcane (P < 0.01), but the latter two did not differ from one another signicantly (P 5 0.86). The three groups of function scores did not differ for Nitrogen/F2 (one-way ANOVA; P 5 0.25), which follows from the manner in which we modeled the hypothetical diets (see text for a detailed description).
13
C3:C4 dieta
MacHomes 2 MacHomes 5 Nansay 6 Duty Free Site Duty Free Site Duty Free Site Duty Free Site Mean:
1 2 3 4
73:27 52:48 68:32 68:32 58:42 63:37 71:29 65:35
MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION consisting of roughly two-thirds C3 foods and one-third sugarcane, and fell completely outside the range of a two-thirds C3/one-third seaweed diet (Fig. 7). Mean Function 1/Carbondiscriminant function scores did not differ between actual bone and sugarcane, whereas both differed signicantly from the hypothetical seaweed diet (one-way ANOVA, P \ 0.01; post hoc Tukey HSD: seaweed vs. bone, P 5 0.03; seaweed vs. sugarcane, P \ 0.01; sugarcane vs. bone, P 5 0.86). In contrast, none of the diets Function 2/Nitrogen means differed (one-way ANOVA, P 5 0.25), which makes sense given that d15N values exert the greatest inuence on Function 2/Nitrogen values, and we did not model dietary differences in d15N values due to sugarcane or seaweed. If our assumptions are valid, then these results suggest that the Saipan population obtained the bulk of its non-C3 dietary component from sugarcane, and not from seaweed. Although no archaeological evidence of sugarcane harvesting exists, the relatively high prevalence of dental caries in some Saipan populations vs. other nearby islands (various sources, discussed in Ambrose et al., 1997:359) appears to corroborate our ndings. There is variation within the group, however, with two individuals (MacHomes 5 and Duty Free 2) falling midway between their hypothetical sugarcane and seaweed diet points. Some individuals may have thus consumed seaweed in addition to sugarcane, but overall our model points to sugarcane as the primary producer of the 13C-enriched signal in the bones of these Saipan islanders.
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CONCLUSIONS
Stable isotope data reect biological processes subject to numerous inuences that result in widely varying outcomes. As such, biometric statistical techniques, which provide probabilistic statements about the relationships between animal tissues and the macronutrients in their diets (e.g., Sokal and Rohlf, 1997; Phillips and Koch, 2002; Bocherens, 2009), offer the best method for understanding, predicting, and interpreting this variation. Recent work applying these techniques to experimental data (e.g., linear regression analysis: Kellner and Schoeninger, 2007; Froehle et al., 2010) is expanded here by applying multivariate cluster analysis and discriminant function analysis to study three isotope variables simultaneously in their relationships to diet. This analysis claried two aspects of diet left ambiguous by previous d13Ccollagen vs. d13Capatite regression models (Kellner and Schoeninger, 2007; Froehle et al., 2010). As expected, the inclusion of d15N data discriminated between marine and C4 protein consumers where carbon-isotope values alone could not. Therefore, in the absence of strong supporting archaeological data, it is important to measure both d13C and d15N from collagen when reconstructing diet in populations from regions where both marine and C4 protein are available. With regard to determining protein sources from positioning relative to the C3- and C4/marine-protein regression lines in the carbon model, the present analysis suggests, at least at the population level, that placement between the lines does indeed indicate mixed protein consumption. This is evident from the placement of Cluster 2 (see Fig. 1b), which included
the Ontario Agricultural and American Bottom populations that clearly obtained both C3 and C4 protein, but is not necessarily true for individuals given considerable scatter around population means even in people with no access to mixed protein, such as Ontario preagriculturalists. The distinction between individuals and populations is an important one for the application of the discriminant function model. While the position of any individual relative to the clusters would provide at least some indication of diet, some overlap between the ranges of diet clusters and their members could weaken any conclusions about individuals. This approach is betterapplied to populations, although samples need not be largefor example, the model was quite capable of making clear dietary distinctions between the northern (N 5 5) and southern (N 5 6) samples from Tierra del Fuego, or between high (N 5 3) and low (N 5 5) status groups from Cahokia. In large populations, this model could reveal nuances of dietary differentiation between sexes, age classes, and social status groups, as well as between regions and over time within societies. Differences within societies, however, may be graded (rather than discreet) or too negrained for the present model to detect, given the wide range of diets included in this sample. In those cases, it would be more productive to apply the same cluster analysis and discriminant function techniques within those populations to look for patterns of distinction. Finally, we should emphasize that the type of negrained, analysis we conducted on the Saipan data requires thorough study of the food web within which a population likely lived (see also Crowley, in press), without which reliable hypothetical diets could not be generated. Contextual information is critical to drawing the strongest conclusions about diet from the discriminant function model, although in the absence of any other information, the model may still have utilityplacement of an individual or population relative to this studys clusters could be used to generate hypotheses, and to direct other, nonisotopic avenues of research. There is considerable room to expand upon and rene this model. The outcomes of cluster analysis depend heavily on the specic sample used, and while the archaeological populations we analyzed here cover a wide range of diets, the addition of data from people eating other diets would likely alter slightly, but strengthen the results. Also, the present model was derived from data on well-preserved bone from healthy adults, potentially limiting the models applicability to other samples, such as children or individuals with clear evidence of malnutrition. Finally, further controlled diet experiments measuring d13Capatite, d13Ccollagen and d15N, would also provide a good test of this studys results, and would allow for the development of a similar model under circumstances of known whole diet and protein d13C and d15N. Continued work in this direction should produce even more effective tools for testing competing hypotheses about prehistoric diets.
ACKNOWLEDGMENTS
The authors thank Alyssa Crittenden and Andrew Somerville for helpful comments on early drafts of this article. They also thank two anonymous reviewers for their thoughtful comments and suggestions. American Journal of Physical Anthropology
TABLE A1. Archaeological bone stable isotope database and cluster centroids
364
Cluster assignment d13Ncollagen % (AIR)b Site name ID F1 Hc Kd DFe F2 Time period
Function scores
American Journal of Physical Anthropology A.W. FROEHLE ET AL.

219.2 219.5 219.9 218.7 221.0 220.4 221.5 220.4 222.2 216.6 221.1 220.6 220.6 220.8 220.2 218.3 218.4 218.5 219.2 219.5 219.0 219.5 219.6 213.3 211.7 212.8 212.1 213.1 210.1 212.2 210.1 211.3 211.2 211.0 210.2 210.8 211.2 210.3 211.3 211.7 212.2 212.2 212.1 211.1 211.5 211.8 11.9 12.5 11.6 11.9 12.7 12.1 12.4 13.9 15.3 11.7 12.4 11.8 12.3 11.9 11.5 13.0 13.4 11.8 11.2 10.8 12.3 11.2 11.2 13.5 11.6 11.1 11.8 11.9 11.6 11.8 10.2 11.0 12.0 10.1 11.0 11.8 10.9 11.4 11.6 12.0 12.3 12.4 12.4 13.9 10.8 12.4 Morrisons Island Donaldson 1 Donaldson 2 Donaldson 2 Levesconte Levesconte Levesconte Levesconte Levesconte Serpent Mounds Serpent Mounds Serpent Mounds Serpent Mounds Serpent Mounds Serpent Mounds Surma Surma Surma Varden Varden Varden Varden Varden Miller Force Force Force Force Fairty Ossuary Fairty Ossuary Uxbridge Uxbridge Uxbridge Uxbridge Uxbridge Uxbridge Uxbridge Uxbridge Uxbridge Kleinberg Ossuary Kleinberg Ossuary Kleinberg Ossuary Ossossane Ossuary Ossossane Ossuary Ossossane Ossuary Ossossane Ossuary MOR R 10 DO 1 V3 BE.28 DO 2 R IA DO 2 R GA LE 1 LE 2 LE 3 LE 4 LE 5 SME D-11-a-100 SMG R OM SMI R 38 SMI 22 SMI 24 SMI 39 SUR 10 SUR 4 SUR 9 VAR 7 VAR 9 VAR 11 VAR 2 VAR 5 MIL 5 FOR 1 FOR 2 FOR R 1 FOR R 2 4 FAI R 2 FAI R 3 1 2 3 4 5 6 7 8 9 2 3 4 2 3 4 5 2300 BCE 555 BCE 5 CE 5 CE 175 CE 175 CE 175 CE 175 CE 175 CE 415 CE 400 CE 400 CE 400 CE 400 CE 400 CE 700 CE 700 CE 700 CE 918 CE 918 CE 918 CE 918 CE 918 CE 1152 CE 1235 CE 1235 CE 1235 CE 1235 CE 1350 CE 1350 CE 1490 CE 1490 CE 1490 CE 1490 CE 1490 CE 1490 CE 1490 CE 1490 CE 1490 CE 1600 CE 1600 CE 1600 CE 1636 CE 1636 CE 1636 CE 1636 CE 1 4 4 4 1 1 1 1 1 4 1 1 1 1 1 4 4 4 4 4 4 4 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 4 4 4 1 1 1 1 1 4 1 1 1 1 1 4 4 4 4 4 4 4 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 4 4 4 1 1 1 1 1 4 1 1 1 1 1 4 4 4 4 4 4 4 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 26.373 25.144 26.405 25.595 28.026 27.786 28.423 27.198 28.297 23.082 27.907 27.836 27.501 27.959 27.579 25.128 24.534 25.182 25.790 25.838 25.919 25.912 27.079 0.516 1.972 0.612 1.660 0.569 3.296 1.555 2.925 1.938 2.197 1.862 2.963 2.348 2.150 2.817 2.005 1.737 1.440 1.429 1.695 2.429 1.620 1.849 1.490 0.212 0.542 1.046 2.377 2.162 2.085 3.046 3.795 20.057 1.863 1.753 1.788 1.788 1.580 1.862 1.940 0.656 0.151 20.452 1.490 20.007 1.316 0.519 21.315 21.222 21.165 20.764 21.298 21.139 22.091 21.399 20.724 21.801 21.528 20.678 21.644 21.096 20.947 20.673 20.553 20.451 20.674 0.785 21.393 20.555
Populationa
d13Capatite % (PDB)b
d13Ccollagen % (PDB)b
SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf SOf
213.6 29.5 211.9 212.3 215.2 215.4 215.1 214.8 215.1 29.1 214.4 214.9 214.2 214.9 214.8 212.5 212.1 211.4 211.3 210.5 212.9 211.0 214.4 26.6 24.4 25.8 24.6 25.8 23.9 24.7 24.1 25.0 25.1 25.3 24.3 25.4 24.5 24.8 25.2 25.4 25.4 25.5 24.9 25.9 25.4 25.1
TABLE A1. (Continued) Cluster assignment d13Ccollagen % (PDB)b Site name ID F1 Hc Kd DFe F2 Time period d13Ncollagen % (AIR)b Function scores
Populationa
SO ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg ABg 11.0 7.9 9.1 8.5 9.0 8.1 9.0 9.4 9.8 9.3 9.1 8.6 10.2 10.2 9.3 9.7 9.2 8.5 8.7 8.2 9.1 9.5 10.3 10.2 10.1 9.3 9.7 9.9 9.0 10.2 9.2 8.3 9.3 9.3 9.6 10.9 9.1 9.4 8.8 8.7 9.5 9.5 9.1 9.6 9.6 8.9 8.9 Ossossane Ossuary ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ ESLSQ Florence St Florence St Florence St Florence St Florence St Florence St Florence St Florence St Range Range Range Range Range Range Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds Corbin Mounds 6 9 17 25 27 39 42 56 77 4 5 45 80 misc 52 14 38 48 67 68 76 78 12 21 29 30 31 34 14 29/9 18 19 13 16 20 14 IC 114 IC 119 IC 116 IC 118 IC 117 IC 46 IC 47 IC 120 IC 123 IC 124 IC 122 1636 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 1275 CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE CE 2 2 2 2h 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2h 2h 5 2 2 2 2 2 2 2 2 2 2 2 2 5 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 2 2 2 2 2 2 2 2 2 2 2 2 2h 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 5h 2 2 2 2 2 2 2 2 2.446 0.471 1.412 20.427 2.338 1.121 1.878 1.262 1.321 1.398 1.058 1.942 1.987 0.335 1.442 3.095 4.000 1.239 2.343 2.184 2.530 2.921 0.354 1.683 2.155 0.448 2.792 1.532 1.892 1.704 2.627 1.464 1.953 2.610 1.661 20.302 21.331 22.084 0.603 1.182 1.181 0.390 0.824 1.266 1.920 2.039 2.750
24.1 26.1 25.8 26.1 24.9 25.1 23.8 25.3 25.9 25.3 24.6 25.6 25.0 26.5 25.8 24.8 23.7 25.9 25.1 24.8 24.1 24.3 25.4 25.7 25.5 26.0 24.2 25.5 24.7 25.4 23.9 25.1 24.5 24.0 25.1 25.1 25.5 28.3 22.9 23.3 24.3 27.0 25.6 25.0 23.4 23.9 23.5
211.0 211.9 211.1 213.3 210.2 211.5 211.3 211.6 211.4 211.4 212.1 210.3 211.0 212.6 211.1 29.4 28.5 211.1 210.0 210.2 210.3 29.8 213.1 211.1 210.5 212.4 210.1 211.3 210.9 211.2 210.3 211.1 211.0 210.3 211.2 214.3 215.0 214.9 213.4 212.4 212.2 212.1 212.0 211.8 211.6 211.0 210.2
21.713 22.968 22.233 22.812 22.517 23.202 23.139 22.347 21.780 22.345 22.875 22.535 21.838 21.480 22.146 22.046 22.651 22.592 22.630 23.085 22.826 22.420 21.898 21.595 21.674 22.197 22.337 21.399 22.695 21.732 22.812 23.000 22.588 22.729 22.248 21.833 22.883 21.551 23.878 23.644 22.727 21.634 22.426 22.342 22.963 23.129 23.179
365
TABLE A1. (Continued)
366
Cluster assignment d13Ncollagen % (AIR)b Site name ID F1 Hc Kd DFe F2 Time period
Function scores
Populationa
d13Ccollagen % (PDB)b
American Journal of Physical Anthropology A.W. FROEHLE ET AL.

29.9 211.0 210.2 212.5 217.7 219.7 215.3 214.8 213.2 212.7 212.3 210.4 220.4 215.3 212.8 218.8 214.3 217.8 218.4 216.0 216.6 218.4 217.2 221.1 220.3 218.6 29.1 211.8 213.3 211.6 212.6 213.3 216.8 218.5 29.9 29.1 211.3 211.1 29.2 210.6 29.7 29.7 210.5 29.7 29.5 211.1 29.1 8.8 9.8 10.7 9.8 9.1 9.4 8.8 8.9 8.3 9.6 7.9 8.8 7.6 8.9 9.9 11.9 7.9 10.2 9.7 8.7 8.7 8.0 7.9 12.6 11.9 10.8 18.0 18.5 15.1 17.2 18.8 17.2 13.2 10.6 19.0 18.6 18.2 19.0 17.2 16.5 19.5 19.2 16.4 16.9 16.4 18.5 18.9 Corbin Mounds Hill Prairie Hill Prairie Hill Prairie Hill Prairie Hill Prairie Schild A Schild A Schild A Schild A Schild A Schild A Schild A Schild A Schild A Mound 72 Mound 72 Mound 72 Mound 72 Mound 72 Mound 72 Mound 72 Mound 72 N. Tierra del Fuego Rio Grande Punta Maria Maria Luisa Caleta Falsa Caleta Falsa Policarpo Ushuaia Hoste Island Hoste Island Navarino Island SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 SNI 40 IC 121 5 6 1 2 3 157 66a 65 62 69a 85 153 149 101a 12 120 162 201 53 142 146 186 1 3 4 5 7 9 10 11 12 13 14 1 2 3 6 9 10 12 16 17 18(257-83) 19 20 257-72 1275 CE 1275 CE 1275 CE 1275 CE 1275 CE 1275 CE 1200 CE 1200 CE 1200 CE 1200 CE 1200 CE 1200 CE 1200 CE 1200 CE 1200 CE 10501150 CE 10501150 CE 10501150 CE 10501150 CE 10501150 CE 10501150 CE 10501150 CE 10501150 CE prehistoric prehistoric post 1500 BCE post 1000 BCE 850 BCE 850 BCE post 1500 BCE post 1500 BCE post 1500 BCE post 1500 BCE post 1500 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2030 BCE 2 2 2 2 4 1 5 2h 2h 2 2 2 5h 5 2 4 2h 4 4 5 5 5 5 1 1 1 3 3 3 3 3 3 4 1 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 4 1 5 5 5 2 2 2 4 5 2 4 5 4 4 5 5 5 5 1 1 1 3 3 3 3 3 3 4 1 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 4 1 5 5 2h 2 2 2 4 5 2 4 5 4 4 5 5 5 5 1 1 1 3 3 3 3 3 3 1h 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3.075 1.537 3.530 20.101 25.312 27.426 22.837 21.067 20.582 0.227 0.403 2.304 26.614 22.782 0.252 24.606 21.469 24.573 25.119 20.888 22.226 23.752 22.644 28.282 27.918 26.569 4.072 1.704 20.421 2.144 0.898 0.006 24.284 26.250 4.096 4.654 2.162 2.805 4.243 2.653 4.254 4.253 3.090 4.264 4.043 2.921 4.720 23.359 21.679 22.531 21.211 20.735 0.001 21.609 23.171 22.663 21.934 23.303 22.883 23.270 21.587 21.850 20.265 23.058 20.796 21.187 24.791 23.770 24.366 24.583 2.453 2.248 1.357 4.466 5.047 3.086 3.557 5.481 4.432 2.539 0.892 4.510 4.289 4.730 4.861 3.444 3.334 4.966 4.700 2.813 2.641 2.671 4.275 4.476
AB ABg ABg ABg ABg ABg IRVi IRVi IRVi IRVi IRVi IRVi IRVi IRVi IRVi CM72j CM72j CM72j CM72j CM72j CM72j CM72j CM72j TDFk TDFk TDFk TDFk TDFk TDFk TDFk TDFk TDFk TDFk TDFk SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg
23.1 26.1 22.3 27.8 211.8 214.0 29.3 25.0 26.7 26.2 25.4 24.4 28.7 29.2 26.1 29.0 26.7 210.0 210.0 21.6 24.4 24.6 23.8 215.7 215.9 214.8 28.1 29.7 210.6 27.9 210.6 210.7 213.4 213.9 26.9 26.7 29.2 28.2 26.8 28.1 27.2 27.0 26.9 25.4 26.2 27.5 26.7
TABLE A1. (Continued) Cluster assignment d13Ccollagen % (PDB)b Site name ID F1 3.564 1.746 3.963 1.988 1.894 2.752 4.906 2.692 3.107 5.029 3.365 3.130 2.005 2.728 2.193 3.610 2.534 5.290 27.567 1.731 3.046 25.276 21.730 A B C: 256-91 256-93 256-98 256-99 256-101 256-104 256-105 25-107.1 25-107 28-F-152 ACC 5-A16 ACC16 2 3 221265 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1350 BCE 1650 CE 1650 CE 1650 CE 1650 CE 1650 CE 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Hc Kd DFe SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI SNI 16 16 16 16 16 16 16 16 16 16 16 16 16 18 18 18 18 18 Time period 210.3 211.6 29.9 211.2 211.6 210.9 28.8 211.1 210.5 28.5 29.7 210.5 210.7 210.6 211.3 210.2 211.2 28.8 17.9 17.0 20.6 15.6 16.5 17.8 16.4 17.6 17.8 16.7 17.5 18.2 14.9 16.4 17.9 19.1 17.8 20.8 12.0 10.1 17.7 11.4 8.8 220.3 211.3 210.5 218.6 215.3 d13Ncollagen % (AIR)b Function scores F2 3.773 3.865 6.095 2.733 3.240 4.063 2.332 3.755 4.038 2.755 4.272 4.365 2.600 3.153 4.426 4.886 4.023 5.776 1.905 21.977 4.013 0.230 22.962
Populationa
SNI SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg SNIg Cluster centroidsl Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5
26.9 29.0 28.4 28.2 28.2 28.0 25.1 27.6 27.8 25.6 28.6 28.0 28.8 27.8 28.9 27.8 28.0 26.9
214.8 25.0 27.8 211.0 25.9
Original sources of data indicated below. Data as reported in original studies, reecting preindustrial atmospheric carbon levels (Marino and McElroy, 1991). From UPGMA agglomerative hierarchical cluster analysis, splitting into ve groups. d From ve-groups k-means cluster analysis. e From discriminant function analysis. f Indicates different classication than obtained using k-means analysis. g Harrison and Katzenberg (2003); SO, Southern Ontario; SNI, San Nicolas Island. h Hedman et al. (2002); AB, American Bottom. i Hedman et al. (2002), originally from Schober (1998); IRV, Illinois River Valley. j Ambrose et al. (2003); CM72, Cahokia Mound 72. k Yesner et al. (2003); TDF, Tierra del Fuego. l Data as determined from the present studys cluster analyses and discriminant function analysis. See Table 1 for descriptions of average diet.
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FROEHLE ET AL. 2012 Multivariate Carbon and Nitrogen

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AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 147:352369 (2012)

WILEY PERIODICALS, INC.

MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION

STABLE ISOTOPE BIOGEOCHEMISTRY

METHODS Archaeological data

Multivariate statistical analysis

RESULTS Cluster analysis

MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION

3.21 9.60 4.28

Discriminant function analysis

Wilks Lambdaa 0.124 0.840 0.142

F 269.315 415.647 231.264

P-value \0.01 \0.01 \0.01

% of Variance 65.1 33.7 1.2

Cumulative % 65.1 98.8 100.0

Canonical correlation 0.965 0.935 0.442

d Capatite 0.597* 20.561 0.573

d13Ccollagen 0.898* 20.047 20.438

d15N 0.192 0.891* 0.411

American Journal of Physical Anthropology

MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION

DISCUSSION Diet classications and archaeological sites

Carbon : F1 0:322 d13 Capatite 0:727 d13 Ccollagen

0:219 d15 N 9:354

MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION

d13Capatite % (PDB)c 214.2 211.9 21.4 26.2 212.0 23.7 24.1

d13Ccollagen % (PDB)c 219.9 213.2 212.2 28.2 219.2 214.5 215.4

d15Ncollagen % (AIR) 9.7 11.1 9.7 11.4 9.6 6.4 9.4

27.561 21.643 2.158 3.893 26.366 20.977 21.103

Experimental animal data

F1 25.177 24.590 25.852 25.705 25.787 25.529 25.301 25.420

F2 21.864 22.886 22.504 22.988 24.082 22.949 21.511 22.683

Foodweb carbon isotope valuesc Plant/Diet Seaweed Sugarcane C3 end-member

Bone apatited 23.7 24.1 0.5 215.3

Bone collagene 214.9 215.2 212.7 221.1

Gracilaria tsudae Gracilaria tsudae Puried cane sugar

213.9 214.3 29.7 225.5

American Journal of Physical Anthropology

MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION

Example application of the model: addressing dietary ambiguity on Saipan

Seaweed : d13 Cdietseaweed %C3 d13 CC3 %C4 d13 Cseaweed

American Journal of Physical Anthropology

A.W. FROEHLE ET AL.

221.2 217.9 220.5 220.4 218.9 219.7 220.9 219.9

211.0 27.7 210.3 210.2 28.7 29.5 210.7 29.7

218.8 217.0 218.4 218.4 217.6 218.0 218.7 218.1

8.4 8.4 7.9 7.1 6.1 7.5 9.2 7.8

26.00 23.67 25.64 25.72 24.92 25.17 25.64 25.25

21.67 22.73 22.20 22.74 23.82 22.70 21.26 22.45

0.84 0.94 0.37 0.25 0.91 0.44 0.42 0.60f

d15N % (AIR)d Seaweedc d Ccollagen % (PDB) d Capatite % (PDB) d Cdiet % (PDB)

222.4 220.0 221.9 221.8 220.8 221.3 222.2 221.5

212.2 29.8 211.7 211.6 210.6 211.1 212.0 211.3

219.4 218.2 219.2 219.1 218.6 218.9 219.3 219.0

8.4 8.4 7.9 7.1 6.1 7.5 9.2 7.8

26.85 25.17 26.63 26.73 26.21 26.31 26.54 26.35

21.28 22.05 21.75 22.27 23.23 22.18 20.85 21.94

73:27 52:48 68:32 68:32 58:42 63:37 71:29 65:35

American Journal of Physical Anthropology

Cluster assignment d13Ncollagen % (AIR)b Site name ID F1 Hc Kd DFe F2 Time period

American Journal of Physical Anthropology A.W. FROEHLE ET AL.

MULTIVARIATE STABLE ISOTOPE DIET RECONSTRUCTION

American Journal of Physical Anthropology