(1) Isotopic analyses of collagen, the main protein preserved in sub fossil bone and tooth, has long provided a powerful tool for the reconstruction of ancient diets and environments. Although isotopic studies of contemporary ecosystems have typically focused on more accessible tissues (e.g., muscle, hair), there is growing interest in the potential for analyses of collagen because it is often available in hard tissue archives (e.g., scales, skin, bone, tooth), allowing for enhanced long-term retrospective studies. The quality of measurements of the stable carbon and nitrogen isotopic compositions of ancient samples are subject to robust and well-established criteria for detection of contaminants and digenesis. Among these quality control (QC) criteria, the most widely utilized is the atomic C:N ratio (C:N_Atomic), which for ancient samples has an acceptable range between  2.9 and 3.6. While this QC criterion was developed for ancient materials, it has increasingly being applied to collagen from modern tissues.

(2) Here we use a large survey of published collagen amino acid compositions (n = 436) from 193 vertebrate species as well as recent experimental isotopic evidence from 413 modern collagen extracts to demonstrate that the C:N_Atomic range used for ancient samples is not suitable for assessing collagen quality of modern and archived historical samples.

(3) For modern tissues, collagen C:N_Atomic falling outside 3.00–3.30 for fish and 3.00–3.28 for mammals and birds can produce systematically skewed isotopic compositions and may lead to significant interpretative errors. These findings are followed by a review of protocols for improving C:N_Atomic criteria for modern collagen extracts.

(4) Given the tremendous conservation and environmental policy-informing potential that retrospective isotopic analyses of collagen from contemporary and archived vertebrate tissues have for addressing pressing questions about long-term environmental conditions and species behaviours, it is critical that QC criteria tailored to modern tissues are established.

Establishing an observed range for vertebrate collagen C:N_Atomic

To establish the natural range of C:N_Atomic for vertebrate collagen we surveyed a wide body of data from the food chemistry literature. Amino acid compositions are from both acid and pepsin solubilized collagens. Comparing C:N_Atomic ratios from studies that performed both methods on collagen from 13 species, Szpak (2011) found that amino acid compositions measured using acid and pepsin solubilized collagen were nearly identical with no statistically significant differences. Data from both types of collagen extraction techniques should therefore produce amino acid compositions that are comparable for our purposes. Because fish collagens are adapted to environmental conditions (Eastoe 1957; Gustavson 1955), we further grouped fish based on habitat preferences using classifications provided on FishBase (Froese and Pauly 2000) as follows: warm water (tropical, subtropical) and cold water (temperate, polar, boreal, and deep-water). Building on Szpak (2011), the survey located 436 amino acid compositions (see Supporting Information [SI] 1, Table S1), which we categorized by tissue type (skin, scale, bone) and by taxonomic class (Table 3). All amino acid compositions were compared as residues per 1000.

Establishing an acceptable range for C:N_Atomic

To establish a C:N_Atomic range within which modern collagen stable isotope compositions have not been meaningfully skewed by contamination with non-collagenous materials, we used δ¹³C and C:N_Atomic data from recent studies (Guiry, et al. 2016c; Szpak and Guiry In Prep) comparing collagen extracted from modern bones prepared following different protocols designed to produce collagen contaminated to varying degrees with residual bone lipids. For fish, data are derived from analyses of four separate collagen extractions (total n=288) from each of 72 bones taken from 35 individual fish representing 17 marine and freshwater taxa. For mammals (n = 25 samples) and birds (n = 25 samples), data are derived from five separate collagen extractions (total n = 250) from high lipid-content bones of 50 individuals representing 19 taxa. Because lipids are carbon-rich and are significantly depleted in ¹³C relative to collagen, lipid contamination is easily detectable by comparing δ¹³C and C:N_Atomic, which will be skewed lower and higher, respectively, when collagen is contaminated with residual lipids. For each set of analyses (i.e., four to five different collagen extractions per bone), we quantified the effect of different amounts of lipid contamination on stable carbon isotope composition by determining the difference in δ¹³C between the sample with the lowest C:N_Atomic (this will be nearest to the theoretical C:N_Atomic observed in collagen (Szpak 2011)) and the other three to four samples. This produced 413 (216 for fish and 197 for birds and mammals) individual comparisons of the relationship between C:N_Atomic and δ¹³C resulting from different levels of contamination with non-collagenous materials (in this case, primarily lipids). We also explored the extent to which δ¹³C may be affected even when samples produce C:N_Atomic values within the acceptable range by quantifying the relationship between positive shifts in C:N_Atomic and skewing of δ¹³C at a smaller scale. To accomplish this we compared Δ¹³Cclean-contaminated (i.e., for each sample, δ¹³C of the extract with the lowest C:N_Atomic subtracted from the δ¹³C of extracts with higher C:N_Atomic) and ΔC:N_Atomic (clean-contaminated) (i.e., for each sample, C:N_Atomic of the extract with the lowest C:N_Atomic subtracted from the C:N_Atomic of extracts with higher C:N_Atomic) within each set of  collagen extractions. This produced 413 (216 for fish and 197 for birds and mammals) individual comparisons of the relationship between Δ¹³Cclean-contaminated and ΔC:N_Atomic (clean-contaminated) for establishing the point at which small deviations in C:NAtomic begin to impact δ¹³C.

Statistics

Statistical analyses were performed with PAST Version 3.22 (Hammer, et al. 2001). For amino acid residue and C:N_Atomic data we used a Shapiro-Wilk test to assess normality of distribution (SI1 Table S2). When comparing groups where one or more samples were not normally distributed, we used a Mann-Whitney U test. For comparisons between group with normal distributions, we used either a Student’s t test (where variances were equal) or a One Way ANOVA followed by either a Dunnett’s (when variances were not equal) or a Tukey’s (when variances were equal) post hoc test. A Levene’s test was used to assess homogeneity of variance. For determining the significance of correlations between bone collagen C:N_Atomic and δ¹³C data we used Spearman’s ρ.

The following documents are included:

Table S1. Amino acid compositions for collagen and non-collagenous proteins collected from published literature. All data are presented as residues/1000. Materials code: 1= bone, 2 = scale, 3=skin. Temperature code: 1 = cold water, 2 = warm water.

Table S2. Statistical results: Shapiro Wilks tests for C:N_Atomic for different taxonomic groups.

Table S3. Results for Spearman’s ρ tests for collagen δ¹³C grouped by ascending C:N_Atomic

Table S4. Extent to which shifts C:N_Atomic (resulting from lipid contamination) affect δ¹³C. Comparison made with data generated by recent studies (Guiry, et al. 2016; Szpak and Guiry In Prep) on the effects of collagen extractions methods on the elemental and isotopic compositions of 122 bones from 85 fish, mammal, and bird specimens from diverse taxa and environments. Four to five extraction procedures were applied to subsamples from each bone. Within each group of four to five samples per bone, the δ¹³C of the sample with the lowest C:N_Atomic was subtracted from the δ¹³C of the other samples. Table shows mean difference in δ¹³C (Δ13C) for analyses binned into groups with ascending C:N_Atomic. Spearman’s ρ tests identify the point at positive shifts in Δ¹³C corresponding with increasing C:N_Atomic become significant. Statistically significant results in bold.

Table S5. Mean amino acid compositions and corresponding C:N_Atomic for vertebrate classes not shown in Table 3

ESM_References: A list of references to the data appearing in Table S1.