DOI: 10.5061/dryad.ffbg79crm
title: Quality control for modern bone collagen stable carbon and nitrogen isotope measurements

description: (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:NAtomic), 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:NAtomic range used for ancient
 samples is not suitable for assessing collagen quality of modern and
 archived historical samples. (3) For modern tissues, collagen C:NAtomic
 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:NAtomic 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.
description: Establishing an observed range for vertebrate collagen
 C:N<sub>Atomic</sub> To establish the natural range of
 C:N<sub>Atomic</sub> 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<sub>Atomic</sub> 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<sub>Atomic</sub> 
 To establish a
 C:N<sub>Atomic</sub> range within which modern collagen stable
 isotope compositions have not been meaningfully skewed by contamination
 with non-collagenous materials, we used δ<sup>13</sup>C and
 C:N<sub>Atomic</sub> 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
 <sup>13</sup>C relative to collagen, lipid contamination is
 easily detectable by comparing δ<sup>13</sup>C and
 C:N<sub>Atomic</sub>, 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 δ<sup>13</sup>C between the sample with the
 lowest C:N<sub>Atomic</sub> (this will be nearest to the
 theoretical C:N<sub>Atomic</sub> 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<sub>Atomic</sub> and
 δ<sup>13</sup>C resulting from different levels of
 contamination with non-collagenous materials (in this case, primarily
 lipids). We also explored the extent to which δ<sup>13</sup>C
 may be affected even when samples produce C:N<sub>Atomic</sub>
 values within the acceptable range by quantifying the relationship between
 positive shifts in C:N<sub>Atomic</sub> and skewing of
 δ<sup>13</sup>C at a smaller scale. To accomplish this we
 compared Δ<sup>13</sup>Cclean-contaminated (i.e., for each
 sample, δ<sup>13</sup>C of the extract with the lowest
 C:N<sub>Atomic</sub> subtracted from the
 δ<sup>13</sup>C of extracts with higher
 C:N<sub>Atomic</sub>) and ΔC:N<sub>Atomic</sub>
 (clean-contaminated) (i.e., for each sample,
 C:N<sub>Atomic</sub> of the extract with the lowest
 C:N<sub>Atomic</sub> subtracted from the
 C:N<sub>Atomic</sub> of extracts with higher
 C:N<sub>Atomic</sub>) within each set of  collagen
 extractions. This produced 413 (216 for fish and 197 for birds and
 mammals) individual comparisons of the relationship between
 Δ<sup>13</sup>Cclean-contaminated and
 ΔC:N<sub>Atomic</sub> (clean-contaminated) for establishing
 the point at which small deviations in C:NAtomic begin to impact
 δ<sup>13</sup>C. Statistics Statistical analyses were performed with PAST Version 3.22 (Hammer, et al. 2001). For amino acid residue and C:N<sub>Atomic</sub> 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<sub>Atomic</sub> and δ<sup>13</sup>C data we used Spearman’s ρ.
description: 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<sub>Atomic</sub> for different taxonomic groups.
 Table S3. Results for Spearman’s <em>ρ </em>tests for
 collagen <em>δ</em><sup>13</sup>C grouped by
 ascending C:N<sub>Atomic</sub> Table S4. Extent to which shifts
 C:N<sub>Atomic</sub> (resulting from lipid contamination)
 affect δ<sup>13</sup>C. 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 δ<sup>13</sup>C of the sample with the
 lowest C:N<sub>Atomic</sub> was subtracted from the
 δ<sup>13</sup>C of the other samples. Table shows mean
 difference in δ<sup>13</sup>C (Δ13C) for analyses binned into
 groups with ascending C:N<sub>Atomic</sub>. Spearman’s ρ tests
 identify the point at positive shifts in Δ<sup>13</sup>C
 corresponding with increasing C:N<sub>Atomic</sub> become
 significant. Statistically significant results in bold. Table S5. Mean amino acid
 compositions and corresponding C:N<sub>Atomic</sub> for
 vertebrate classes not shown in Table 3 ESM_References:
 A list of references to the data appearing in Table S1.