Small tails tell tall tales - intra-individual variation in the stable isotope values of fish fin

RESEARCH ARTICLE Small Tails Tell Tall Tales –Intra-Individual Variation in the Stable Isotope Values of Fish Fin Brian Hayden* 1 , David X. Soto 1,2 , Tim D. Jardine 1,3 , Brittany S. Graham 1,4 , Richard A. Cunjak 1 , Atso Romakkaniemi 5 , Tommi Linnansaari 1 1Biology Department, University of New Brunswick, New Brunswick, Canada, 2Environment Canada, Saskatoon, Saskatchewan, Canada, 3School of Environmental and Sustainability, Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, 4National Institute of Water and Atmospheric Research (NIWA), Greta Point, Wellington, New Zealand, 5Natural Resources Institute Finland, Oulu, Finland *[email protected] Abstract Background Fish fin is a widely used, non-lethal sample material in studies using stable isotopes to assess the ecology of fishes. However, fish fin is composed of two distinct tissues (ray and membrane) which may have different stable isotope values and are not homogeneously distributed within a fin. As such, estimates of the stable isotope values of a fish may vary according to the section of fin sampled. Methods To assess the magnitude of this variation, we analysed carbon (δ 13 C), nitrogen (δ 15 N), hydrogen (δ 2 H) and oxygen (δ 18 O) stable isotopes of caudal fin from juvenile, riverine stages of Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). Individual fins were sub-sectioned into tip, mid and base, of which a further subset were divided into ray and membrane. Findings Isotope variation between fin sections, evident in all four elements, was primarily related to differences between ray and membrane. Base sections were 13 C depleted relative to tip (~ 1 ‰) with equivalent variation evident between ray and membrane. A similar trend was evident in δ 2 H, though the degree of variation was far greater (~ 10 ‰). Base and ray sections were 18 O enriched (~ 2 ‰) relative to tip and membrane, respectively. Ray and membrane sections displayed longitudinal variation in 15 N mirroring that of composite fin (~ 1 ‰), indicating that variation in 15 N values was likely related to ontogenetic variation. Conclusions To account for the effects of intra-fin variability in stable isotope analyses we suggest that researchers sampling fish fin, in increasing priority, 1) also analyse muscle (or liver) tissue PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 1/18 OPEN ACCESS Citation: Hayden B, Soto DX, Jardine TD, Graham BS, Cunjak RA, Romakkaniemi A, et al. (2015) Small Tails Tell Tall Tales –Intra-Individual Variation in the Stable Isotope Values of Fish Fin. PLoS ONE 10(12): e0145154. doi:10.1371/journal.pone.0145154 Editor: Robert Britton, Bournemouth University, UNITED KINGDOM Received: September 16, 2015 Accepted: December 1, 2015 Published: December 15, 2015 Copyright: © 2015 Hayden et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All the stable isotope data associated with this project are included in S1 File. Funding: This work was funded by the FinnishSwedish Transboundary River Commission (grant number: Ä 13/7) and by Canadian Research Chair (grant number: 1-132477-45-01) and NSERC Discovery (grant number: 1-550006-40-01) grants to RC. Competing Interests: The authors have declared that no competing interests exist. from a subsample of fish to calibrate their data, or 2) standardize sampling by selecting tissue only from the extreme tip of a fin, or 3) homogenize fins prior to analysis. Introduction The application of stable isotope-based approaches to ecological research is growing at a near exponential rate [1]. Stable isotopes are used to examine trophic relationships, physiology and migration history across all levels of biological organisation [2–4]. The stable isotopes of carbon ( 13 C) and nitrogen ( 15 N) are the most commonly applied in ecological research [1]. As the δ 13 C and δ 15 N values of a consumer are related to the same ratios in their prey, researchers can use these markers to reconstruct the resource use of an organism, predator–prey interactions or even the entire food-web structure [5,6]. In contrast, stable isotopes of hydrogen (deuterium, hereafter 2 H) can differentiate between allochthonous and autochthonous resource use in freshwater consumers [7,8], and are particularly useful to re-create migration history or to assign point of origin to material, for which they can be used in conjunction with stable isotopes of oxygen ( 18 O) [9]. Furthermore as different body tissues turnover at different rates, analysis of the isotope ratios of different tissues can provide an outline of temporal variability in the ecology of an organism [10,11]. One of the principal disadvantages of stable isotope based ecological research is that sampling is often destructive. Muscle is the most commonly analysed animal tissue in these studies and in the majority of cases obtaining a sufficient sample requires the animal to be sacrificed [1,10]. This has obvious ethical implications for the field especially in relation to studies involving rare or threatened species [12,13]. In recent years, the number of studies employing nonlethal sampling has increased. This is most evident in ornithological studies where destructive samples have been replaced with non-lethal sampling of blood or non-invasive collection of feathers. In studies examining fur bearing mammals, hair and vibrissae samples have begun to replace muscle [14–16]. In fish biology, the focus of the current study, scales [17] and fin clips [18,19] represent non-lethal alternatives to commonly sampled muscle and liver[20]. Fish scales are somewhat troublesome, due their heterogeneous structure which characterises the entire lifetime of the fish [21,22]. Fin clips, however, are routinely taken during fish population surveys and their isotopic turnover rate is similar to that of muscle [11,23]. This has led to fin clips being proposed as an alternative to muscle for stable isotope studies of fish [24– 27]. However, fish fin is not a homogeneous structure. Fins are composed of bone and cartilage rays interspersed between a thin epithelial skin membrane [28]. These tissues will likely have a different isotopic turnover time and may have different isotope ratios [11]. Furthermore, the relative proportion of ray and membrane components of a fishes’fin varies from predominantly ray at the base to predominantly membrane at the tip [29]. If such variation exists within fins, it may be sufficient to confound estimates of resource use or movements based on stable isotope analysis of fish fin. Due to the ethical benefits of using fish fin rather than muscle, an understanding of such confounding effects would be of great utility to the field. To address these issues we assessed the levels of intra-fin variability in four commonly used stable isotopes (δ 13 C, δ 15 N, δ 2 H and δ 18 O) in wild-caught, juvenile anadromous Atlantic salmon (Salmo salar; hereafter salmon) and brown trout (Salmo trutta, hereafter trout) sampled in the Tornionjoki river system in northern Finland. We formulated the study around three principal hypotheses, which tested the sources of variation within fins: Firstly, as fish fin is not homogenous in structure [29], we predicted (hypothesis 1; H 1 ) that the isotopic Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 2/18 composition within an individual fin would not be homogenous. The remaining two hypotheses (H 2 and H 3 ) explored the source of variation observed within fins. We predicted (H 2 ) that variation within fins was a reflection of the relative abundance of isotopically distinct ray and membrane sections, and that longitudinal isotope variation from the tip to the base sections of the fin would mirror the variation between ray and membrane. An alternate hypothesis (H 3 ) related to fin regeneration. Fish, particularly juvenile salmon and trout, reside in turbulent environments. As a consequence their fins are constantly regenerating due to damage and abrasion with the substrate [30]. If a fish changes its diet or location while fins are regenerating this may be reflected as variation in the isotope ratio between base and regenerated tip sections. If this regeneration hypothesis (H 3 ) explained the heterogeneous patterns in fish fin we expected that longitudinal isotope variation from tip to base of ray and membrane analysed in isolation would be equivalent to that observed from tip to base sections of composite (ray + membrane) fin. Method Field sampling The fin samples used in this study were collected as a part of a salmon and trout smolt monitoring program conducted in the River Tornionjoki by the Natural Resources Institute of Finland. The Tornionjoki River system (drainage area 40,000 km 2 ) forms a 500-km long border between Finland and Sweden and flows into the Baltic Sea with an annual mean discharge of approximately 380 m 3 s -1 (Fig 1). In the last two decades, the river has had a dramatic increase in returns of adult salmon and is currently one of the most productive salmon rivers in the world, with annual returns exceeding 100,000 adult salmon [31]. Juvenile salmon and trout were sub-sampled in 2006 from the smolt catch of a large fykenet (100m leader width, set at 2—5m depth in center of 800m wide channel) used when estimating the total smolt run of the Tornionjoki River. The sampling location was approximately 5 km upstream from the river mouth (65°52'23" N; 24° 8'22" E; Fig 1). The total smolt run in 2006 was estimated at approximately 830,000 salmon and 13,000 trout [31]. Salmon (sampling dates from 17 May to 3 July) and trout (sampling dates from 18 May to 13 June) were randomly selected from the smolt catch. Fish were removed from the nets several times per day during the peak smolt run and daily at all other times. Each fish was measured (mm), weighed (0.1g) and a section of caudal fin was collected. Fin samples were frozen (-20°C) within 12 hours of sampling and later oven-dried (60°C; 24-h). Samples were shipped to the Stable Isotopes in Nature Laboratory (New Brunswick, Canada) for further preparation and analysis. For stable isotope analysis, 108 salmon and 19 trout were selected from this sample. The total length and weight of the analysed salmon ranged from 117 mm to 188 mm (avg. 142 ± 13 mm SD) and from 10.2 g to 48 g (avg. 20.3 ± 6.6 g SD), respectively, with a median age of 3 years. The total length and weight of the analysed trout ranged from 134 mm to 214 mm (avg. 182 ± 19 mm SD) and from 17.6 g to 73.0 g (avg. 48.4 ± 14.5 g SD), respectively, with a median age of 3 years. Ethics statement Permission for sampling was sought and granted under licence from the Lapland County Administrative of the Ministry of Forestry and Agriculture as well as the Finnish-Swedish Transboundary River Commission and informally from private water owners at the sampling site. Salmon and trout are classified as protected fishes in Finland and are subject to a national monitoring program. A portion of this monitoring program consists of lethal sampling of smolts throughout the annual migration period to monitor the sex ratio of the migrating fish. Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 3/18 The fin samples for this study required an atypically large section of caudal fin to ensure each sample included base, midand tip sections of the caudal fin. All the samples for this study were opportunistically collected from fish that were sacrificed for the purposes of the sex determination as part of a regular national monitoring program and no fish were sacrificed solely for the purposes of this study. The sampling procedure was approved by the Finnish Animal Ethics Committee. Licences and documentation, in Finnish, are available from the authors on request. Laboratory analysis The fins were sub-sectioned into three groups (Fig 2). The first group (n = 60; Tip-Mid-Base; hereafter TMB) were cut into three equidistant sections representing the distal tip, mid and base section of each fin. To account for the potential biasing effects of lipids within the fins [32,33] we also analysed fins from which lipids had been chemically removed. We assumed that if variation between fin sections was similar in lipid extracted and non-extracted samples then this variation was related to differences in the isotopic composition of the fin rather than Fig 1. Map of Study Region. The River Tornionjoki drainage forms the northern boundary between Finland and Sweden. The main rivers and the smolt sampling location near the river mouth are highlighted. doi:10.1371/journal.pone.0145154.g001 Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 4/18 a variation in the proportion of lipids within the fin. The lipid extracted group (TMB.LE) were treated with repeated (n = 3) immersion (30 minutes) in a chloroform: methanol (2: 1) solution to remove lipids. Following the final immersion, each fin was dried at 60°C for 24 hours. These lipid-extracted fins (n = 60) were subsequently cut into three equidistant sections as outlined above. The final group (hereafter TMB.RM) were cut into three equidistant sections and each section was further dissected into ray and membrane components. The sample size of trout (n = 19) was too small to facilitate analysis of TMB.LE or TMB.RM groupings for this species. Hence, trout fins were analysed solely as TMB. Sufficient tissue to facilitate all isotope analyses was collected from a few individuals, but generally samples for different experimental groups were obtained from separate fish. For carbon and nitrogen stable isotope analysis, 1.0 ± 0.1mg subsamples of each fin section were placed in tin-foil cups. Samples were combusted and analyzed in a Delta Plus continuousflow, isotope-ratio mass spectrometer (Thermo Finnegan GmbH, Bremen, Germany) connected to a Carlo Erba NC2500 elemental analyzer (ThermQuest S.p.A., Milan, Italy). Carbon and nitrogen isotope ratios are reported relative to the international standards Vienna PeeDee Belemnite carbonate (V-PDB) and atmospheric nitrogen (AIR), respectively. Analytical error was calculated as 0.1 ‰for both δ 13 C and δ 15 N based on repeat analyses of an in-house standard. For hydrogen and oxygen isotope analysis, 0.2 ± 0.05 mg subsamples of each fin section were placed in silver foil cups. Stable-hydrogen and oxygen isotope measurements for animal tissues were normalized to the international standard VSMOW (Vienna Standard Mean Ocean Water) using standards EC1 and EC2 (Environment Canada, Saskatoon, Canada). We determined the non-exchangeable δ 2 H of samples using the comparative equilibration approach [34] with two secondary standards (EC1 and EC2). These standards were previously calibrated to account for the hydrogen exchangeability between the hydrogen atoms of ambient water vapor and tissues [34,35]. Samples and standards were allowed to exchange with local atmospheric hydrogen for a minimum of 72 hours prior to analysis. Samples were combusted and analysed using a High Temperature Conversion Elemental Analyser (Thermo Scientific GmbH, Bremen, Germany) connected to a Delta Plus XP continuous flow isotope-ratio mass spectrometer (Thermo Scientific GmbH, Bremen, Germany). Repeat analysis of an in house Fig 2. Subsections of fin analysed. Diagram of brown trout highlighting the relative locations of tip, mid and base sections sampled on each fin. Image provided courtesy of P. Antti-Poika. doi:10.1371/journal.pone.0145154.g002 Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 5/18 standard, keratin (Spectrum-Porcine #SJ1400), indicated that analytical precision was better than 2 ‰and 0.5 ‰for δ 2 H and δ 18 O, respectively. Data analysis The stable isotope data associated with this work are provided in the Supporting Information file with this paper (S1 File). The natal and home range of each fish sampled was not known, and as the river basin covers >40, 000 km 2 along >500 km North-South gradient, they likely forage in numerous habitats that encompassed different isotopic baselines. As such, a direct comparison between fish would likely be affected by variation associated with the isotopic baseline of each individual’s home range. To avoid this, we used paired analyses to assess the differences within individuals. Paired Welch t-tests (a non-parametric equivalent of the paired Student’s t-test) were used to examine the variation in δ 13 C, δ 15 N, δ 18 O, δ 2 H values between sections of each fin. Pairwise comparisons were made between the tip, mid and base sections of each fin to test for heterogeneity in the isotope values within fins from both non-treated (TMB) and lipid-extracted (TMB. LE) salmon and all trout (H 1 ). To determine the effect of lipid extraction on intra-fin variability, a Welch t-test was used to compare the difference between tip and base sections of nontreated and lipid-extracted salmon (H 1 ). To test H 2 , that the isotope ratios of ray and membrane would differ, pairwise comparisons of the isotope ratios of ray and membrane were conducted across tip, mid and base sections of all TMB.RM fins. Finally, to test H 3 and determine whether isotopic variation within fins was due to isotopic shifts associated with fin regeneration we used paired Welch t-tests to examine the variation between tip, mid and base sections separately for ray and membrane tissues in TMB.RM group fish. If the heterogeneity within fins was related to variation between the isotope ratios of ray and membrane we expected that variation observed between ray and membrane would reflect that observed between base and tip sections of composite (ray + membrane) fins. In contrast, if the variation related to fin regeneration then we expected that variation between tip, mid and base sections of ray and membrane independently would mirror that observed in composite fins. A Chi-square test was used to test whether the variation between the isotope ratios of tip and base sections was due to variation between membrane and ray or longitudinal variation within each tissue. Variation between tip and base was classed as tip enriched whereby tip sections were enriched in a specific isotope relative to base sections (option 1), or base enriched, whereby base sections were enriched relative to tip (option 2). The count of options 1 and 2 was then compared with variation between membrane and ray (option 1: membrane enriched, whereby membrane was enriched relative to ray; option 2: ray enriched, whereby ray was enriched relative to membrane), tip and base of fin membrane (option 1: tip enriched; option 2: base enriched), and finally tip and base of fin ray (option 1: tip enriched; option 2: base enriched) using Pearson’s Chi-square test of a 2 × 2 matrix. The test was conducted in R [36]. Results Hypothesis 1 –Variation between tip, mid and base sections of composite fin Salmon. In the TMB group, tip sections were 13 C enriched (1.0 ± 0.6 ‰SD) relative to base sections (Fig 3a). This variation was equally distributed between the tip, mid and base sections (Table 1). A similar trend was observed in the TMB.LE group; tip sections were enriched (1.2 ± 0.6 ‰) relative to base sections with mid sections intermediate between both extremes (Fig 4a;Table 1). Tip sections were 15 N depleted relative to base sections in both Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 6/18 TMB (1.2 ± 0.6 ‰;Fig 3b) and TMB.LE (0.8 ± 0.4 ‰;Fig 4b) fins. This variation was equally dispersed between the tip, mid and base sections (Table 1). Intra-individual variation in δ 2 H exceeded that evident in the other three isotopes, in the most extreme cases differences of greater than 30 ‰were observed between tip, mid and base sections of a single fin. On average, tip sections were 2 H enriched relative to base sections by 10 ± 10 ‰and 12 ± 6 ‰in TMB (Fig 3c) and TMB.LE (Fig 4c) samples, respectively. Tip sections were 18 O depleted relative to tip sections by 1.9 ± 0.7‰in TMB and by 1.8 ± 0.8‰in TMB.LE groups (Figs 3d and 4d; Table 1). Mid sections were intermediate between tip and base (Table 1). The removal of lipids had no effect on the degree of variation between tip and base sections in carbon (t = 1.52; d.f. = 60.842; P = 0.134), oxygen (t = 0.46; d.f = 28.58; P = 0.647) or hydrogen (t = 1.01; d.f. = 56.11; P = 0.313) stable isotope ratios. Statistically significant variation was evident in stable isotopes of nitrogen (t = 3.31, d.f. = 56.18; P = 0.001) but the actual variation in isotope ratios (mean ± SD; non treated: 1.2 ± 0.6; treated: 0.8 ± 0.4) was small and unlikely to confer any biological significance. Trout. The variation in tip-mid-base comparisons of trout mirrored that evident in salmon. Tip sections were 13 C enriched (0.8 ± 0.9 ‰) and 15 N depleted (1.3 ± 0.5 ‰) relative Fig 3. Variation of isotope ratios in salmon fin (TMB). Comparison of the (a) carbon (δ 13 C), (b) nitrogen (δ 15 N), (c) hydrogen (δ 2 H) and (d) oxygen (δ 18 O) stable isotope ratios of base and tip sections of non-treated Atlantic salmon fins. 1:1 lines are added to each plot for visualisation purposes. doi:10.1371/journal.pone.0145154.g003 Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 7/18 to base (Fig 5a–5b;Table 1). This variation was equally dispersed between tip, mid and base sections (Table 1). Tip sections were 2 H enriched relative to base sections (17.6 ± 7.2 ‰;Fig 5c), with the variation equally split between tip, mid and base comparisons (Table 1). Due to sample size limitations, oxygen isotope ratios of trout fins were not analysed. Hypothesis 2 –Variation between ray and membrane The δ 13 C, δ 15 Nδ 2 H and δ 18 O isotope ratios of ray and membrane differed in tip, mid and base sections. Fin membrane was 13 C enriched relative to ray in tip (0.5 ± 0.5 ‰), mid (0.8 ± 0.4 ‰) and base (0.7 ± 0.6 ‰) sections (Fig 6a;Table 2). Variation in δ 15 N values between membrane and ray, though statistically significant (Table 2), was too small to be biologically meaningful (Fig 6b); membrane was 15 N enriched relative to ray in tip (0.1 ± 0.3 ‰), mid (0.2 ± 0.3 ‰) and base sections (0.3 ± 0.2 ‰;Table 2). Fin membrane was 2 H enriched relative to ray in all sections (Fig 6c) though the difference was most pronounced in mid (21 ± 11 ‰) and base (22 ± 15 ‰;Table 2) sections. Fin membrane was 18 O depleted relative to ray (Fig 6d) and this variation was evident in tip (0.9 ± 0.8 ‰), mid (1.4 ± 0.8 ‰) and base (1.7 ± 0.9 ‰) sections (Table 2). Hypothesis 3 –Longitudinal variation in ray and membrane When assessed independently, the variation between tip, mid and base sections of fin ray and membrane was minimal, and only evident in certain stable isotope values (Fig 7;Table 3). Variation in δ 13 C values between tip, mid and base sections of both ray and membrane was not significant (Fig 7a,Table 3). Tip sections of membrane (0.7 ± 0.5 ‰) and ray (0.9 ± 0.3 ‰) were 15 N depleted relative to base sections (Fig 7b;Table 3). This variation was equally dispersed between tip, mid and base sections of both tissues (Table 3). Variation in δ 2 H values was Table 1. Intra-fin variation in carbon (δ 13 C), nitrogen (δ 15 N), hydrogen (δ 2 H) and oxygen (δ 18 O) stable isotope ratios. Tip—Base Tip—Mid Mid—Base Isotope n Mean (SD) t (1, n-1) PMean (SD) t (1, n-1) PMean (SD) t (1, n-1) P Salmon—non treated δ 13 C32 1.0 (0.6) -9.7 <0.001 0.4 (0.5) -4.9 <0.001 0.5 (0.5) -5.5 <0.001 δ 15 N32 -1.2 (0.6) 11.9 <0.001 -0.6 (0.5) 6.4 <0.001 -0.6 (0.3) 11.8 <0.001 δ 2 H34 9.5 (10.3) -5.38 <0.001 4.9 (8.5) -3.4 <0.001 4.5 (7.5) -3.5 <0.001 δ 18 O14 -1.9 (0.7) 9.9 <0.001 -1.4 (0.8) 6.8 <0.001 -0.6 (0.7) 3.2 <0.001 Salmon—lipid extracted δ 13 C31 1.2 (0.6) -11.5 <0.001 0.7 (0.5) -9.2 <0.001 0.5 (0.5) -5.4 <0.001 δ 15 N31 -0.8 (0.4) 10.7 <0.001 -0.3 (0.3) 5.2 <0.001 -0.5 (0.4) 6.4 <0.001 δ 2 H31 11.6 (6.4) -10.1 <0.001 7.4 (4.6) -9.1 <0.001 4.1 (4.9) -4.7 <0.001 δ 18 O31 -1.8 (0.8) 12.1 <0.001 -1.1 (0.8) 7.9 <0.001 -0.7 (0.7) 5.7 <0.001 Trout δ 13 C9 0.8 (0.9) -2.7 0.033 0.8 (1.1) -2.1 0.072 0.1 (0.6) -0.1 0.998 δ 15 N9 -1.3 (0.5) 7.2 <0.001 -0.8 (0.5) 4.8 <0.001 -0.5 (0.4) 3.6 <0.001 δ 2 H10 17.6 (7.2) -7.7 <0.001 11.2 (7.8) -4.5 <0.001 6.4 (5.7) -3.6 <0.001 δ 18 O-- -- - -- - -- Number of fins analysed (n) and mean (‰) difference between tip-base, tip-mid and mid-base comparisons of stable isotope ratios in non-treated (TMB) and lipid extracted (TMB.LE) Atlantic salmon and non-treated anadromous brown trout. Standard deviation of difference is presented in parentheses. Results of paired Welch t-test of the difference is also presented, degrees of freedom for each test are n–1. doi:10.1371/journal.pone.0145154.t001 Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 8/18 evident between tip, mid and base sections of fin membrane (Fig 7c,Table 3), but not fin ray (Fig 7c). In contrast to the variation observed between tip, mid and base sections of composite fin (TMB), tip sections of membrane were 2 H depleted (10.6 ± 11.6 ‰) relative to base sections (Table 3). Variation in the δ 18 O values of membrane was not significant (Fig 7d,Table 3), but tip sections of ray were 18 O enriched relative to mid (0.8 ± 0.9 ‰) and base (0.8 ± 1.2 ‰). Chi-square tests of carbon and oxygen isotope ratios indicated that variation between tip and base sections was most similar to variation between membrane and ray (Table 4). Tip— base variation in nitrogen in composite (membrane + ray) was significantly different from variation between membrane and ray, but similar to tip—base variation in membrane and ray (Table 4). Tip-base variation in hydrogen differed from membrane ray and tip-base variation of membrane but not from tip-base variation in fin ray. A slight modification of the test, characterising the enriched / depleted cut off at +4 / -4 ‰respectively rather than 0, which limited the potential error associated with slightly enriched or depleted samples, indicated that tip— base variation in hydrogen was similar to variation between membrane and ray (χ 2 = 1.555, P= 0.212). Fig 4. Variation of isotope ratios in lipid-treated salmon fin (TMB.LE). Comparison of the (a) carbon (δ 13 C), (b) nitrogen (δ 15 N), (c) hydrogen (δ 2 H) and (d) oxygen (δ 18 O) stable isotope ratios of base and tip sections of Atlantic salmon fins from which lipids have been extracted. 1:1 lines are added to each plot for visualisation purposes. doi:10.1371/journal.pone.0145154.g004 Stable Isotope Analysis of Fish Fin PLOS ONE | DOI:10.1371/journal.pone.0145154 December 15, 2015 9/18 2. Layman CA, Araujo MS, Boucek R, Hammerschlag-Peyer CM, Harrison E, Jud ZR, et al. Applying stable isotopes to examine food-web structure: an overview of analytical tools. Biol Rev. 2012; 87: 545– 562. doi: 10.1111/j.1469-185X.2011.00208.x PMID: 22051097 3. Hobson KA, Van Wilgenburg SL, Wassenaar LI, Powell RL, Still CJ, Craine JM. A multi-isotope (δ13C, δ15N, δ2H) feather isoscape to assign Afrotropical migrant birds to origins. Ecosphere. 2012; 3: art44. doi: 10.1890/ES12-00018.1 4. Reitsema LJ. 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