Transactions of the American Fisheries Society 134:1103–1110, 2005 q Copyright by the American Fisheries Society 2005 DOI: 10.1577/T04-124.1
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Stable Isotope Variability in Tissues of Temperate Stream Fishes TIMOTHY D. JARDINE* Department of Biology, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada
MICHELLE A. GRAY Department of Zoology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
SHERISSE M. MCWILLIAM
AND
RICHARD A. CUNJAK
Department of Biology, University of New Brunswick, Fredericton, New Brunswick E3B 5A3, Canada Abstract.—Previous measurements of stable isotope ratios in fishes have typically used white muscle, but potential applications exist for the use of other tissues. We tested three tissues (liver, fin, and gonad) in three freshwater species (juvenile Atlantic salmon Salmo salar, slimy sculpin Cottus cognatus, and brook trout Salvelinus fontinalis) to investigate potential ecological applications of stable isotopes in tissues other than muscle. Caudal fin tissue correlated closely with muscle tissue for Atlantic salmon and brook trout for d13C (r 5 0.96 and 0.94, respectively) and d15N (r 5 0.80 and 0.74). Liver d13C values were tightly linked to muscle values, and differences were due to lipid effects. Associations between liver and muscle d15N suggested subtle changes in nutritional status. Isotope ratios of gonads differed markedly between male and female slimy sculpin; these differences were probably governed by differences in the allocation of specific nutrients. Knowledge of isotopic fractionation among tissues will aid fish biologists in nonlethal sampling of fishes for stable isotope analysis and in using stable isotopes to assess nutritional status and the allocation of nutrients to reproduction.
Stable isotope analysis (SIA) has become a popular method used by ecologists to quantify the dietary habits of animals in the wild (Peterson and Fry 1987). The relative isotopic similarity of 13C/ 12 C between diet and consumer (0–1‰; DeNiro and Epstein 1978; Post 2002) allows identification of an animal’s diet source, and the incremental increase of 15N/14N (3–5‰) at successive trophic levels (DeNiro and Epstein 1981; Minagawa and Wada 1984; Post 2002) allows determination of an animal’s trophic position in a food web. What remains somewhat speculative in ectothermic or* Corresponding author:
[email protected]. Received July 19, 2004; accepted April 7, 2005 Published online August 10, 2005
ganisms, however, is the suitability of a particular tissue for giving a representative picture of the whole animal’s dietary history (Tieszen et al. 1983; Hobson and Clark 1992a, 1992b; Pinnegar and Polunin 1999). Most researchers choose to sample white muscle because of its low lipid content and ease of homogenization (Pinnegar and Polunin 1999). However, other tissues may hold potential for nonlethal sampling and may aid in assessing nutrient allocation and nutritional status. Readily accessible tissues that can be sampled in a nondestructive manner are advantageous for species of conservation concern, such as several populations of salmonid fishes (COSEWIC 2003). Fish fin clips are one such tissue that can be sampled nonlethally in a fashion similar to that used previously for bird feathers and blood (Hobson and Clark 1993). Previous studies on freshwater species have assumed that fin and muscle tissue are comparable in reconstructing past diets (Rounick and Hicks 1985; Doucett et al. 1999a; Finlay et al. 2002). Metabolic processes can lead to isotopic fractionation of 13C and 15N in endothermic and ectothermic species (Hobson et al. 1993; Doucett et al. 1999b). Discrimination against 13C during lipogenesis (DeNiro and Epstein 1977) has been proposed as the mechanism leading to depletion of the heavier isotope in lipid-rich tissues such as red muscle, liver, and eggs (McConnaughy and McRoy 1979; Kline et al. 1993; Doucett et al. 1999b; Pinnegar and Polunin 1999; McCarthy and Waldron 2000; Grey 2001). Transamination during protein recycling (Gaebler et al. 1966; Macko et al. 1986) results in increases in 15N that can be tissue specific during periods of food deprivation (Hobson et al. 1993; Doucett et al. 1999b; Jardine
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et al. 2004). Stable isotope analysis can therefore be useful in understanding nutritional status and metabolic processes in fishes at capture. These factors can also impact interpretation of isotope data when different tissues are used within or across studies. The objective of this study was to determine the degree of isotopic similarity among tissues in three fish species collected from New Brunswick, Canada, rivers: migrating Atlantic salmon Salmo salar smolts captured in the Northwest Miramichi River; resident slimy sculpin Cottus cognatus from the Little River; and brook trout Salvelinus fontinalis native to the Pointe Wolfe River. These results could then be used to guide future studies in choosing comparable tissues, avoiding tissues that are subject to confounding influences, and using tissue pairs to understand fish nutritional status and energy allocation. Methods In June 2002, Atlantic salmon smolts (n 5 24) were collected at a Fisheries and Oceans Canada trap net in the Northwest Miramichi River (468569N, 658479W; Jardine et al. 2004). Slimy sculpin were sampled by backpack electroshocking in the Little River (478059N, 678459W) in November 2000 (n 5 19) and April 2001 (n 5 16) (Gray et al. 2004). Brook trout were collected opportunistically in a headwater lake of the Pointe Wolfe River (458339N, 658019W) by Parks Canada personnel in May and June of 1996 (n 5 13) and 1997 (n 5 10). All fishes were kept frozen (2208C) prior to isotope analysis. White muscle and liver tissue were excised from all species. Gonadal tissue (eggs and testes) was removed from slimy sculpin only, and caudal fin clips were taken from salmonid species. All tissues were oven dried at 608C for 48 h. Muscle, liver, and gonad samples were ground with a mortar and pestle to a fine, uniform powder, and fin clips were cut to appropriate weights. All samples were combusted in a NC2500 elemental analyzer connected via continuous flow to a Thermo Finnigan Mat Delta Plus mass spectrometer. Isotope ratios are reported in delta notation (d, in ‰) relative to international standards for C (Pee Dee belemnite; Craig 1957) and N (air; Mariotti 1983). Analytical error was calculated as described by Jardine and Cunjak (in press). Acting as internal standards, single slimy sculpin and hatcheryreared Atlantic salmon muscle samples were run alongside each set of unknown samples. Mean values (6SD) for the slimy sculpin standard were
227.82 6 0.13‰ for d13C and 9.05 6 0.16‰ for d15N. Means for the Atlantic salmon standard were 219.79 6 0.14‰ for d13C and 8.82 6 0.31‰ for d15N. A commercially available organic analytical standard (Acetanilide; Elemental Microanalysis, Ltd.) had a mean (61 SD) d13C value of233.56 6 0.19‰ and a mean d15N value of 22.95 6 0.28‰ during the study period. Within a given analytical run, one standard deviation of sample repeats was never greater than 0.18‰ for d13C or 0.30‰ for d15N. All statistical analyses were conducted by use of MINITAB software (Ryan and Joiner 2000); separate analyses were computed for each species and each response variable (C and N). Assumptions for an analysis of variance (ANOVA), including independence, homogeneity of variances, and normality, were tested by use of experimental design, Cochran’s test, and histograms, respectively. For all three species, the data for each response variable were nonindependent with equal variances; C data were nonnormal, while N data were normal. Because ANOVA is robust to violations of the normality assumption (Underwood 1997), we did not transform the data. To account for the nonindependence, a split-plot general linear model (a form of ANOVA) was constructed for each variable (Underwood 1997). Fish (Atlantic salmon and brook trout), year (brook trout), and season, sex, and fish (slimy sculpin) were the plot factors, while tissue type was the split-plot factor in these statistical models. To test for the effect of fish in these models, we first had to test the assumption that the interaction term between tissue type and fish was not significant (Underwood 1997). Tukey’s test for nonadditivity was performed to determine the significance of the interaction term. To determine where the effect of tissue type occurred, we performed a comparisonwise post hoc test (Tukey’s test; Underwood 1997). Correlations between tissues and means in figures are presented separately when significant differences were identified and together when no effects of season or sex were found. Regression analyses were used to determine the influence of fish size (independent variable) on the difference between fin clip and muscle d15N (dependent variable) for Atlantic salmon and brook trout.
Results There was a strong correlation between caudal fin tissue and muscle d13C for both Atlantic salmon and brook trout (Table 1; Figure 1A). For stable N ratios, however, the association was weaker and
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TABLE 1.—Muscle tissue stable C and N isotope ratios in three fishes from New Brunswick streams and the relative difference between values for fin, liver, or gonad tissue and muscle (with correlation coefficients r). A positive sign indicates enrichment of the heavier isotope relative to the value for muscle, and a negative sign indicates depletion relative to muscle. An asterisk indicates a significant difference from the value for muscle (M 5 male; F 5 female). Carbon
Species
Tissue (n)
Atlantic salmon
Muscle (24)
Brook trout
Fin (24) Liver (24) Muscle (23)
Slimy sculpin
Fin (23) Liver (23) Muscle, Nov (19) Gonad, M (8) Gonad, F (11) Liver, M (8) Liver, F (11) Muscle, Apr (16) Gonad, M (7) Gonad, F (9) Liver, M (7) Liver, F (9)
d13C 6 SD (Minimum, maximum) 223.84 6 1.89 (228.41, 221.76) 230.01 6 2.64 (241.10, 227.76) 232.65 6 0.89 (234.12, 230.35)
233.11 6 0.68 (234.09, 231.62)
Nitrogen
Difference 6 SD
r
d15N 6 SD (Minimum, maximum)
Difference 6 SD
r
20.80 6 0.35* 21.22 6 0.39*
0.796 0.793
10.31 6 0.42 20.99 6 1.97*
0.742 0.937
22.04 20.39 21.07 20.59
6 6 6 6
0.46* 0.36 0.51* 0.19*
0.313 0.584 0.884 0.499
22.63 11.03 10.22 20.86
6 6 6 6
0.46* 0.45* 0.31 0.42*
0.537 0.137 0.823 0.378
8.66 6 0.54 (7.71, 9.90) 10.21 6 0.38 21.64 6 0.56*
0.965 0.969
10.65 6 0.94 21.86 6 1.21*
0.940 0.960
10.81 20.46 20.89 20.38
6 6 6 6
0.60* 0.58 0.27* 0.52
0.697 0.873 0.941 0.920
12.46 21.75 21.70 21.35
6 6 6 6
0.64* 0.55* 0.36* 0.44*
the two species showed contrasting patterns. Atlantic salmon smolt fin tissue was depleted in 15N relative to muscle, whereas brook trout fin tissue and muscle 15N values were statistically equivalent (Table 1; Figure 1B). Some of the variability in fractionation between fin clips and muscle in the two species was accounted for by fish size. Smaller (and presumably younger) fish had less 15N in caudal fins relative to muscle, while the pattern seemed to reverse in larger fish (brook trout: fin d15N–muscle d15N 5 0.0748·fork length2 0.786, r2 5 0.343, P 5 0.001; Atlantic salmon smolts: fin d15N–muscle d15N 5 0.2078·fork length2 3.5646, r2 5 0.438, P , 0.001). Liver tissue was consistently depleted in 13C relative to muscle in all three species except female sculpin sampled in November, and liver and muscle values were strongly correlated within all species (Table 1). Lipid normalization with C:N (McConnaughy and McRoy 1979) and lipid extraction with chloroform–methanol (Bligh and Dyer 1959) eliminated differences between muscle and liver d13C for all species (T.D.J., unpublished data). Liver tissue was also consistently depleted in 15N relative to muscle in all species and groups except male sculpin sampled in April (Table 1). Gonad tissue of male sculpin showed enrichment in 13C and depletion in 15N relative to muscle
9.37 6 0.64 (6.85, 10.15) 8.97 6 0.36 (8.22, 9.39)
9.35 6 0.45 (8.43, 10.18) 0.616 0.679 0.880 0.817
in both November and April (Table 1; Figure 2). Females had equivalent gonad and muscle d13C and d15N values in November, but in April gonads were enriched in 15N and depleted in 13C relative to muscle values (Table 1; Figure 2). Lipid normalization and extraction failed to eliminate differences in d13C between female gonad and muscle. Discussion Tissue–isotope pairs presented a variety of scenarios, illustrating potential complications in the use of different tissues across studies but also revealing the potential applications of analyzing tissue pairs to understand nutritional status and reproductive nutrient allocation in wild fishes. For example, fin d13C in Atlantic salmon and brook trout was equivalent to muscle d13C over a wide range of values, while the correlation for d15N of the same tissue pair was not as strong and fish size was a potential confounding factor. These results differ slightly from previously published studies that found equivalency between fin and muscle tissue for both d13C and d15N in brown trout Salmo trutta (McCarthy and Waldron 2000) and humpback chub Gila cypha (Shannon et al. 2001). Meanwhile, 13C depletion in liver tissue relative to muscle is consistent with previous observations made
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FIGURE 1.—Caudal fin versus muscle tissue with respect to (A) d13C and (B) d15N for wild Atlantic salmon smolts (circles) and brook trout (squares) collected from New Brunswick streams in 1996 and 1997 (brook trout) and 2002 (Atlantic salmon).
by Hesslein et al. (1993), Kline et al. (1993), Peterson et al. (1993), Johnson et al. (2002), and Bunn et al. (2003). The predictable patterns in stable C ratios among fin, liver, and muscle tissue allow flexibility in choosing a particular tissue for analysis (Hesslein et al. 1993). The larger volume provided by tissues such as muscle and liver would allow for more replicate samples to be run (Jardine and Cunjak, in press) and would provide the possibility for conducting other measurements (e.g., analysis of contaminants or other isotopes) on the same animal. In contrast, the ability to sample fin tissue nonlethally provides distinct advantages over muscle tissue, which can only be sampled nonlethally in larger-sized animals (Shannon et al. 2001); nonlethal sampling is particularly important for those animals that are threatened or endangered (COSEWIC 2003). The relatively tight correlation between fin and muscle isotope ratios suggests that
fin sampling is an appropriate means of assessing fish diet in a nonlethal manner. However, caution should be taken when comparing fin and muscle across a wide range of ages or sizes within a species, as the relative difference between the two tissues changes slightly with size. This size effect may be related to differences in turnover rates as fishes change diets during ontogeny and requires further clarification via laboratory-based dietswitch experimentation. The overwhelming majority of stable N measurements in the livers of fishes have yielded 15N values that are lower than those of muscle from the same individual (Peterson et al. 1993; Pinnegar and Polunin 1999; Johnson et al. 2002; Bunn et al. 2003; this study). However, under extreme circumstances, liver may become enriched in 15N relative to muscle in fishes, such as during long periods of food deprivation (Doucett et al. 1999b) or severe weight loss prior to mortality (Jardine et al.
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FIGURE 2.—Seasonal relationships between stable isotopes (d13C and d15N) in muscle (sexes combined) and gonad tissue (sexes separate; F 5 female, M 5 male) for slimy sculpin collected from the Little River, New Brunswick in 2000 and 2001. All values are means 6 95% confidence intervals.
2004; Figure 3). Male slimy sculpin sampled in April were the only group that showed this 15N enrichment pattern in the current study, suggesting that they may be in poor nutritional condition prior to the spawning period in May. This is probably a consequence of a cessation or reduction in feeding during the establishment of territories and nest guarding by males (Goto 1990), as both liver and muscle became enriched in 15N between the No-
vember and April sampling periods, in a similar fashion to that exhibited by fasting female Ross geese Chen rossi (Hobson et al. 1993). Female slimy sculpin in the current study showed no significant 15N enrichment in liver and muscle over the same period, suggesting that their prey consumption rates may remain high throughout the winter as they prepare to spawn in spring. Our observation that slimy sculpin eggs were
FIGURE 3.—Influence of growth rate on the difference between liver and muscle d15N in Atlantic salmon fed a hatchery diet. Modified from Jardine et al. (2004).
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enriched in 15N is consistent with the results of Bilby et al. (1996), who showed a similar pattern in a limited sample of spawning coho salmon Oncorhynchus kisutch. However, Grey (2001) found that brown trout eggs were slightly 15N-depleted relative to muscle (by less than 1‰), while McCarthy and Waldron (2000) found no differences in d15N between brown trout muscle and eggs. Slight differences within and among species may be related to the timing of sampling, as illustrated in the current study; only immediately prior to spawning was a d15N difference between eggs and muscle detected. The 13C content of fish egg tissue is universally less than that of muscle (Kline et al. 1993; McCarthy and Waldron 2000; Grey 2001), typically because of the high proportion of lipids that are 13 C-depleted (DeNiro and Epstein 1977). However, lipid extraction of slimy sculpin eggs did not eliminate differences from muscle in the current study (T.D.J., unpublished data). For fish testes, our results agree well with those of Kline et al. (1993), who found 13C-enrichment in male sockeye salmon O. nerka gonads relative to muscle. The shunting of specific amino acids with different stable isotope signatures (Gaebler et al. 1966; Fantle et al. 1999; Fogel and Tuross 2003) to gonads during gametogenesis may explain the differences we observed in male and female slimy sculpin. Muscle–gonad differences observed in this study may prove useful for future investigations of reproductive nutrient allocation in fishes, similar to previous investigations with birds (Gauthier et al. 2003). For example, slimy sculpin in this study had no access to the eggs of anadromous fishes (Foote and Brown 1998), as they were captured above an impassable waterfall. Therefore, we have estimated the muscle–gonad difference when feeding on a diet of benthic macroinvertebrates with typical freshwater signatures (M.A.G., unpublished data). A shift in gonad tissues toward a marine signature at sites with spawning anadromous fishes could indicate consumption of eggs and allocation of those nutrients to slimy sculpin reproduction. The measurements presented here, coupled with those of other studies on fish tissue variability (Rounick and Hicks 1985; Pinnegar and Polunin 1999; Satterfield and Finney 2002; Perga and Gerdeaux 2003), increase the potential applications of SIA in fisheries research. An understanding of the mechanisms governing isotopic fractionation among tissues will provide insight into nutrient allocation and metabolism in fishes and the reli-
ability of using nondestructive tissues to quantify fish diet with SIA. Acknowledgments We are indebted to Fisheries and Oceans and Parks Canada personnel, Peter Batt, and Kyle Vodjani for their collective assistance in gathering and processing samples. Staff at SINLAB (Fredericton, New Brunswick) conducted all analyses. Funding was provided by Parks Canada, the Canada Research Chairs Program, and the Toxic Substances Research Initiative. An earlier version of this manuscript was improved by comments from Daniel Hayes and two anonymous reviewers. Special thanks to Sarah McKim and Rick Doucett for advice on lipid extractions, Myriam Barbeau for statistical advice, and Sandra Brasfield and Rachel Keeler for fruitful discussions about the fish of the future. References Bilby, R. E., B. R. Fransen, and P. A. Bisson. 1996. Incorporation of nitrogen and carbon from spawning coho salmon into the trophic system of small streams: evidence from stable isotopes. Canadian Journal of Fisheries and Aquatic Sciences 53:164– 173. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37:911– 917. Bunn, S. E., P. M. Davies, and M. Winning. 2003. Sources of organic carbon supporting the food web of an arid zone floodplain river. Freshwater Biology 48: 619–635. COSEWIC (Committee on the Status of Endangered Wildlife in Canada). 2003. Canadian species at risk, November 2003. COSEWIC, Ottawa. Craig, H. 1957. Isotopic standards for carbon and oxygen and correction factors for mass-spectrometric analysis of carbon dioxide. Geochimica et Cosmochimica Acta 12:133–149. DeNiro, M. J., and S. Epstein. 1977. Mechanism of carbon isotope fractionation associated with lipid synthesis. Science 197:261–263. DeNiro, M. J., and S. Epstein. 1978. Influence of the diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42:495– 506. DeNiro, M. J., and S. Epstein. 1981. Influence of the diet on the distribution of nitrogen isotopes in animals. Geochimica et Cosmochimica Acta 45:341– 351. Doucett, R. R., W. Hooper, and G. Power. 1999a. Identification of anadromous and nonanadromous brook trout and their progeny in the Tabusintac River, New Brunswick, by means of multiple-stable-isotope analysis. Transactions of the American Fisheries Society 128:278–288.
NOTE
Doucett, R. R., R. K. Booth, G. Power, and R. S. McKinley. 1999b. Effects of the spawning migration on the nutritional status of anadromous Atlantic salmon (Salmo salar): insights from stable isotope analysis. Canadian Journal of Fisheries and Aquatic Sciences 56:2172–2180. Fantle, M. S., A. I. Dittel, S. M. Schwalm, C. E. Epifanio, and M. L. Fogel. 1999. A food web analysis of the juvenile blue crab, Callinectes sapidus, using stable isotopes in whole animals and individual amino acids. Oecologia 120:416–426. Finlay, J. C., S. Khandwala, and M. E. Power. 2002. Spatial scales of carbon flow in a river food web. Ecology 83:1845–1859. Fogel, M. L., and N. Tuross. 2003. Extending the limits of paleodietary studies of humans with compound specific carbon isotope analysis of amino acids. Journal of Archaeological Science 30:535–545. Foote, C. J., and G. S. Brown. 1998. Ecological relationship between freshwater sculpins (genus Cottus) and beach-spawning sockeye salmon (Oncorhynchus nerka) in Iliamna Lake, Alaska. Canadian Journal of Fisheries and Aquatic Sciences 55:1524– 1533. Gaebler, O. H., T. G. Vitti, and R. Vukmirovich, R. 1966. Isotope effects in metabolism of 14N and 15N from unlabeled dietary proteins. Canadian Journal of Biochemistry 44:1249–1257. Gauthier, G., J. Bety, and K. A. Hobson. 2003. Are greater snow geese capital breeders?: new evidence from a stable isotope model. Ecology 84:3250– 3264. Goto, A. 1990. Alternative life history styles of Japanese freshwater sculpins revisited. Environmental Biology of Fishes 28:101–112. Gray, M. A., R. A. Cunjak, and K. R. Munkittrick. 2004. Site fidelity of slimy sculpin (Cottus cognatus): insights from stable carbon and nitrogen analysis. Canadian Journal of Fisheries and Aquatic Sciences 61:1717–1722. Grey, J. 2001. Ontogeny and dietary specialization in brown trout (Salmo trutta L.) from Loch Ness, Scotland, examined using stable isotopes of carbon and nitrogen. Ecology of Freshwater Fish 10:168–176. Hesslein, R. H., K. A. Hallard, and P. Ramlal. 1993. Replacement of sulfur, carbon, and nitrogen in tissue of growing broad whitefish (Coregonus nasus) in response to a change in diet traced by d34S, d13C, and d15N. Canadian Journal of Fisheries and Aquatic Sciences 50:2071–2076. Hobson, K. A., and R. G. Clark. 1992a. Assessing avian diets using stable isotopes, I. Turnover of 13C in tissues. Condor 94:181–188. Hobson, K. A., and R. G. Clark. 1992b. Assessing avian diets using stable isotopes, II. Factors influencing diet2tissue fractionation. Condor 94:189–197. Hobson, K. A., R. T. Alisauskas, and R. G. Clark. 1993. Stable-nitrogen isotope enrichment in avian tissues due to fasting and nutritional stress: implications for isotopic analyses of diet. Condor 95:388–394. Hobson, K. A., and R. G. Clark. 1993. Turnover of 13C in cellular and plasma fractions of blood: implica-
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tions for nondestructive sampling in avian dietary studies. Auk 110:638–641. Jardine, T. D., D. L. MacLatchy, W. L. Fairchild, R. A. Cunjak, and S. B. Brown. 2004. Rapid carbon turnover during growth of Atlantic salmon (Salmo salar) smolts in seawater, and evidence for reduced food consumption by growth stunts. Hydrobiologia 527: 63–75. Jardine, T. D., and R. A. Cunjak. In press. Analytical error in stable isotope ecology. Oecologia. Johnson, B. M., P. J. Martinez, and J. D. Stockwell. 2002. Tracking trophic interactions in coldwater reservoirs using naturally occurring stable isotopes. Transactions of the American Fisheries Society 131: 1–13. Kline, T. C., Jr., J. J. Goering, O. A. Mathisen, P. H. Poe, P. L. Parker, and R. S. Scalan. 1993. Recycling of elements transported upstream by runs of Pacific salmon, II. d15N and d13C evidence in the Kvichak River watershed, Bristol Bay, Southwestern Alaska. Canadian Journal of Fisheries and Aquatic Sciences 50:2350–2365. Macko, S. A., M. L. Fogel Estep, M. H. Engel, and P. E. Hare. 1986. Kinetic fractionation of stable nitrogen isotopes during amino acid transamination. Geochimica et Cosmochimica Acta 50:2143–2146. Mariotti, A. 1983. Atmospheric nitrogen is a reliable standard for natural 15N abundance measurements. Nature (London) 303:685–687. McCarthy, I. D., and S. Waldron. 2000. Identifying migratory Salmo trutta using carbon and nitrogen stable isotope ratios. Rapid Communications in Mass Spectrometry 14:1325–1331. McConnaughy, T., and C. P. McRoy. 1979. Food-web structure and the fractionation of carbon isotopes in the Bering Sea. Marine Biology 53:257–262. Minagawa, M., and E. Wada, E. 1984. Stepwise enrichment of 15N along food chains: further evidence and the relation between d15N and animal age. Geochimica et Cosmochimica Acta 48:1135–1140. Perga, M. E., and D. Gerdeaux. 2003. Using the d13C and d15N of whitefish scales for retrospective ecological studies: changes in isotope signatures during the restoration of Lake Geneva, 1980–2001. Journal of Fish Biology 63:1197–1207. Peterson, B. J., and B. Fry. 1987. Stable isotopes in ecosystem studies. Annual Review of Ecology and Systematics 18:293–320. Peterson, B., B. Fry, L. Deegan, and A. Hershey. 1993. The trophic significance of epilithic algal production in a fertilized tundra river system. Limnology and Oceanography 38:872–878. Pinnegar, J. K., and N. V. C. Polunin. 1999. Differential fractionation of d13C and d15N among fish tissues: implications for the study of trophic interactions. Functional Ecology 13:225–231. Post, D. M. 2002. Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology 83:703–718. Rounick, J. S., and B. J. Hicks. 1985. The stable carbon isotope ratios of fish and their invertebrate prey in
1110
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four New Zealand rivers. Freshwater Biology 15: 207–214. Ryan, B. F., and B. L. Joiner. 2000. MINITAB handbook, 4th edition. Duxbury Press, New York. Satterfield, F. R., and B. P. Finney. 2002. Stable isotope analysis of Pacific salmon: insight into trophic status and oceanographic conditions over the last 30 years. Progress in Oceanography 53:231–246. Shannon, J. P., D. W. Blinn, G. A. Haden, E. P. Benenati, and K. P. Wilson. 2001. Food web implications of d13C and d15N variability over 370 km of the reg-
ulated Colorado River, USA. Isotopes in Environmental and Health Studies 37:179–191. Tieszen, L. L., T. W. Boutton, K. G. Tesdahl, and N. A. Slade. 1983. Fractionation and turnover of stable carbon isotopes in animal tissues: implications for d13C analysis of diet. Oecologia 57:32–37. Underwood, A. J. 1997. Experiments in ecology: their logical design and interpretation using analysis of variance. Cambridge University Press, Cambridge, UK.
