About 10 years ago, reviews of the use of stable isotopes in animal ecology predicted explosive growth in this field and called for laboratory experiments to provide a mechanistic foundation to this growth. They identified four major areas of inquiry: (1) the dynamics of isotopic incorporation, (2) mixing models, (3) the problem of routing, and (4) trophic discrimination factors. Because these areas remain central to isotopic ecology, we use them as organising foci to review the experimental results that isotopic ecologists have collected in the intervening 10 years since the call for laboratory experiments. We also review the models that have been built to explain and organise experimental results in these areas.

Summary Ten years ago Gannes et al. (1997, Stable isotopes in animal ecology: assumptions, caveats, and a call for laboratory experiments. Ecology , 78 , 1271-1276, 1998) identified four major areas requiring further research in experimental animal isotopic ecology: (i) the dynamics of isotopic incorporation, (ii) mixing models, (iii) the problem of routing, and (iv) trophic discrimination factors. 2. Differences in isotopic incorporation rates among tissues seem to be explained by variation in protein turnover. The application of multi-compartment models to isotopic incorporation data has revealed that different inferences can be derived between these and one-compartment models. 3. A variety of mixing models of varying degrees of complexity and realism are used to find the contribution of isotopic sources to the elements in an organism's tissues. The use of these models demands the use of tissue to diet discrimination factors that are rarely measured experimentally. 4. Mixing models assume that assimilated nutrients are disassembled into their elemental components and that these elements are reassembled into biomolecules. This assumption is unrealistic as macromolecules are routed differentially into tissues. Isotopic routing is an area that isotopic ecologists have neglected in their experimental and modelling research. 5. Isotopic ecologists are just beginning to understand why 15 N biomagnifies along trophic chains, and to explore the factors that determine the degree of 15 N biomagnification. We review the hypotheses that explain why 15 N biomagnifies up trophic chains. 6. The use of compound-specific isotopic analyses is opening new fruitful areas of research at the intersection of nutritional and isotopic ecology.

Animals with high metabolic rates are believed to have high rates of carbon and nitrogen isotopic incorporation. We hypothesized that (1) chronic exposure to cold, and hence an increase in metabolic rate, would increase the rate of isotopic incorporation of both 13C and 15N into red blood cells; and (2) that the rate of isotopic incorporation into red blood cells would be allometrically related to body mass. Two groups of sparrows were chronically exposed to either 5 or 22 degrees C and switched from a 13C-depleted C3-plant diet to a more 13C-enriched C4-plant one. We used respirometry to estimate the resting metabolic rate (VO2) of birds exposed chronically to our two experimental temperatures. The allometric relationship between the rate of 13C incorporation into blood and body mass was determined from published data. The (VO2) of birds at 5 degrees C was 1.9 times higher than that of birds at 22 degrees C. Chronic exposure to a low temperature did not have an effect on the rate of isotopic incorporation of 15N save for a very small effect on the incorporation of 13C. The isotopic incorporation rate of 13C was 1.5 times faster than that of 15N. The fractional rate of 13C incorporation into avian blood was allometrically related to body mass with an exponent similar to -1/4. We conclude that the relationship between metabolic rate and the rate of isotopic incorporation into an animal's tissues is indirect. It is probably mediated by protein turnover and thus more complex than previous studies have assumed.

Understanding rates of isotopic incorporation and discrimination factors between tissues and diet is an important focus of ecologists seeking to use stable isotopes to track temporal changes in diet. We used a diet-shift experiment to measure differences among tissues in (13)C incorporation rates in house sparrows (Passer domesticus). We predicted faster incorporation rates in splanchnic than in structural tissues. We also assessed whether isotopic incorporation data were better supported by the one-compartment models most commonly used by ecologists or by multi-compartment models. We found large differences in the residence time of (13)C among tissues and, as predicted, splanchnic tissues had faster rates of isotopic incorporation and thus shorter retention times than structural tissues. We found that one-compartment models supported isotopic incorporation data better in breath, excreta, red blood cells, bone collagen, and claw tissues. However, data in plasma, intestine, liver, pectoralis muscle, gizzard, and intestine tissues supported two-compartment models. More importantly, the inferences that we derived from the two types of models differed. Two-compartment models estimated longer (13)C residence times, and smaller tissue to diet differences in isotopic composition, than one-compartment models. Our study highlights the importance of considering both one- and multi-compartment models when interpreting laboratory and field isotopic incorporation studies. It also emphasizes the opportunities that measuring several tissues with contrasting isotopic residence times offer to elucidate animal diets at different time scales.

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