Accurate interpretation of isotopic differences within or among animal tissues is dependent on information on three sources of isotopic variation: (1) the isotopic composition of the potential inputs, (2) an understanding of the isotopic fractionations that occur between these sources and animal tissues, and (3) an understanding of how long it takes for the isotopic value of these sources to be reflected in a tissue (often referred to as isotopic turnover). Here, we focus on the latter two sources of variation: tissue-to-source isotopic fractionation and isotopic turnover rates. For tissue-to-source fractionations, we consider carbon and nitrogen, which are supplied by diet, separately from oxygen, which is largely supplied by ingested water. We lay out general patterns that might be expected from studies of other mammals and birds, but highlight whenever possible studies of marine mammals.
Tissue-to-Diet Nitrogen and Carbon Isotope Discrimination in Marine Mammals
A clear understanding of the tissue-to-diet isotope discrimination for a species is critical for interpreting ecological information from tissue isotope values. The magnitude of these fractionations can vary as a result of differences in metabolic routing of dietary components between tissues (e.g., lipids, proteins, and carbohydrates), variation in an animal's growth rate and the nutritional quality of its diet, differences in the amino acid or lipid composition of tissues, and the interplay between these factors and temporal variation in the ecology and physiology of marine mammals. We discuss the impact of each of these factors on nitrogen and carbon isotope tissue-to-diet discrimination below.
The dominant source of nitrogen in marine mammals is dietary protein. An increase in δ15N value with each trophic step has been recognized across taxonomic groups and food webs (typically +2‰–+5‰ for each increase in trophic level; Minagawa and Wada 1984, Kelly 2000, Vanderklift and Ponsard 2003). Trophic discrimination is thought to relate to excretion of urea and other nitrogenous wastes that are 15N-depleted relative to body nitrogen pools. Isotopic fractionation of nitrogen occurs during deamination and transamination reactions flowing into and out of the TCA cycle and in the recycling of urea within the body (see review and modeling study by Balter et al. 2006). Dietary protein quantity and quality can also influence the magnitude of isotopic fractionation (Robbins et al. 2005); both models and limited data suggest that Δ15Ntissue-diet decreases with increasing dietary protein quality, but increases with increasing dietary protein quantity (Martínez del Rio et al. 2009). Based on differences in protein quantity, we might expect higher discriminations in carnivorous marine mammals (cetaceans, pinnipeds) than in herbivorous species (sirenians). Predictions related to differences in protein quantity vs. quality are more difficult to generate within these broad feeding categories.
Δ15Ntissue-diet values for pinnipeds, the only group of marine mammals on which controlled feeding experiments have been conducted, are relatively consistent across taxa and are in the +3‰–+5‰ range commonly observed in studies of terrestrial carnivores (Table 3). Analyzing different tissues in captive phocids fed an isotopically homogenous diet, Hobson et al. (1996) found that Δ15N values range from 1.7‰ for red blood cells to 3.1‰ for liver. Focusing on just blood and fur of captive phocids, Lesage et al. (2002) found a similar range in Δ15N values. In contrast, Kurle (2002) found that Δ15N values for various blood components in captive northern fur seals (Callorhinus ursinus) ranged from 4.1‰ to 5.2‰. Focusing on blood serum, Zhao et al. (2006) also found relatively large Δ15Nserum-diet values for captive harbor seals (Phoca vitulina), ranging from 3.9‰ to 4.6‰. Recently, Newsome et al. (in review) found a mean Δ15Nvibrissae-diet value of 3.5‰ for a wild population of California sea otters (Enhydra lutris nereis).
Table 3. Summary of diet-tissue discrimination factors observed for various species in controlled feeding experiments or inferred from wild populations. Numbers in parentheses indicate the mean Δtissue-diet value for carbon and nitrogen; see references for associated variance.
|Citation||Species||Δ13Ctissue-diet discrimination||Δ15Ntissue-diet discrimination||Lipid extracted|
|Hobson et al. 1996||P. groenlandicus||RBC (+1.7)||RBC (+1.7)||Y|
|P. vitulina||Fur (+2.8)||Fur (+3.0)||N|
|P. hispida||Liver (+0.6)||Liver (+3.1)||N|
| ||Muscle (1.3)||Muscle (2.4)||Y|
| ||Nails (+2.8)||Nails (+2.3)||N|
| ||Skin (+2.8)||Skin (+2.3)||N|
| ||Vibrissae (+3.2)||Vibrissae (+2.8)||N|
|Prey|| || ||Y|
|Kurle 2002||C. ursinus||RBC (+1.3)||RBC (+4.1)||N|
|Plasma (+1.0)||Plasma (+5.2)||N|
|Serum (+0.6)||Serum (+5.2)||N|
|Prey|| || ||Y|
|Lesage et al. 2002||H. grypus||RBC (+1.5)||Blood-RBC (+1.7)||N|
|P. groenlandicus||Serum (+0.8)||Blood-Serum (+3.1)||N|
|P. vitulina||Fur (+2.3)||Fur (+2.3)||N|
|Prey|| || ||N|
|Zhao et al. 2006||P. vitulina||Serum (−0.6–1.7)||Serum (+3.9–+4.6)||N|
|Prey|| || ||Y|
|Newsome et al. in review||E. lutris||Vibrissae (+2.2)||Vibrissae (+3.5)||N|
|Prey|| || ||N|
|Clementz et al. 2007||D. dugon||Bioapatite (+11–+14)|| ||NA|
|H. gigas|| || || |
|T. inunguis|| || || |
|T. manatus|| || || |
|Prey|| || ||N|
Whereas the nitrogen in an animal's diet is mainly sourced from the proteins it consumes, the carbon for an animal's tissues is supplied by dietary proteins, lipids, and carbohydrates, which may differ in their carbon isotope composition. In addition, carbon occurs in tissues composed of materials other than protein, such as bioapatite and lipids, which have a greater isotopic range than that observed for nitrogen from protein-rich tissues. In terrestrial mammals, the δ13C value of bioapatite reflects that of bulk diet, whereas that of proteins and lipids is often biased toward the protein or lipid portion of the diet, respectively, as a result of dietary routing of these components. For most lipids, there is usually a balance between routing of dietary lipids to tissue and de novo synthesis of new lipids; bone cholesterol is the one lipid that strongly reflects bulk diet (Jim et al. 2003). For proteins, there is a similar balance between routing of amino acids—particularly indispensible amino acids that cannot be produced through de novo synthesis—and production of the R-groups of dispensable amino acids from bulk diet or carbohydrate and lipid carbon (Howland et al. 2003, Jim et al. 2006). For pinnipeds, cetaceans and otters, which consume protein-rich diets with variable amounts of fat, the δ13C value of body protein should closely track that of bulk diet, but perhaps with different tissue-to-diet fractionations depending on dietary lipid content. Herbivorous sirenians would receive bulk dietary carbon from carbohydrates along with a smaller quantity of proteins from plants or protein-rich epizooans, which should, in turn, reflect plant-derived carbon.
Measured tissue-to-diet isotope discriminations for bioapatite, lipids and proteins are significantly different. For bioapatite, tissue-to-diet isotope fractions in terrestrial mammals differ between carnivores (+9‰) and herbivores (+12‰–+14‰) (reviewed in Koch 2007). The Δ13Capatite-diet value has been measured in manatees (Trichechus manatus latirostrius) on controlled diets and is +14‰ (MacFadden et al. 2004). While Δ13Capatite-diet values have not been determined experimentally for other marine mammals, field studies suggest they are similar to values for land carnivores (Clementz and Koch 2001, Clementz et al. 2007). In contrast, bulk consumer lipid is 13C-depleted by 2‰–5‰ relative to bulk diet (DeNiro and Epstein 1978, Tieszen et al. 1983, Howland et al. 2003) and controlled feeding studies of captive pinnipeds show that trophic Δ13C values for consumer proteins range from +0.5‰ to +2.0‰ for most tissues (Table 3), except those composed of keratin (e.g., fur, vibrissae), which range from +2‰ to +3‰. The only study of a wild marine mammal population found that mean Δ13Cvibrissae-diet values of California sea otters was 2.2‰ (Newsome et al., in review), within the range found for captive pinnipeds (Table 3). Unfortunately, there are no controlled studies in which collagen has been measured, so most workers assume a value of +5‰, as seen in other mammals and birds.
Along with preferential routing of dietary components into different tissues, nutritional status and growth rate have been shown to affect tissue-to-diet isotope fractionation, particularly trophic 15N enrichment (Vanderklift and Ponsard 2003, Robbins et al. 2005). With the exception of sirenians, all marine mammals are carnivores that consume prey with a high nitrogen concentration; lipid-extracted marine mammal prey typically have atomic C/N ratios of 3–4. Because urea δ15N values can be up to 10‰ lower than serum (see review by Balter et al. 2006), theoretical considerations and empirical data suggest that a higher fractional loss of nitrogen as urea—which typically correlates positively with both the rate of protein intake and the rate of urea loss—will lead to higher body δ15N values (reviewed and modeled by Martínez del Rio and Wolf 2005 and Martínez del Rio et al. 2009). Zhao et al. (2006) found that captive harbor seals fed a protein-rich diet of pollock had slightly higher Δ15N values (4.6‰vs. 3.9‰, Table 2) than animals that consumed a relatively protein-poor diet of herring. While subtle, this pattern agrees with findings on other taxa that show nitrogen isotope fractionation can be influenced by protein quantity. These findings suggest that trophic Δ15N values for sirenians—herbivores that consume low protein food—might be lower than the range seen in carnivorous marine mammal species.
Different amino acids in a single tissue can vary in δ13C and δ15N values by more than 15‰ (e.g., Hare et al. 1991). As different proteins contain distinct proportions of amino acids, differences in the protein composition among tissue types can yield dissimilar isotopic compositions irrespective of changes in diet. For example, Kurle (2002) found differences in the 15N-enrichment of various tissues relative to the diet of captive northern fur seals that were fed a strict diet of known isotopic composition. Red blood cells had δ15N values approximately 4.1‰ higher than diet, whereas plasma and serum were enriched by approximately 5.2‰ relative to diet. This discrepancy in trophic discrimination among tissue types was interpreted as a consequence of differences in amino acid composition between these tissues. Stegall et al. (2008) found that vibrissae δ13C values were approximately 2‰ higher than serum from Steller sea lion (Eumetopias jubatus) pups and juveniles but found no significant differences in δ15N values between these tissues. Again, the observed differences in δ13C values likely result from differences in the amino acid composition of blood serum vs. vibrissae keratins. It has been long recognized that another commonly analyzed tissue, bone collagen, has a distinctive amino acid composition that produces larger than normal diet-tissue δ13C fractionation. While “soft” tissues such as muscle, liver, and skin are 13C-enriched by only 1‰–2‰ relative to diet, bone collagen typically has δ13C values that are 4‰–5‰ higher than diet (Koch 2007). Accurate interpretation of intertissue isotopic differences requires careful consideration of such tissue-dependent discrimination patterns.
Many marine mammals experience seasonal cycles in food intake and energy demands that may impact the physiological processes that govern isotopic fractionation during metabolism and tissue synthesis. For example, many pinniped and mysticetes are capital breeders, storing vast amounts of fat to provide energy during reproduction and nursing. Some of these animals also undertake long migrations during which food intake may be limited. Because blubber is primarily composed of 13C-depleted lipids, it has a significantly lower δ13C value than a piscivorous (pinniped) or planktonic (mysticete) diet. An animal that relies on blubber stores to maintain metabolism will be “consuming” a food source with a lower δ13C value than its regular diet and have a Δ13Ctissue-diet value that is lower than when it is not relying on fat.
Such factors may influence Δ15Ntissue-diet values as well. Catabolism of protein from lean tissues (e.g., muscle) during periods of nutritional stress may cause δ15N values to rise as the animal continues to shed waste that is 14N-enriched relative to the body. Furthermore, the nitrogen source for any additional protein deposition is body tissue, which is already 15N-enriched relative to dietary sources. A number of laboratory and a few field experiments have explored the utility of stable isotopes as proxies of nutritional stress (e.g., Hobson et al. 1993, Polischuck et al. 2001, Cherel et al. 2005). For experiments in which no exogenous protein was supplied to subjects, significant bulk tissue or whole body 15N-enrichments of 0.5‰–2.5‰ were observed. In a wild population, Cherel et al. (2005) found significant 15N-enrichments in the plasma, red blood cells, and feathers of fasting penguins, which rely exclusively on endogenous protein when breeding and molting. Finally, in a longitudinal study tracking pregnant women, in those with severe morning sickness who entered negative nitrogen balance, hair δ15N values rose by 0.4‰–1.2‰ (Fuller et al. 2004, 2005). Overall, such effects would lead to increased Δ15Ntissue-diet values for animals in nutritional stress.
Isotopic consequences of growth, pregnancy, and lactation have received little study. We might expect that growing, pregnant, or nursing animals might lose relatively less body nitrogen as urinary waste and therefore have lower Δ15N values. While not designed to study such patterns in mothers, early work on human nursing did not detect an isotopic effect in lactating women (Fogel et al. 1997). In contrast, a study of wild horses showed that lactating females had lower δ15N values than other adults (males, nonlactating females) and used mass balance calculations to argue that this 15N-depletion is the expected result of the nitrogen balance perturbations associated with lactation (Koch 1997). Further support for this trend was reported in Kurle (2002), where blood δ15N values of a single lactating northern fur seal were approximately 1‰ lower than those for nulliparous females. Fuller et al. (2004) reported δ15N variations among pregnant human females. They found that δ15N values dropped from conception to birth, and that the magnitude of the drop correlated to the birth weight of the baby as well as the amount of weight gained by the mother. If these phenomena occur in marine mammals, they would reduce Δ15Ntissue-diet values for growing or pregnant females. Expectations for lactating females are more complex and may depend on whether animals feed or fast while lactating (i.e., income vs. capital breeders).
Oxygen Isotope Fractionation in Marine Mammals
The δ18O value of a biomineral depends on the temperature at which it forms and the 18O value of the body fluid from which it precipitates (discussion below based on Clementz and Koch 2001 and Koch 2007). For mammals there is a constant offset between the 18O value of body water and phosphate (∼+18‰), and between the phosphate and carbonate components of bioapatite (∼+8‰), close to values predicted for isotopic equilibrium at typical body temperatures.
Physiology affects the 18O value of body water by altering the fluxes of oxygen into and out of the body, as well as fractionations associated with transport and/or transformation of oxygen-bearing compounds. Ingested water is a major flux of oxygen into marine mammals and includes preformed water in food, seawater consumed incidentally when eating, and water taken by active drinking (mariposia). The proportion of water gained from these sources varies widely among marine mammals (Ortiz 2001), yet as these processes do not strongly fractionate oxygen, these fluxes should all have 18O values close to that of seawater (0‰ V-SMOW). Metabolic water generated by oxidation of food dry matter may contribute to marine mammal body water. This water may be 18O-enriched relative to ingested water, as atmospheric O2 is much heavier than ingested water (∼+21‰ V-SMOW). Finally, there is evidence in cetaceans for a substantial flux of water across the skin (Hui 1981, Andersen and Nielsen 1983); it is unlikely that this process greatly fractionates oxygen isotopes, though the issue has not been studied. Fluxes of oxygen out of the body include respired carbon dioxide (which strongly fractionates), water and organic matter in waste, and water lost during exhalation.
Overall, the 18O value of marine mammal body water is similar to that of environmental water, as their bioapatite phosphate and carbonate form in near isotopic equilibrium with environmental water. Clementz and Koch (2001) noted that there is a systematic difference in apatite 18O values between pinnipeds and cetaceans. Pinnipeds have values expected for equilibrium with seawater at body temperature, whereas cetacean values are about 2‰ higher. They speculated on potential causes for this difference, but were unable to explain the difference. Clementz and Koch (2001) also noted that bioapatite 18O values from aquatic mammal teeth showed little within-population variability, presumably because body water 18O values vary little within an individual during its lifetime or among individuals in populations.
Isotopic turnover rates can vary within or among individuals as a function of body size, growth rate, and protein turnover. A simple single-component box model shows that the rate of isotopic turnover is approximately equal to the net rate of influx of new material divided by the size of the pool of the element in the tissue. Because of the large daily fluxes of oxygen into and out of mammals, turnover times are rapid, on the scale of a week to a month, and are well established from the literature on isotope dilution and measurement of metabolic rate (Nagy and Costa 1980, Ortiz 2001). For carbon and nitrogen in tissues, the rate of elemental incorporation is approximately proportional to body mass (mb) to the 3/4 power (Martinez del Rio and Wolf 2005, Martinez del Rio et al. 2009), whereas the mass of animal tissues usually scales isometrically with mb. Thus, isotopic turnover of metabolically active tissues is proportional to mb−1/4 (i.e., mb3/4/mb). This prediction has only been empirically tested on a single tissue (red blood cells) from a few small bird species (Carleton and Martínez del Rio 2005).
In addition to overall body size, both the growth of new tissue and the amount of tissue replacement due to catabolic turnover play fundamental roles in determining isotopic turnover rates. In short, the isotopic turnover rate equals the sum of the growth rate and the allometric effect of body size on catabolic turnover (mb−1/4). Most marine mammals undergo determinate growth, so for adults that are not nutritionally stressed, the growth term is zero; thus isotopic turnover rates should scale allometrically with mb−1/4. Like most endotherms, marine mammals only experience exponential growth during the first year of life and thus the growth of new tissue need only be considered for this ontogenetic stage. During this phase, mass-specific growth rate also scales with mb−1/4 because maximal growth rate (in units of mass per unit time) scales with mb3/4 (Martinez del Rio and Wolf 2005, Martinez del Rio et al. 2009). Therefore the contributions of catabolic turnover and growth on isotopic incorporation both scale allometrically with mb−1/4 for very young animals.
In addition to factors related to body size and growth rate, isotopic turnover rates vary among tissue types. Carleton and Martínez del Rio (2005) hypothesized that protein turnover is the primary determinant of isotopic turnover rate for the most commonly used tissues in isotopic ecology, especially since samples are typically lipid-extracted prior to analysis. While this prediction has not been tested by simultaneously measuring protein turnover and isotopic turnover in the same organism, there are data from the laboratory and field studies that suggest a close link between these processes. The first is the observation that splanchnic organs (e.g., liver) and plasma proteins, which have relatively high rates of protein turnover, also have higher isotopic turnover rates than structural elements (e.g., collagen, striated muscle). Second, several studies have shown that protein intake, or the amount of dietary nitrogen is positively correlated with isotopic turnover rates. Because pinnipeds, cetaceans, and sea otters consume high quality, nitrogen-rich carnivorous diets, protein intake rate is not likely to be an important source of variation in isotopic turnover. Diet quality could be an important factor for sirenians, which consume nitrogen-poor sea grass and algae.
A relatively new contribution to the discussion of isotopic turnover is the concern that multiple isotope pools may exist within an organism and each of these pools may have different turnover rates. Ayliffe et al. (2004) were the first to discuss this issue when interpreting carbon isotope turnover in tail hair and breath CO2 from domestic horses. They were able to isolate three carbon pools with distinct turnover rates ranging from fast (t1/2 ∼ 0.2–0.5 d) to slow (t1/2 ∼ 50–140 d). Cerling et al. (2007) refined this approach further by presenting the “reaction-progress variable” as a method for determining whether isotopic turnover was best expressed using a single exponential function or by using multiple linear functions, an approach that has been effectively used in geochemical studies. Martínez del Rio and Anderson-Sprecher (2008) and Carleton et al. (2008) have evaluated the necessity of this approach by quantifying the uncertainty inherent in estimates of isotope retention by multicompartment models and by testing whether multicompartment models are more effective than single-compartment models. They argued that the appropriate model may depend upon the type of tissue. The significance of the these findings has yet to be determined for isotopic incorporation studies for marine mammals; turnover rates are determined by diet-switching experiments, which are difficult to perform on marine mammals, so few studies have produced data on isotopic turnover for metabolically active tissues (Table 1, Zhao et al. 2006, Newsome et al. 2006, Orr et al. 2009). Future switching experiments on marine mammals should be designed to develop allometric relationships between body size and isotopic turnover in tissues such as muscle and blood components, as well as to test whether single- or multicompartment models are appropriate.
While isotopic turnover rates are important for the interpretation of tissues that undergo catabolic replacement, other tissues are metabolically inert and do not experience continual exchange once synthesized. For such tissues, there will still be an isotopic turnover time for the pool from which the tissue is synthesized. Four types of metabolically inert and continually growing tissues have proven useful in studies of marine mammal ecology: (1) fur or vibrissae (keratin), (2) baleen (keratin and bioapatite), (3) tooth dentin (collagen and bioapatite), and (4) tooth enamel (bioapatite). When interpreting data from fur, vibrissae, and baleen, consideration of tissue growth rate is a much more important issue than isotopic turnover. For teeth, the critical factor is the time of tissue formation. Tooth enamel, even on permanent dentition, forms early in life, and for many cetaceans and pinnipeds enamel on many teeth begins to form prior to weaning (Perrin and Myrick 1980, Modig et al. 1997, Stewart et al. 1998). Tooth dentin, in contrast, may deposit within the crown and root of a tooth for decades. Annual lamellae are pronounced in many species, providing material for the construction of ontogenetic time series of isotope values.