Isotopic fractionation and turnover in captive Garden Warblers ( Sylvia borin ): implications for delineating dietary and migratory associations in wild passerines

1630 NOTE / NOTE Isotopic fractionation and turnover in captive Garden Warblers (Sylvia borin): implications for delineating dietary and migratory associations in wild passerines Keith A. Hobson and Franz Bairlein Abstract: There is currently a great deal of interest in using stable-isotope methods to investigate diet and migratory connections in wild passerines. To apply these methods successfully, it is important to understand how stable isotopes discriminate or change between diet and the tissue of interest and what the element-turnover rates are in metabolically active tissues. Of particular use are studies that sample birds non-destructively through the use of blood and feathers. We investigated patterns of isotopic discrimination between diet and blood and feathers of Garden Warblers (Sylvia borin) raised on an isotopically homogeneous diet (48% C, 5% N) and then switched to one of two experimental diets, mealworms (56.8% C, 8.3% N) and elderberries, Sambucus niger (47.4% C, 1.5% N). We established that the discrimination factors between diet and blood appropriate for stable carbon (δ13C) and nitrogen (δ15N) isotopes are +1.7‰ and +2.4‰, respectively. For feathers, these values were +2.7‰ and +4‰, respectively. Turnover of elemental nitrogen in whole blood was best approximated by an exponential-decay model with a half-life of 11.0 ± 0.8 days (mean ± SD). Corresponding turnover of carbon was estimated to range from 5.0 ± 0.7 to 5.7 ± 0.8 days. We conclude that this decoupling of nitrogen- and carbon-turnover rates can be explained by differences in metabolic routing of dietary macromolecules. Our results suggest that tracking frugivory in migratory passerines that switch diets between insects and fruits may be complicated if only a trophic-level estimate is made using δ15N measurements. Résumé : L’utilisation des méthodes d’isotopes stables pour étudier le régime alimentaire et les liens migratoires chez les passereaux sauvages suscite actuellement beaucoup d’intérêt. Pour appliquer ces méthodes avec succès, il est important de comprendre comment les isotopes stables permettent la discrimination et comment ils changent entre le régime alimentaire et le tissu étudié, ainsi que de savoir quel est le taux de remplacement des éléments dans les tissus métaboliquement actifs. Les études des oiseaux vivants par prélèvement de sang ou de plumes sont particulièrement utiles. Nous avons déterminé les patterns de discrimination isotopique entre la régime alimentaire, d’une part, et le sang et les plumes, d’autre part, chez des fauvettes des jardins (Sylvia borin) élevées à un régime alimentaire isotopiquement homogène (48 % C, 5 % N), puis transférées à deux régimes expérimentaux composés de vers de farine (56,8 % C, 8,3 % N) ou de baies de sureau, Sambucus niger (47,4 % C, 1,5 % N). Les facteurs de discrimination entre le régime alimentaire et le sang correspondant aux isotopes stables de carbone (δ13C) et d’azote (δ15N) sont respectivement de +1,7 ‰ et de +2,4 ‰. Dans le cas des plumes, les valeurs respectives sont de +2,7 ‰ et de +4 ‰. Le taux de remplacement de l’azote élémentaire dans le sang entier se décrit le mieux par un modèle exponentiel de décomposition avec une demi-vie de 11,0 ± 0,8 jours (moyenne ± écart type). Le taux de remplacement correspondant pour le carbone varie de 5,0 ± 0,7 à 5,7 ± 0,8 jours. La dissociation des taux de remplacement de l’azote et du carbone s’explique, selon nous, par des différences dans le cheminement métabolique des macromolécules alimentaires. Nos résultats laissent croire que l’identification de la frugivorie chez les passereaux migrateurs qui changent leur régime alimentaire d’insectes pour un régime de fruits peut s’avérer difficile, si on ne fait qu’estimer le niveau trophique à l’aide de mesures de δ15N. [Traduit par la Rédaction] Hobson and Bairlein 1635 Received 10 March 2003. Accepted 16 July 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 8 October 2003. K.A. Hobson.1 Prairie and Northern Wildlife Research Centre, Canadian Wildlife Service, 115 Perimeter Road, Saskatoon, SK S7N 0X4, Canada. F. Bairlein. Institut für Vogelforschung, Vogelwarte Helgoland, An der Vogelwarte 21, D-26386, Wilhelmshaven, Germany. 1 Corresponding author (Keith.Hobson@ec.gc.ca). Can. J. Zool. 81: 1630–1635 (2003) doi: 10.1139/Z03-140 © 2003 NRC Canada Hobson and Bairlein Introduction The use of stable-isotope methods to elucidate several aspects of avian ecology and ecophysiology is rapidly expanding (Gannes et al. 1998; Hobson 1999). Applications include delineation of relative proportions of diets gleaned from two isotopically distinct sources (e.g., Hobson 1986, 1990; Mizutani et al. 1990; Hobson and Sealy 1991; Hobson et al. 2000b) to the reconstruction of habitat use and origins of migratory species (Chamberlain et al. 1997; Hobson and Wassenaar 1997; Marra et al. 1998; Rubenstein et al. 2002). This field is based on the phenomenon that food webs show a range of stable-isotope signatures that depend largely, but not exclusively, on photosynthetic pathway and climate, and these signatures are passed on to higher level consumers. The measurement of stable-isotope ratios in avian tissues can thus be used to infer spatial and dietary information that is difficult if not impossible to obtain by more conventional methods. There are two fundamental aspects to the successful application of the stable-isotope technique to avian studies. The first is the establishment of diet–tissue-discrimination (previously “fractionation”) factors that describe how isotope signatures change between a bird’s diet and a specific tissue of interest. The second is establishing the period of time that tissue isotope values reflect diet, since elemental turnover occurs in metabolically active tissues. Tissues with high turnover rates, such as liver and plasma, reflect recent diet, whereas tissues with slower turnover rates, such as blood cells and muscle, reflect diet over longer terms (Hobson and Clark 1993). While stable-isotope analyses have been used widely to interpret the diets of free-living bird populations, few controlled laboratory studies have been performed to determine isotope-turnover rates and determine how stable isotopes discriminate or change once they are incorporated into avian tissues (Hobson and Clark 1992a, 1992b; Gannes et al. 1997). Moreover, recent developments in the use of isotopemixing models to derive quantitative estimates of dietary contributions of isotopically distinct components specifically require precise estimates of diet–tissue discrimination (Phillips and Greg 2001). Avian field studies using stableisotope analyses have thus far assumed that the results of controlled laboratory studies on captive Japanese Quail (Coturnix japonica), Ring-billed Gulls (Larus delawarensis), and American Crows (Corvus brachyrhynchos) (Hobson and Clark 1992 a, 1992b, 1993) are representative of a wide taxonomic range of bird species, and interpreted their results on the basis of turnover rates and diet–tissue-discrimination factors derived from these captive species. Haramis et al. (2001) and Bearhop et al. (2002) recently provided information on both stable carbon- and nitrogen-isotope turnover in whole blood from wintering Canvasbacks (Aythya valisineria) and captive Great Skuas (Catharacta skua), respectively. These studies were the first to report patterns of isotope-turnover rates for stable isotopes of both carbon (δ13C) and nitrogen (δ15N) simultaneously, and they suggested that such patterns were similar. Evans-Ogden et al. (2003) conducted similar investigations on captive Dunlin (Calidris alpina pacifica) but the isotope-turnover rates and discrimination factors that they determined differed from those from these previous studies on larger bodied waterfowl 1631 and seabirds. Most recently, Pearson et al. (2003) examined carbon- and nitrogen-isotope discrimination and turnover in blood and feathers of Yellow-rumped Warblers (Dendroica coronata) and reported the fastest isotope-turnover rates to date and a dependence of diet–tissue-discrimination factors on the elemental composition of the diet. This suggests a complexity to the application of estimates of stable-isotope discrimination and turnover that was not previously considered (Bearhop et al. 2002). Here we present the results of a diet switch and derivation of diet–tissue-discrimination factors for feathers and blood of captive Garden Warblers (Sylvia borin) and discuss the relevance of our results to current isotopic investigations of migratory songbirds. Methods Avian sample Ours was an opportunistic study that made use of dietswitching experiments planned for Garden Warblers as part of a general nutritional study. We were therefore unable to evaluate the isotope values of the various diets prior to the diet switching. Nonetheless, because our control group of birds were maintained on a constant diet for several months and through feather growth, this did not influence our determination of diet–tissue-discrimination factors per se. Retrospectively, we were able to determine that only two of three diet switches allowed suitable evaluation of turnover rates for each isotope and we report on those two manipulations here. Birds were housed in captivity for 6 months at the Institute of Avian Research, Wilhelmshaven, Germany. Prior to our experiment, birds were maintained on a standardized semi-synthetic control diet (14.2% protein, 10% fat, 5% sugars by wet mass; for details see Bairlein 1986). At the time of flight-feather molt we randomly selected groups of 5 or 6 individuals each representing a control group of birds that were maintained on this original diet and two experimental groups, one that was switched to a diet of meal worms and the other to a diet of black elderberries (Sambucus nigra) supplemented with a small amount (2.5 g) of the control diet. Blood sampling was conducted 0, 2, 4, 9, 16, 23, and 30 days following the diet switch by puncturing the brachial vein and collecting blood in a small capillary tube. Blood was then transferred to vials containing equal volumes of 70% ethanol. Our experiment followed the guidelines recommended by the Canadian Council on Animal Care. Stable-isotope analyses Stable-isotope analyses were performed at the University of Saskatchewan, Saskatoon, Canada. Blood was freezedried and ground to a fine powder in an analytical mill, and then directly analyzed isotopically. Feathers were rinsed in a 2:1 chloroform:methanol solution and air-dried in a fume hood. For diet samples, subsamples were obtained to represent the whole diet, the diet with lipids removed, and the lipids themselves. Lipids were removed following drying using successive rinses in a 2:1 chloroform:methanol solvent. We did not perform lipid extraction on whole-blood samples because of the typically low proportion of lipids in bird blood (see references in Bearhop et al. 2002). Stable carbon and nitrogen isotope assays for all tissues were performed on 1-mg subsamples of homogenized materials by loading © 2003 NRC Canada 1632 Can. J. Zool. Vol. 81, 2003 Table 1. Elemental and isotopic composition of control and experimental diets used in this study of Garden Warblers (Sylvia borin) and the isotopic differences between these diet types. Diet n Control Control minus lipid Control lipid Mealworms Mealworms minus lipid Mealworm lipid Elderberries Elderberries minus lipid Elderberry lipid 6 6 3 6 6 3 6 6 3 %C 48 44.0 69.2 55.8 48.2 74.9 47.4 46.3 36.3 ± ± ± ± ± ± ± ± ± 2.0 1.3 1.0 2.5 4.0 1.2 0.9 1.3 1.0 %N 5.0 6.0 na 8.3 11.8 na 1.5 1.6 na ± 1.0 ± 0.9 ± 1.6 ± 1.3 ± 0.1 ± 0.04 δ13C (‰) ∆δ13C control (‰) δ15N (‰) ∆δ15N control (‰) –25.8 –24.2 –29.1 –28.0 –25.6 –31.4 –28.8 –29.0 –28.1 na na na –2.2 –1.4 –2.3 –3.0 –4.8 +1.0 6.5 6.2 na 4.3 4.6 na 6.7 6.9 na na na na –2.2 –1.6 na +0.2 +0.7 na ± 0.23 ± 0.3 ± 0.2 ± 0.04 ± 0.1 ± 0.3 ± 1.3 ± 0.2 ± 0.2 ± 0.1 ± 0.1 ± 0.1 Table 2. Diet–tissue isotope-discrimination factors (∆δ13C and ∆δ15N) for whole blood and feathers derived from controlled feeding trials of Garden Warblers and comparisons with the results of other studies. Mean diet–tissue discrimination Blood Garden Warbler Yellow-rumped Warbler Dunlin Great Skua Japanese Quail Canvasback Feathers Garden Warbler Yellow-rumped Warbler Great Skua Quail Fish-eating birds ∆δ13C (‰) ∆δ15N (‰) Source 1.7 –1.2 to 2.2 1.3 1.1 to 2.3 1.2 1.5 2.4 1.7 to 3.0 2.9 2.8 to 4.2 2.2 3.0 This study Pearson et al. 2003 Evans-Ogden et al. 2003 Bearhop et al. 2002 Hobson and Clark 1992a Haramis et al. 2000 2.7 1.9 to 4.3 2.1 to 2.2 1.4 3.3 4.0 3.2 to 3.5 4.6 to 5.0 1.6 4.4 This study Pearson et al. 2003 Bearhop et al. 2002 Hobson and Clark 1992a Mizutani et al. 1992 them into tin cups and combusting them at 1800 °C in a Robo-Prep elemental analyzer. Resultant CO2 and N2 gases were then analyzed using an interfaced Europa 20:20 continuous-flow isotope ratio mass spectrometer with every five unknowns separated by two laboratory standards. Stable-isotope abundances are expressed in δ notation as the deviation from standards in parts per thousand (‰) according to δX = [(Rsample/Rstandard) – 1] × 1000, where X is 13C or 15 N and R is the corresponding 13C/12C or 15N/14N ratio. The standard values were based on the Vienna PeeDee Belemnite for δ13C measurements and atmospheric N2 for δ15N measurements. Hundreds of replicate assays of internal laboratory standards (albumen) indicate measurement errors (SD) of ±0.1‰ and ±0.3‰ for stable carbon and nitrogen isotope measurements, respectively. Results Diet–tissue isotope discrimination The results of our isotopic and elemental analyses of dietary samples and how they compared with the control diet are given in Table 1. Birds maintained on the control diet showed remarkably consistent isotope values for blood and feathers while they were grown on this diet (Table 1) and this facilitated accurate estimation of diet–tissue-discrimination factors for these two tissues (Table 2). Because birds switched to a new diet had not reached equilibrium after 30 days, we did not calculate diet–tissue-discrimination factors for diets other than the control. As expected, the lipid component of diets was generally more depleted in 13C than in whole diets or lipid-extracted diets (Table 1). Turnover rates We obtained suitable isotopic dietary shifts to allow us to model turnover rates for one case involving both δ15N and δ13C values. The exponential decay curve Y = Y0 + a × exp(–bt) provided the best fit to the data for the shift from the control to the mealworm diet. Here Y0 represents the initial tissue isotope value, a and b are derived constants, and t is time since the diet switch. To calculate the half-life of each element without risk of pseudoreplication (O’Brien et al. 2000), we fitted exponential decay curves for data for each individual and then averaged these values. We determined a half-life (defined as ln(0.5)/b) of 11.0 ± 0.8 days (n = 5) for δ15N values and 5.0 ± 0.7 days (n = 5) for δ13C values. A similar half-life of 5.7 ± 0.8 days (n = 6) was found for δ13C values following a switch to elderberries, but the negative exponential regression was not significant. For purely illustrative purposes, we depicted element-turnover patterns for nitrogen and carbon by fitting a single decay © 2003 NRC Canada Hobson and Bairlein 1633 Fig. 1. Exponential decay curves for δ15N (A) and δ13C (B) in blood of captive Garden Warblers (Sylvia borin) switched on day 0 from the control diet to the mealworm diet. Error bars represent the standard deviation of the mean for all experimental birds. For illustrative purposes only; half-life estimates given in the text are based on means for all individuals calculated separately. Fig. 2. Exponential decay curves for δ15N (A) and δ13C (B) in blood of captive Garden Warblers switched on day 0 from the control diet to the elderberry diet. Error bars represent the standard deviation of the mean for all experimental birds. For illustrative purposes only; half-life estimates given in the text are based on means for all individuals calculated separately. curve to the means of the combined data for all individuals for the control-to-mealworm switch (Fig. 1; δ15N: r2 = 0.99, F[2,4] = 199.1, p < 0.001; δ13C: r2 = 0.90, F[2,4] = 17.8, p = 0.01) and for the control-to-elderberry switch (Fig. 2; δ13C: r2 = 0.38, F[2,4] = 1.3, p = 0.38). ets more typical of fruit- or plant-based diets than for highprotein (or higher trophic level) diets. This suggests that while general discrimination factors thus far established can be used in cases where birds consume a known plant- or animal-based diet, omnivorous species that switch between diets which differ in elemental composition need to be dealt with using concentration-dependent mixing models (Phillips and Koch 2002). Using the equations of Pearson et al. (2003) relating isotopic discrimination to the percentage of carbon and nitrogen in diets, we found a generally poor fit to our data (∆dt15N = 1.9‰ vs. 2.4‰; ∆dt13C = 0.6‰ vs. 1.7‰) and so further tests of the generality of the effect of elemental composition on discrimination factors are required. In addition to elemental composition, we believe that differential metabolic routing of elements from diet to tissues can contribute to variation in derived diet–tissue-discrimination factors. For example, 13C is typically more depleted in lipids than in other macromolecules (e.g., Table 1). Since carbon from the lipid fraction of the diet may not contribute to the carbon Discussion Diet–tissue discrimination Our study provides additional insight into factors influencing diet–tissue isotope discrimination factors for blood and feathers of birds. As shown in Table 2, these values are generally similar to those found for other species of diverse taxonomy, with the possible exception of domestic quail (Coturnix japonica) raised on a plant-based diet and Great Skuas raised on a beef versus fish diet (Bearhop et al. 2002). Pearson et al. (2003) recently established that isotopediscrimination factors in Yellow-rumped Warblers are sensitive to the elemental composition of diets. Specifically, less discrimination was found for low-carbon or low-nitrogen di- © 2003 NRC Canada 1634 content of proteins in blood or keratin in feathers, differential lipid contents of food can contribute to variation in diet– tissue-discrimination factors. For this reason, it is often difficult to compare the results of studies that do not present the lipid-extracted δ13C values of the diet. Elemental turnover Our opportunistic evaluation of element-turnover rates in the blood of a small passerine confirmed the findings of Pearson et al. (2003), who reported half-life estimates of 3.9–6.1 days for δ13C in Yellow-rumped Warblers. Unfortunately, those authors were unable to establish reliable turnover-rate estimates for δ15N. In our study, the switch from the control diet to the mealworm diet corresponded to a δ15N change in dietary substrate (e.g., ~2‰) sufficient to provide an approximation of 11.0 days for the mean half-life of nitrogen in blood. This value is extremely close to that derived for Japanese Quail (11.4 days; Hobson and Clark 1992b) and captive Dunlin (9.6 and 10.4 days using δ15N and δ13C values, respectively; Evans-Ogden et al. 2003). Bearhop et al. (2002) reported blood half-life estimates of 14.4–15.7 days for Great Skuas and Haramis et al. (2001) reported estimates of 17.6–19.8 days (here derived from their turnover-time constants for whole blood) for Canvasbacks switched to a high-carbohydrate food (celery and tubers) and 25.3–26.3 days for birds switched to a high-protein (clam) diet. These results support the hypothesis that smaller birds with correspondingly higher metabolic rates have a higher element-turnover rate in blood (Tieszen et al. 1983; Pearson et al. 2003). There is considerable interest in using stable-isotope measurements of blood in small migratory passerines arriving in spring in North America and elsewhere to infer geographic location or habitat use by individuals on the wintering grounds. Marra et al. (1998) used isotope measurements of muscle tissue in spring-arriving male American Redstarts (Setophaga ruticilla) to determine that later arriving birds came from more xeric, 13C-isotope-enriched habitats than early arrivals, which apparently occupied more favorable mesic habitats. Their study was based on the assumption that isotopic signals from the wintering grounds were detectable following long-distance migration. Although we provide information only on whole-blood isotope turnover, our results can be used to model the appropriate window during which wintering ground signals might be detectable in such metabolically active tissues in small migratory passerines. Such detectability will depend on the magnitude of the isotopic difference between wintering-ground food webs and those experienced during migration. In addition, it is important to realize that migratory passerines do not have ad libitum access to food but replenish primarily lipid and some protein reserves at stopover sites en route. The isotopic signal available at the spring arrival grounds will depend in part, then, on the number of stopovers the individual has made prior to arrival. Hobson (2003a) has modeled such isotopic changes for a hypothetical migratory bird for varying degrees of protein use en route. Our estimate of relatively rapid turnover rates of elements in small passerines provides some evidence that such isotope tracking will be applicable only for birds moving between biomes with relatively large isotopic differences, and perhaps in as little as just a few weeks. Recently, Can. J. Zool. Vol. 81, 2003 Bearhop et al. (2003) suggested that isotopic measurements of bird claws can provide more useful information on wintering-ground biomes, owing to their slow growth rates. The magnitude of the carbon-isotope shift from the control diet to the experimental diets was relatively small compared with results from previous studies using the shifts from C-3 to C-4 (Hobson and Clark 1992b) or from terrestrial to marine sources (Bearhop et al. 2002). Our estimate of a half-life of about 5–5.7 days for carbon in blood, derived from two independent dietary-shift experiments, should therefore be considered with caution. However, such estimates concur with those made by Pearson et al. (2003). If these relatively rapid turnover estimates are accurate, they may correspond to a decoupling of the nitrogen and carbon metabolic pathways during blood-cell formation. For dietary protein, a macromolecule readily incorporated into the body protein of the consumer, we can expect reasonably close coupling between patterns of turnover of carbon and nitrogen. However, for carbohydrates and fats, metabolic pathways for carbon and nitrogen can become decoupled (Hobson and Stirling 1997; Hobson et al. 2000a). The faster uptake of carbon following the switch to a mealworm diet may reflect more immediate metabolism of the lipid component of this new diet compared with slower uptake of proteins. Similarly, the switch to elderberries from the control diet could reflect rapid uptake of new carbon from the carbohydrate versus the protein component of this fruit diet. If true, our results have significant implications for tracing the importance of frugivory in migratory birds. In such studies, reliance on δ15N values to indicate a trophic shift from insects to fruit may underestimate the relative importance of fruits in the weeks following blood sampling (e.g., Hererra et al. 2001). Pearson et al. (2003) similarly advised caution, based on expected changes in isotope-discrimination factors, as birds may switch between high- and low-nitrogen diets. The results of our study underline the need for further experimental manipulations of the diet of captive birds to evaluate the intricacies of how dietary substrate influences both diet–tissue isotopic discrimination and elemental turnover in blood. Currently, we still do not have a good idea of how metabolic-rate changes expected during migration or other energetically taxing periods for wild birds influence these parameters. Hobson (2003b) recently suggested that isotopic manipulations of the diet of birds conditioned to use a wind tunnel would be an optimal means of investigating these questions. Acknowledgments This study was supported in part by an operating grant to K.A.H. from the Canadian Wildlife Service. We thank Patricia Healy for assistance with stable isotope sample preparation. Stable-isotope analyses were performed at the Department of Soil Science, University of Saskatchewan, Saskatoon. 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