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Turnover rates of stable isotopes in avian blood and toenails: Implications for dietary and migration studies
Journal of Experimental Marine Biology and Ecology 472 (2015) 89–96
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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Turnover rates of stable isotopes in avian blood and toenails: Implications for dietary and migration studies Pedro M. Lourenço a,⁎, José P. Granadeiro b, João L. Guilherme c, Teresa Catry a a b c
Centro de Estudos do Ambiente e do Mar (CESAM)/Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Lisboa, Portugal Centro de Estudos do Ambiente e do Mar (CESAM)/Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Lisboa, Portugal
a r t i c l e
i n f o
Article history: Received 14 November 2014 Received in revised form 7 July 2015 Accepted 8 July 2015 Available online xxxx Keywords: Calidris alpina Captive study Diet switch Isotopic discrimination Half-life Shorebirds
a b s t r a c t Most stable isotope (SI) applications in avian ecology are based on the analysis of feathers and blood, whereas toenails are much less used. These structures grow slowly and continuously, thus integrating information over comparatively longer periods which may be useful for migration connectivity studies, while avoiding some of the difficulties posed by incomplete information on feather molt. In spite of this, interpretation of data from toenails is limited by lack of accurate figures on their turnover rates and discrimination values. To improve our understanding of SI ratios in toenails and surpass these drawbacks we measured the change in carbon (δ13C) and nitrogen (δ15N) SI ratios in plasma, red blood cells and toenails of captive dunlins Calidris alpina after a controlled diet switch. Discrimination values were estimated by comparing isotopic values at the end of the experiment with those of artificial food. We also estimated toenail growth rates in this species. Toenails showed much lower SI turnover rates than blood components, with half-lives of 27 days and 35 days for δ13C and δ15N, respectively. Isotopic ratios in toenails reached equilibrium with the new diet after 100–120 days, roughly coinciding with the duration of toenail replacement. The discrimination values of 2.74 ± 0.68‰ for δ13C and 4.06 ± 0.27‰ for δ15N found for toenails are higher than those of both blood components. These values are also higher than most published values for blood in other avian species, but similar to those previously reported for feathers which, like toenails, are metabolically inert after synthesis and are mainly composed of keratin. This study highlights the potential role of toenails as sources of SI data for ecological studies, particularly to determine geographic origin of birds migrating between isotopically distinct environments. Indeed, their long turnover rates might allow for the detection of SI signals from the wintering or breeding ranges, by sampling individuals at their staging sites. Isotopic turnover in toenails follow their replacement rates, and these seem to be rather similar across several avian taxa. Thus, the turnover rates described here may potentially be used to interpret SI data for other birds. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Stable isotope (SI) ratios are increasingly used in ecological studies, particularly to study trophic interactions and migratory connectivity (Rubenstein and Hobson, 2004; Inger and Bearhop, 2008). Isotopic ratios in body components reflect the diet of an animal during their synthesis, which means that after a diet switch these ratios will progressively converge towards the isotopic ratio of the new diet (Hobson and Clark, 1992a; Bearhop et al., 2002). The rate at which this process occurs varies among different tissues and structures of an animal (Tieszen et al., 1983; Bearhop et al., 2002; Klaassen et al., 2010), a feature that ⁎ Corresponding author at: Museu Nacional de História Natural e da Ciência, Rua da Escola Politécnica 58, 1250-102 Lisboa, Portugal. E-mail address: [email protected] (P.M. Lourenço).
http://dx.doi.org/10.1016/j.jembe.2015.07.006 0022-0981/© 2015 Elsevier B.V. All rights reserved.
has been used to gather information on dietary changes (and/or migratory movements) over a particular time window. Many studies of avian migration have derived their conclusions from the analysis of SI in feathers (Hobson and Wassenaar, 1996; Inger and Bearhop, 2008), relying on their inert nature which depicts the isotopic landscape where the feather was grown. Although feathers are a very useful source of isotopic information, interpreting their SI signature requires detailed knowledge of the timing and “geography” of the molt process (Ramos et al., 2009), which is not available for many species. Also, in many migratory birds, feather molt may occur over a rather protracted period, across several different locations (Battley et al., 2006), which may preclude assigning a given feather to a particular location. Blood (either whole blood, or separating plasma and red blood cells) is used to determine arrival dates and residence periods at stopovers, as this metabolically active tissue has a fast turnover rate
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thus providing information over a short time window of a few days to weeks before sampling (Hobson and Barlein, 2003; Klaassen et al., 2010; Oppel and Powell, 2010). Stable isotopes in bird toenails received comparatively rather less attention than in both feathers and blood. Toenails grow continuously and a few studies suggest that they integrate isotopic information over a time window of several weeks to a few months (Bearhop et al., 2003), making them ideal, for instance, to infer the geographic origin of migrant birds (Clark et al., 2006; Catry et al., 2012; Hopkins et al., 2013). Despite their potential as a target structure for SI work on avian migration, very few studies attempted to estimate SI turnover rates in bird toenails. The proposed time window for the information this structure integrates was estimated based on toenail growth rates (Bearhop et al., 2003; Hahn et al., 2014) and the only study we are aware of that attempted to measure the turnover rate of SI in toenails through a controlled diet switch experiment reported no shift in isotopic values even after five months (Barquete et al., 2013). Also, nail growth in higher vertebrates is a complex and not necessarily linear process (Ethier et al., 2010; Hahn et al., 2014), and some evidence suggests that toenail growth during migration may limit the use of SI data in toenails for determining breeding and wintering origins in ground foraging birds due to differences between the base and the tip of the toenail (Mazerolle and Hobson, 2005). These issues emphasize the need for more controlled experiments in order to correctly interpret SI data from bird toenails. The isotopic discrimination values, i.e. the difference between SI ratios in animals and in their diet, can also vary among tissues and structures of an animal (Hobson et al., 1996; Bearhop et al., 2002). These differences may constrain the analysis and interpretation of SI data (Bond and Diamond, 2011), but have not been described for bird toenails, limiting the use of these structures for dietary studies. In order to enhance our ability to interpret SI data derived from bird toenails, we studied the change in carbon (δ13C) and nitrogen (δ15N) stable isotope ratios of captive dunlins Calidris alpina (L.) after a controlled diet switch, aiming to 1) describe the turnover rates of δ13C and δ15N in toenails; 2) compare the turnover rates of δ13C and δ15N in toenails, plasma and red blood cells; and 3) calculate the isotopic discrimination values between diet and toenails.
2. Material and methods 2.1. Bird capture and experimental procedure We captured 40 dunlins at a high-tide roost in the southern bank of the Tejo estuary, Portugal (38°49′N, 8°57′W), using mist-nets. Birds were caught in four different events during the highest spring tides, between 20-Nov-2013 and 5-Jan-2014. Post-breeding migrant dunlins arrive in August–September in the Tejo estuary (Alves et al., 2011), where they feed mainly on the polychaete Hediste diversicolor Malmgren, the bivalve Scrobicularia plana (da Costa) and the gastropod Hydrobia ulvae (Pennant) (Santos et al., 2005; Martins et al., 2013). Therefore, we assumed that dunlins had been on this diet for ca. 3–4 months before being captured so that they were in equilibrium with the SI values of their prey. After capture, all birds were weighed (to the nearest 0.1 g), and ringed with an individual combination of one metal and four color rings. We collected the distal portion from all toenails (ca. 2 mm long) on the right foot of each bird. We also made a small incision near the base of the central toenail on the left foot, using a scalpel, and the distance between the incision and the base of the toenail was measured to the nearest 0.1 mm with a caliper, in order to estimate toenail growth rate during the experiment. For a subsample of eight birds (two groups of four individuals captured in two different dates) we also collected 150 μl of blood from the brachial vein. Blood was centrifuged for 10 min at 3400 rpm within 2 h of collection, in order to separate the
cellular and plasma components. The separated blood components were frozen at −20 °C until further analysis. Within a few hours from capture, birds were transported by car in cardboard boxes to the housing facilities of the Lisboa Wild Animal Recovery Centre (LxCRAS), located in the Monsanto Forest Park, a large forested area within the city of Lisboa, ca. 50 km from the catching area. This facility provided advice as well as logistic and veterinary support, whenever necessary. Birds were divided in three groups, each kept in an outdoor aviary (roughly l × w × h: 3 × 2 × 2 m) under natural light and temperature conditions. The ground was covered with coarse sand to mimic a natural substrate, and each aviary had a pool with sloping banks where the birds could drink and bathe. The pool was filled with fresh water and frequently flushed to minimize the risk of diseases. The walls of the cages were padded with a soft mesh to minimize the risk of injury and visits to the cages were kept to a minimum to avoid stressing the birds. During the whole captive period birds were fed ad libitum with trout pellets (T-2 Optiline-sf 1P; Skretting España S.A., Spain), which have a different isotopic value from that of the natural prey taken by birds in the estuary (see results). These pellets are manufactured from soy, fish meal, rapeseed flour, fish oil, vegetable oil, wheat, vegetable proteins, vitamins and minerals, and most of their macromolecular composition consists of protein (44%) and fat (21%). All used pellets originated from a single stock, so differences in SI values among pellets should be minimal. Food was placed in several trays per aviary and always mixed with some water to soften the pellets. During the first 2–3 days of captivity we also used live mealworms Tenebrio molitor L. to train birds to feed from the trays. Birds that were sampled for blood were only given mealworms in the first day of captivity to minimize any effect on blood isotopic values due to its fast turnover rate. To evaluate temporal variation in SI values in toenails, groups of five birds were sequentially sampled over increasing periods of exposure to the new diet. The first group was sampled after 10 days, the second after 22 days, the third after 34 days and the remaining groups each after an additional 15 days, with the last group being sampled 109 days after capture. In each case, sampling was performed as at capture, but toenails were collected from the left foot. The eight birds initially sampled for blood were subsequently resampled after 3, 6, 11, 16, 21, 26, 34, 41, 48 and 55 days of captivity. During the experiment, all birds were weighed at least once every two weeks and every time they were sampled, in order to monitor their response to artificial food. Simultaneously, to monitor toenail growth, we measured the distance between the incision made upon capture and the base of the toenail to the nearest 0.1 mm. In a subsample of birds we also measured the total length of the central toenail at the end of the captive period, just before sampling the toenail for SI. For comparison purposes, we also measured the total length of the central toenail on the left foot of 28 dunlins caught in the Tejo estuary, in April 2014. After the final toenail/blood sampling each group of birds was released back in the estuary, during low tide, in an area where dunlin flocks are frequently seen foraging near the coast. This experiment was carried out in strict accordance with the current national ethical and legal regulations. The protocol was approved by the responsible ethical and legal authority, the Portuguese Institute of Nature Conservation and Forests (ICNF) and performed under official permits 385/2013/CAPT, 386/2013/CAPT and 387/2013/ CAPT. In order to compare SI values in bird blood and toenails with that of their natural food, we collected 10–20 individuals of their three main prey, with sizes within the range known to be consumed by dunlin (H. ulvae, all available sizes; H. diversicolor, 15–50 mm total length; and S. plana, 4–8 mm shell width) in a mudflat located 1 km south of the roost where the birds were captured and thus a likely foraging location (Dias et al., 2006). The animals were kept in estuarine water for 24 h to release their gut contents and afterwards divided in 2–3 samples and frozen until further analysis. A 2 g sample of the trout pellets
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used during the experiment and 10 mealworms were also stored for SI analysis at the beginning of the experiment.
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3. Results 3.1. Stable isotope values of food samples
2.2. Stable isotope analysis Toenail samples were washed in double baths of 0.25 N sodium hydroxide solution alternated with baths of double distilled water to remove adherent contamination (Catry et al., 2012). All samples of blood, toenails and food items were dried at 50 °C for 48 h and then ground to powder and homogenized. In order to extract lipids, food samples were immersed in a 2:1 chloroform/methanol solution with a solvent volume 3–5 times larger than sample volume (Logan et al., 2008). Samples were then mixed for 30 s, left undisturbed for approximately 30 min, centrifuged for 10 min at 3400 rpm, and the supernatant containing solvent and lipids was removed. We repeated this process at least three times until the supernatant was clear and colorless after centrifugation. Samples were re-dried at 60 °C for 24 h to remove any remaining solvent. Subsamples of H. ulvae and S. plana were acidified with several drops of 10% hydrogen chloride to remove carbonates from any shell remains. After bubbling stopped, the sample was centrifuged, the acid removed and, as a final step, the sample was washed with distilled water. This process was repeated three times (Vinagre et al., 2008). A non-acidified subsample was kept for nitrogen isotope analysis. Between 0.60 and 1.20 mg of each sample was stored in tin cups and combusted at 1000 °C in a Euro EA Elemental Analyzer, at the Stable Isotopes and Instrumental Analysis Facility of the Faculty of Sciences, University of Lisbon. Resultant CO2 and N2 gases were analyzed using a continuous-flow isotope ratio mass spectrometer IsoPrime (MicroMass), with unknowns separated by laboratory standards. Results are presented conventionally as δ values in parts per thousand (‰) relative to the Vienna Pee Dee Belemnite (PDB) for δ13C, and atmospheric nitrogen (N2) for δ15N, with a precision of 0.11 to 0.25‰ for δ13C and 0.05 to 0.17‰ for δ15N (SD), calculated from standards. 2.3. Data analysis We used Generalized Linear Mixed Models (GLMM) through R package ‘lme4’ (Bates et al., 2014) to analyze toenail growth rates and the variation in body mass during the experiment, using individual as a random factor to take into account the fact that birds were sampled repeatedly while in captivity and for a different number of times. We used t-tests to compare the average length of toenails in birds at the end of captivity with that of birds captured in the wild. We compared SI values and isotopic discrimination values from different sample types (red cell, plasma and nails) and dates, using Kruskal–Wallis and Mann–Whitney tests. The change in the isotopic value of an animal following a diet switch can be described by an exponential decay curve as y = a.e− bx + c, where y is the isotopic value of the sampled body component at time x after the diet switch (in days), a is the change in isotope values associated with the change in diet, c is the isotope value at x = ∞ (the difference between isotope values at equilibrium and those at x = ∞ are likely to be very small, so this equals to the asymptotic condition), and b is the fractional turnover rate (Bearhop et al., 2002; Hobson and Barlein, 2003; Ogden et al., 2004). Turnover rates for δ13C and δ15N were estimated for toenails and both blood components by fitting the general equation using least squares nonlinear regression in the non-linear fitting module of Statistica 8.0. The half-life was calculated using −ln(0.5)/b for each case (Bearhop et al., 2002). The isotopic discrimination values (Δdt) of δ13C and δ15N for toenails and both blood fractions were calculated using the equation: Δdt = δtissue − δdiet, based on the SI signals from the last sampling dates. Unless stated otherwise, all statistical analyses were performed in R 2.14 (R Development Core Team, 2012). Throughout the text data are presented as means ± SD, unless stated otherwise.
The three invertebrate prey species collected in the estuary had similar isotopic values (H. diversicolor: δ13C − 14.6 ± 0.4‰ and δ15N 14.6 ± 0.3‰ (n = 3); S. plana: δ13C − 14.8 ± 0.3‰ and δ15N 14.8 ± 0.4‰ (n = 2); H. ulvae: δ13C − 12.9 ± 0.5‰ and δ15N 14.9 ± 0.7‰ (n = 3)), which were clearly different from the values of the trout pellets used during the experiment (δ13C − 23.4‰ and δ15N 5.3‰), with both isotopes showing more enriched values in the estuarine invertebrates. The mealworm isotopic values (δ13C −27.2‰ and δ15N 8.3‰) were also impoverished when compared to the estuarine invertebrates.
3.2. Body mass and toenail growth rates Dunlins had an average body mass of 46.6 ± 4.1 g (n = 40) at capture, and most birds lost weight in the first days after capture, reaching a minimum average body mass of 42.5 ± 6.7 g (n = 12) after three days in captivity. Afterwards they began gaining weight, eventually stabilizing about 12 days after capture at an average body mass of 51.0 ± 3.7 g (n = 14). There was no significant change in average body mass from day 12 until the end of the experiment (GLMM: t = 2.02; P N 0.1; R2 = 0.01). Toenail length, measured as the distance between the incision and the base of the toenail, increased linearly during the experiment (GLMM: t = 58.6; P b 0.01; R2 = 0.96), at a rate of 0.041 mm·day−1 (± 0.001 SE; Fig. 1). The average total length of the central toenail measured at the end of the captivity period was 4.9 ± 0.4 mm (range: 4.1–5.7; n = 29), indicating that it takes 120 days (i.e. 4.9 mm divided by a growth rate of 0.041 mm·day−1) to replace the whole toenail. However, the average total length of the central toenail in dunlins caught in the Tejo estuary in April 2014 was 4.3 ± 0.5 mm (range: 3.0–5.3; n = 28), which is significantly lower than in captivity (t = 4.4; n = 29 in captivity and 28 in the wild; P b 0.001). Assuming the same growth rate, it would take 105 days to replace the whole toenail in the wild. The two groups did not vary in terms of wing size (t = 1.74; n = 29 and 28; P N 0.05) or tarsus length (t = 1.41; n = 29 and 28; P N 0.10).
Fig. 1. Toenail length, measured as the distance between the incision and the base of the toenail over the captivity period. The black line is the linear fit between toenail length and time evidencing an average growth rate (i.e. slope) of 0.041 mm·day−1.
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3.3. Isotopic value of toenails, plasma and red blood cells of dunlins There were no significant differences among the initial SI values of birds caught at different dates in either toenails (Kruskall–Wallis test (KW) — δ13C: H(3,40) = 4.16; P N 0.10 and δ15N: H(3,40) = 4.78; P N 0.10) or red blood cells (Mann–Whitney test (MW) — δ13C: Z = 2.01; P N 0.05 and δ15N: Z = 1.73; P N 0.10). There were also no significant differences in δ15N in plasma among catching dates (MW: Z = 1.73; P N 0.10), although the difference in δ13C in plasma between catching dates was marginally significant (MW: Z = 2.30; P = 0.042). In animals sampled both for toenails and blood (Table 1), we found no significant differences in δ15N among different sample types (KW: H(2,24) = 1.99; P N 0.10) at the time of capture, but toenails tended to have higher average values. The value of δ13C varied among sample types (KW: H(2,24) = 8.02; P b 0.05), with significantly higher values in toenails in relation to plasma. After the diet switch, δ13C and δ15N values in all sampled body components declined towards the values observed in the trout pellets, showing a strong fit to the exponential decay curves (Table 2). The turnover rate in plasma was the highest, with half-lives of 1.3 days (1.0–2.2; 95% CI) for δ13C and 2.8 days (2.5–3.2; 95% CI) for δ15N (Fig. 2A). Turnover in red blood cells was intermediate, with half-lives of 8.6 days (7.2–10.8; 95% CI) for δ13C and 10.2 days (9.1–11.4; 95% CI) for δ15N (Fig. 2B). Toenails had the lowest turnover rate with half-lives of 26.7 days (19.8–40.8; 95% CI) for δ13C and 34.7 days (26.7–46.2; 95% CI) for δ15N (Fig. 2C). Turnover tended to be faster for δ13C than for δ15N (Fig. 2), but the 95% confidence intervals for each isotope overlapped in both red blood cells and toenails. The isotopic discrimination (Δdt) of captive birds showed significant differences between sample types (KW: δ13C: H(2,21) = 15.7; P b 0.001 and δ15N: H(2,21) = 17.6; P b 0.001), with higher values for toenails (Table 3). Both blood fractions had significantly lower Δ13C than toenails, but only the red blood cells had a significantly lower Δ15N than toenails. The Δ15N estimated for birds on a natural diet at capture in relation to average values for their three main prey, are very similar to those derived from the captivity experiment (plasma: 3.52 ± 1.64‰, n = 8, MW: Z = 1.34, P N 0.10; red blood cells: 3.20 ± 0.82‰, n = 8, MW: Z = 1.60, P N 0.10; toenails: 3.37 ± 1.32‰, n = 40, MW: Z = 0.52; P N 0.5). Surprisingly, Δ13C values for birds on a natural diet were negative and significantly different from those obtained during the experiment (plasma: − 3.83 ± 1.28‰, n = 8, MW: Z = 3.00, P b 0.01; red blood cells: − 2.25 ± 0.60‰, n = 8, MW: Z = 3.24, P b 0.01; toenails: −1.85 ± 1.88‰, n = 40, MW: Z = 3.27, P b 0.01). 4. Discussion In this study we provide novel information concerning estimated SI turnover rates of several body components in shorebirds under captivity, with particular emphasis on toenails, a continuously-growing structure. By sampling individuals over increasingly longer periods of exposure to an isotopic distinctive food source, we were able to establish a “time-response” curve of δ13C and δ15N, a key calibration tool to use these structures in ecological studies and particularly in ascertaining the geographic origins of migratory species. Birds caught in the wild at different dates all showed similar isotopic values, with the exception δ13C in plasma, strongly suggesting that they were in
Table 1 Average (±SD) δ13C and δ15N (‰) values in plasma, red blood cells and toenails of eight dunlins sampled at the time of capture. Different letters in superscript indicate significant differences (P b 0.05) between samples types in post-hoc Dunn tests. Sample type Plasma Red blood cells Toenails
Δ13C
Δ15N a
−18.95 ± 2.62 −17.35 ± 2.33ab −15.86 ± 1.08b
17.66 ± 2.32a 16.73 ± 2.38a 18.16 ± 1.32a
Table 2 Turnover rates of δ13C and δ15N in plasma, red blood cells and toenails of captive dunlins after the diet switch, obtained from the fitted exponential decay models. We present the turnover rate (day−1), 95% confidence intervals (95% CI), model fit and the percentage of variation in the data explained by the model (Expl. variance). (***P b 0.001). Sample type
Isotope
Turnover rate
95% CI
Model fit
Expl. variance
Plasma
δ13C δ15N δ13C δ15N δ13C δ15N
0.522 0.244 0.081 0.068 0.020 0.026
0.315–0.730 0.215–0.273 0.064–0.097 0.061–0.076 0.017–0.035 0.015–0.026
T84 = 5.0*** T84 = 1.7*** T84 = 9.7*** T84 = 16.7*** T80 = 5.9*** T80 = 7.5***
68% 92% 72% 89% 60% 67%
Red blood cells Toenails
equilibrium with their natural diet in the Tejo estuary. As expected, both toenails and the two blood components exhibited a decline in δ13C and δ15N values towards the isotopic value of the artificial food, but the turnover rates were different among sample types. The two blood components showed a much higher turnover rate than toenails, in line with previous studies (Bearhop et al., 2003; Ogden et al., 2004; Oppel and Powell, 2010), and we now have reliable estimates for the half-lives of δ13C and δ15N in dunlin toenails: 27 and 35 days, respectively, which represents the time-window to which isotopic information obtained from toenails refers to. Our data suggest that the isotopic value of dunlin toenails reach equilibrium with the new diet after 100–120 days, which fits very well with our observations of toenail growth rates. Toenail growth in captive dunlins (0.041 ± 0.001 mm·day− 1) is very similar to that measured in several European passerines (0.04 ± 0.01 mm·day−1 in Bearhop et al., 2003; 0.034 ± 0.001 mm·day−1 in Hahn et al., 2014). Although smaller than dunlins, these passerines have similar sized toenails (range 3.8–5.3 mm in Bearhop et al., 2003) indicating that the time required to replace a whole toenail should be similar. The much larger African penguin Spheniscus demersus has also been recorded to require 126 days to replace much larger toenails (16.1 ± 1.6 mm, Barquete et al., 2013) hinting for some consistency across avian taxa in the time required to replace toenails and, potentially, in the time required for toenail isotopic values to reach equilibrium with their diet. The fact that all these studies show a linear growth of the toenail while the isotope depletion curves are better modeled as negative exponentials, and that we can detect change in isotopic values just a couple of weeks after the dietary shift (even though we only sample the distal half of the nail), both indicate that not all parts of the toenail grow equally. Actually, avian toenails show conical growth (i.e. both longitudinal and lateral) which causes any part of the toenail to contain a blend of materials formed at different times (Hahn et al., 2014). Also, our toenail growth estimate only refers to the larger toenail. Since the smaller toenails are likely to have similar growth rates (Bearhop et al., 2003), this means they will be replaced faster influencing the incorporation of isotopic signals. However, due to their very small size, several nails had to be sampled from each individual to achieve the mass required for the isotopic determination, thus precluding any analysis of how the nails from different toes incorporate isotopic information. The length of the central toenail in wild caught dunlins was on average lower than that observed in our captive birds at the end of the experiment. Since this disparity is not likely to be due to allometric differences (as the two groups had similar wing length and tarsus length), it seems likely that captive dunlins suffer less wear to their toenails because they have easier access to food and spend much less time walking about. This suggests that it is likely we slightly overestimated the time dunlins require to replace their toenails. There are no published data on the half-lives of stable isotopes in each of the two blood components in dunlins. One study showed that the half-lives of δ13C and δ15N in whole blood samples of dunlins were similar to what we found for the red blood cell fraction (Ogden et al., 2004; Table 4). The half-lives of δ13C in captive red knots Calidris canutus
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Fig. 2. Temporal variation of δ13C and δ15N values in the plasma (A), red blood cells (B) and toenails (C) of captive dunlins sampled at increasing periods after the diet switch. In each sampling date black squares represent the average and whiskers show the standard deviation. The black curves are the fitted exponential decay models used to estimate turnover rates (see Table 3 for more details). The gray horizontal lines indicate the isotopic value of the artificial food pellets.
were found to be higher than our estimates for dunlin (Klaassen et al., 2010) and the same is true for the half-lives of δ15N in African penguins (Barquete et al., 2013) and of both isotopes on whole blood samples of great skua Catharacta skua (Bearhop et al., 2002; Table 4). In fact, the short half-lives recorded in our study are more similar to what is described for passerines such as the yellow-rumped warbler Dendroica coronata (Pearson et al., 2003; Podlesak et al., 2005) and the garden warbler Sylvia borin (Hobson and Barlein, 2003; Table 4).
Table 3 Isotopic discrimination values (Δdt) of δ13C and δ15N (‰) in plasma, red blood cells and toenails of captive dunlins in relation to the artificial diet used during the experiment. For both blood components n = 8 birds, while for toenail n = 4 birds. Different letters in superscript indicate significant differences (P b 0.05) between sample types in post hoc Dunn tests. Sample type Plasma Red blood cells Toenails
Δ13C
Δ15N a
0.32 ± 0.16 0.94 ± 0.24a 2.74 ± 0.68b
3.30 ± 0.20ab 2.30 ± 0.28a 4.06 ± 0.27b
Dunlins are small shorebirds, roughly 1/3 the mass of a red knot, and consequently have a higher metabolic rate (Kersten and Piersma, 1987). Several studies have suggested that isotopic turnover rates are correlated with metabolic rate and body size (Bearhop et al., 2002; MacAvoy et al., 2006), an indirect relationship probably mediated by protein turnover (Carleton and Martínez del Rio, 2005), which may explain why the isotopic turnover rate in dunlins is more similar to a passerine than to its larger congener or other larger birds. In our study, the turnover rate of δ13C tended to be faster than that of δ15N, a result consistent with some studies (Hobson and Barlein, 2003; Pearson et al., 2003), but not with others (Bearhop et al., 2003; Ogden et al., 2004). Such differences in the incorporation of the isotopic ratios of each element into animal tissues can be related with diet quality and the C:N ratio in food (Mirón et al., 2006), so any comparison between the turnover rates of different isotopes in different experiments would only be valid if the same food was used in all cases. The observed discrimination values for δ15N in our dunlin samples lay within the range 2–4‰ that have been described in most SI studies (Bearhop et al., 2002; Inger and Bearhop, 2008). Toenails tended to have higher Δ15N than blood, although this difference was only
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Table 4 Half-lives of δ13C and δ15N (in days) in blood samples of a range of bird species with different body masses. Body mass ranges (in g) were obtained from (del Hoyo et al., 2013). Species are ordered by increasing body mass. Species
Body mass
Analyzed tissue
Half-life of δ13C
Half-life of δ15N
References
D. coronata
10–17
Pearson et al. (2003)
10–17 16–22 33–85
C. alpina C. canutus
33–85 85–220
C. skua S. demersus
1100–1700 2100–3700
0.4–0.7 3.9–6.1 1.0 5.0–5.7 1.0–2.2 7.2–10.8 11.2 ± 0.8 6.03 15.07 14.4
0.5–0.7 7.5–27.7
D. coronata S. borin C. alpina
Plasma Whole blood Plasma Whole blood Plasma Red blood cells Whole blood Plasma Red blood cells Whole blood Plasma Red blood cells
significant when compared with red blood cells. The only study that compared discrimination values between blood and toenails actually found a lower Δ15N in toenails (Barquete et al., 2013), which could indicate that our values were overestimated, possibly because our last measurements were yet to reach asymptotic values. However, the fact that δ15N values show virtually no change between the two last sampling dates suggest this is not the case (after 94 days: 9.69 ± 1.14‰, n = 4; after 109 days: 9.48 ± 0.27‰, n = 4). Also, the Δ15N at the end of the experiment was similar to what was estimated for birds on a natural diet, supporting the idea that our estimates for Δ15N in toenails are not biased. Stable carbon isotope ratios usually undergo fairly limited increase between trophic levels, in most cases with Δ13C falling in the range 0–1‰ (Inger and Bearhop, 2008), which is consistent with what we observed for the two blood fraction in dunlins. However, the observed value for toenails was again significantly higher. Although in the case of δ13C the estimated values for birds on a natural diet were not consistent with what was observed during the experiment, as dunlin samples at capture had lower δ13C than their main prey, Δ13C was also higher (in this case less negative) for toenails at capture. These unexpected negative discrimination values possible derive from the fact that we cannot measure discrimination in the wild as precisely as in captivity, because the natural diet may suffer variations and natural prey may have spatial–temporal patterns of variation in their signatures that may scramble the SI values. To our knowledge, no studies have compared Δ13C in toenails and blood, but work in great skuas (Bearhop et al., 2002) and garden warblers (Hobson and Barlein, 2003) have reported higher Δ13C and Δ15N in feathers than in blood, with values for feathers (Δ13C: 2.1–2.7‰, Δ15N: 4.0–5.0‰.) that are similar to what we describe for toenails in dunlins. Studies in king Aptenodytes patagonicus and rockhopper Eudyptes chrysocome penguins (Cherel et al., 2005) and in California condors Gymnogyps californianus (Kurle et al., 2013) have also reported higher discrimination values in feathers than in blood, although not as high as we found in dunlin toenails. On the other hand, no difference was observed in Japanese quails Coturnix japonica and ring-billed gulls Larus delawarensis (Hobson and Clark, 1992b). Avian feathers and toenails are structures that become metabolically inert after being synthesized and are both mainly composed of keratin (Gill, 2007), so it seems reasonable to assume that they will have similar isotopic discrimination values, which in many cases seem to be higher than that of blood. 4.1. Implications for the use of isotopic data from avian toenails in ecological studies Feathers and blood can be collected without excessive intrusion to birds, and to a large extent this explains the extensive use of these body components in studies of avian ecology. However, toenails can
11.0 2.5–3.2 9.1–11.4 10.0 ± 0.6
15.7 ± 2.1 7.6 ± 0.7 14.3 ± 1.6
Podlesak et al. (2005) Hobson and Barlein (2003) This study Ogden et al. (2004) Klaassen et al., 2010 Bearhop et al. (2002) Barquete et al. (2013)
also be easily collected and may have a greater potential for the study of avian migration, particularly for determining the geographic origin of migratory birds. Toenails integrate isotopic information over a longer period than blood and, unlike feathers, their constant growth can deliver information during the whole yearly cycle. This means that isotopic values of the distal half of toenails from birds at a staging area are likely to be related with those found at the wintering or breeding areas from where they originate. Mapping the isotopic values at several potential breeding and wintering areas can help us identify their most likely geographic origin (Catry et al., 2012). Of course, such data needs to be interpreted carefully, as populations with uncommonly short breeding or wintering periods may not stay in an area long enough to fully incorporate the local isotopic values, while populations with very long intermediate staging periods prior to capture may mask the original SI values. Still, due to the asymptotic nature of the incorporation of isotopic information into body tissues, this should only be a problem in rather extreme cases. In order to apply toenail SI data in ecological studies, we require an in-depth knowledge on the rate at which isotopic information is incorporated, as well as on how isotope ratios change between ingested food and toenails. Initial work with SI in toenails assumed that the turnover rate would follow the growth rate of the toenails (Bearhop et al., 2003), but there was no empirical data to confirm this and the only experimental work on this topic was unable to detect isotopic turnover in toenails (Barquete et al., 2013). Our data shows a turnover rate of 0.020.day− 1 for δ13C and 0.026.day− 1 for δ15N in dunlin toenails, which indicate a period of about four months to reach asymptotic equilibrium values with their diet, translating into half-lives of about one month. Since toenail growth data also indicate a period of about four months to replace the whole toenails in dunlins, this confirms that the isotopic turnover in avian toenails indeed follows the growth rate of the nails, although one process is linear and the other is not. Moreover, in species belonging to three widely distinct avian groups (passerines, shorebirds and penguins) the time required to replace toenails is remarkably similar. Even though more data would be required to support this assumption, and toenail wear possibly varies considerably among species with different behavior and habitat use, we can speculate that the turnover rates we measured and the four months window for incorporation of isotopic information into toenails can be used when interpreting SI data for other bird species. Although our study design offers no insights into isotopic variations within individual toenails, previous studies have shown that such differences can be found in continuously growing structures such as hairs, teeth, baleen, otholits and mammalian nails, with potential applications for the study of temporal variations in SI values (revision in Dalerum and Angerbjȯrn, 2005). In fact, at least one study reported isotopic differences between the tip and base of toenails in birds (Mazerolle and Hobson, 2005), which may indicate there is some potential for
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using different sections of avian toenails to study temporal variations in diet or foraging location. However, since avian toenails grow both longitudinally and laterally (Hahn et al., 2014), such data needs to be treated with great caution until further experiments determine precisely how isotopic differences within individual toenails are originated. Toenail isotopic values appear to have a higher discrimination value than that of blood, and these values may fall above the usual figures used in most dietary studies, particularly in the case of δ13C. This is an important consideration for interpreting SI data from avian toenails, particularly in dietary studies. However, looking through the literature, it is clear that discrimination values vary considerably among species, tissues and diets (Hobson and Clark, 1992b; Pearson et al., 2003; Podlesak et al., 2005). Since we only studied individuals of a single species being fed a single diet, we must recommend caution when using the discrimination values found for dunlin toenails in other species. Glossary
Half-life the amount of time required for the amount of something to fall to half its initial value. Half-life is used to describe a quantity undergoing exponential decay, and is constant over the lifetime of the decaying quantity. High-tide roost locations, such as beaches, marshes or saltpans, where shorebirds and other intertidal foraging birds rest during the periods when their foraging areas are submerged by the tide. Isotopic discrimination enrichment of one isotope relative to another in a chemical or physical process. Migratory connectivity a term used to describe the relationship between populations of animals (especially birds) and geographic locations at different time points during the year. Stable isotope An isotope (i.e. one of several nuclides having the same number of protons in their nuclei, and hence having the same atomic number but differing in the number of neutrons) of an element that shows no tendency to undergo radioactive breakdown. Acknowledgments We would like to thank LxCRAS, and particularly Nuno Ventinhas, Manuela Mira and their staff for help preparing the housing facilities for the birds, as well as helping in the daily care and providing veterinary support. Thanks are also due to the volunteers who helped catching the birds, and especially Camilo Carneiro who also helped preparing samples for stable isotope assays. We would also like to acknowledge Sr. Almiro Sousa who gave us permission to access the private shorebird roosting site in the Tagus estuary where the bird captures took place. Two anonymous reviewers provided several useful comments on an earlier version of this paper. The authors were funded by Fundação para a Ciência e Tecnologia (FCT) through Project “Invisible Links” (PTDC/MAR/119920/2010) and postdoctoral grants to PML (SFRH/BPD/84237/2012) and TC (SFRH/BPD/102255/2014).[SS] References Alves, J.A., Dias, M., Rocha, A., Barreto, B., Catry, T., Costa, H., Fernandes, P., Ginja, B., Glen, K., Jara, J., Martins, R.C., Moniz, F., Pardal, S., Pereira, T., Rodrigues, J., Rolo, M., 2011. Monitoring waterbird populations on the Tagus, Sado and Guadiana estuaries: 2010 report. Anu. Ornitol. 8, 118–133. Barquete, V., Strauss, V., Ryan, P.G., 2013. Stable isotope turnover in blood and claws: a case study in captive African penguins. J. Exp. Mar. Biol. Ecol. 448, 121–127. Bates, D., Maechler, M., Bolker, B., Walker, S., 2014. R Package Version 1.1–7. http://CRAN. R-project.org/package=lme4 (Accessed 14 June 2014). Battley, P.F., Rogers, D.I., Hassell, C.J., 2006. Prebreeding moult, plumage and evidence for a presupplemental moult in the great knot Calidris tenuirostris. Ibis 148, 27–38.
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Bearhop, S., Waldron, S., Votier, S.C., Furness, R.W., 2002. Factors that influence assimilation rates and fractionation of nitrogen and carbon stable isotopes in avian blood and feathers. Physiol. Biochem. Zool. 75, 451–458. Bearhop, S., Furness, R.W., Hilton, G.M., Votier, S.C., Waldron, S., 2003. A forensic approach to understanding diet and habitat use from stable isotope analysis of (avian) claw material. Funct. Ecol. 17, 270–275. Bond, A.L., Diamond, A.W., 2011. Recent Bayesian stable-isotope mixing models are highly sensitive to variation in discrimination factors. Ecol. Appl. 21, 1017–1023. Carleton, S.A., Martínez del Rio, C., 2005. The effect of cold-induced increased metabolic rate on the rate of 13 C and 15 N incorporation in house sparrows (Passer domesticus). Oecologia 144, 226–232. Catry, T., Martins, R.C., Granadeiro, J.P., 2012. Discriminating geographic origins of migratory waders at stopover sites: insights from stable isotope analysis of toenails. J. 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Distance to hightide roosts constrains the use of foraging areas by dunlins: implications for the management of estuarine wetlands. Biol. Conserv. 131, 446–452. Ethier, D.M., Kyle, C.J., Kyser, T.K., Nocera, J.J., 2010. Variability in the growth patterns of the cornified claw sheath among vertebrates: implications for using biogeochemistry to study animal movement. Can. J. Zool. 88, 1043–1051. Gill, F.B., 2007. Ornithology. third ed. W.H. Freeman & Co, New York. Hahn, S., Dimitrov, D., Rehse, S., Yohannes, E., Jenni, L., 2014. Avian claw morphometry and growth determine the temporal pattern of archived stable isotopes. J. Avian Biol. 54, 202–207. Hobson, K.A., Barlein, F., 2003. Isotopic fractionation and turnover in captive garden warblers (Sylvia borin): implication for delineating dietary and migratory associations in wild passerines. Can. J. Zool. 81, 1630–1635. Hobson, K.A., Clark, R.G., 1992a. Assessing avian diets using stable isotopes I: turnover of 13 C in tissues. Condor 94, 181–188. Hobson, K.A., Clark, R.G., 1992b. Assessing avian diets using stable isotopes II: factors influencing diet-tissue fractionation. Condor 94, 189–197. Hobson, K.A., Wassenaar, L.I., 1996. Linking brooding and wintering grounds of neotropical migrant songbirds using stable hydrogen isotopic analysis of feathers. Oecologia 109, 142–148. Hobson, K.A., Schell, D.M., Renouf, D., Noseworthy, E., 1996. Stable carbon and nitrogen isotopic fractionation between diet and tissues of captive seals: implications for dietary reconstructions involving marine mammals. Can. J. Fish. Aquat. Sci. 53, 528–533. Hopkins, J.B., Cutting, K.A., Warren, J.M., 2013. Use of stable isotopes to investigate keratin deposition in the claw tips of ducks. PLoS ONE 8, e81026. http://dx.doi.org/10.1371/ journal.pone.0081026. Inger, R., Bearhop, S., 2008. Applications of stable isotope analyses to avian ecology. Ibis 150, 447–461. Kersten, M., Piersma, T., 1987. High levels of energy expenditure in shorebirds: metabolic adaptations to an energetically expensive way of life. Ardea 75, 175–187. Klaassen, M., Piersma, T., Korthals, H., Dekinga, A., Dietz, M.W., 2010. Single-point isotope measurements in blood cells and plasma to estimate the time since diet switches. Funct. Ecol. 24, 796–804. Kurle, C.M., Finkelstein, M.E., Smith, K.R., George, D., Ciani, D., Koch, P.L., Smith, D.R., 2013. Discrimination factors for stable isotopes of carbon and nitrogen in blood and feathers from chicks and juveniles of the California condor. Condor 115, 492–500. Logan, J.M., Jardine, T.D., Miller, T.J., Bunn, S.E., Cunjak, R.A., Lutcavage, M.E., 2008. Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods. J. Anim. Ecol. 77, 838–846. MacAvoy, S.E., Arneson, L.S., Bassett, E., 2006. Correlation of metabolism with tissue carbon and nitrogen turnover rate in small mammals. Oecologia 150, 190–201. Martins, R.C., Catry, T., Santos, C.D., Palmeirim, J.M., Granadeiro, J.P., 2013. Seasonal variations in the diet and foraging behaviour of dunlins Calidris alpina in a South European estuary: improved feeding conditions for northward migrants. PLoS ONE 8, e81174. http://dx.doi.org/10.1371/journal.pone.0081174. Mazerolle, D.F., Hobson, K.A., 2005. Estimating origins of short distance migrant songbirds in North America: contrasting inferences from hydrogen isotope measurements of feathers, claws, and blood. Condor 107, 280–288. Mirón, L.L., Herrera, L.G., Ramírez, N., Hobson, K.A., 2006. Effect of diet quality on carbon and nitrogen turnover and isotopic discrimination in blood of a New World nectarivorous bat. J. Exp. Biol. 209, 541–548. Ogden, L.J.E., Hobson, K.A., Lank, D.B., 2004. Blood isotopic (δ13C and δ15N) turnover and diet tissue fractionation factors in captive dunlin (Calidris alpina pacifica). Auk 121, 170–177. Oppel, S., Powell, A.N., 2010. 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Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Turnover rates of stable isotopes in avian blood and toenails: Implications for dietary and migration studies Pedro M. Lourenço a,⁎, José P. Granadeiro b, João L. Guilherme c, Teresa Catry a a b c
Centro de Estudos do Ambiente e do Mar (CESAM)/Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Lisboa, Portugal Centro de Estudos do Ambiente e do Mar (CESAM)/Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal Museu Nacional de História Natural e da Ciência, Universidade de Lisboa, Lisboa, Portugal
a r t i c l e
i n f o
Article history: Received 14 November 2014 Received in revised form 7 July 2015 Accepted 8 July 2015 Available online xxxx Keywords: Calidris alpina Captive study Diet switch Isotopic discrimination Half-life Shorebirds
a b s t r a c t Most stable isotope (SI) applications in avian ecology are based on the analysis of feathers and blood, whereas toenails are much less used. These structures grow slowly and continuously, thus integrating information over comparatively longer periods which may be useful for migration connectivity studies, while avoiding some of the difficulties posed by incomplete information on feather molt. In spite of this, interpretation of data from toenails is limited by lack of accurate figures on their turnover rates and discrimination values. To improve our understanding of SI ratios in toenails and surpass these drawbacks we measured the change in carbon (δ13C) and nitrogen (δ15N) SI ratios in plasma, red blood cells and toenails of captive dunlins Calidris alpina after a controlled diet switch. Discrimination values were estimated by comparing isotopic values at the end of the experiment with those of artificial food. We also estimated toenail growth rates in this species. Toenails showed much lower SI turnover rates than blood components, with half-lives of 27 days and 35 days for δ13C and δ15N, respectively. Isotopic ratios in toenails reached equilibrium with the new diet after 100–120 days, roughly coinciding with the duration of toenail replacement. The discrimination values of 2.74 ± 0.68‰ for δ13C and 4.06 ± 0.27‰ for δ15N found for toenails are higher than those of both blood components. These values are also higher than most published values for blood in other avian species, but similar to those previously reported for feathers which, like toenails, are metabolically inert after synthesis and are mainly composed of keratin. This study highlights the potential role of toenails as sources of SI data for ecological studies, particularly to determine geographic origin of birds migrating between isotopically distinct environments. Indeed, their long turnover rates might allow for the detection of SI signals from the wintering or breeding ranges, by sampling individuals at their staging sites. Isotopic turnover in toenails follow their replacement rates, and these seem to be rather similar across several avian taxa. Thus, the turnover rates described here may potentially be used to interpret SI data for other birds. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Stable isotope (SI) ratios are increasingly used in ecological studies, particularly to study trophic interactions and migratory connectivity (Rubenstein and Hobson, 2004; Inger and Bearhop, 2008). Isotopic ratios in body components reflect the diet of an animal during their synthesis, which means that after a diet switch these ratios will progressively converge towards the isotopic ratio of the new diet (Hobson and Clark, 1992a; Bearhop et al., 2002). The rate at which this process occurs varies among different tissues and structures of an animal (Tieszen et al., 1983; Bearhop et al., 2002; Klaassen et al., 2010), a feature that ⁎ Corresponding author at: Museu Nacional de História Natural e da Ciência, Rua da Escola Politécnica 58, 1250-102 Lisboa, Portugal. E-mail address: [email protected] (P.M. Lourenço).
http://dx.doi.org/10.1016/j.jembe.2015.07.006 0022-0981/© 2015 Elsevier B.V. All rights reserved.
has been used to gather information on dietary changes (and/or migratory movements) over a particular time window. Many studies of avian migration have derived their conclusions from the analysis of SI in feathers (Hobson and Wassenaar, 1996; Inger and Bearhop, 2008), relying on their inert nature which depicts the isotopic landscape where the feather was grown. Although feathers are a very useful source of isotopic information, interpreting their SI signature requires detailed knowledge of the timing and “geography” of the molt process (Ramos et al., 2009), which is not available for many species. Also, in many migratory birds, feather molt may occur over a rather protracted period, across several different locations (Battley et al., 2006), which may preclude assigning a given feather to a particular location. Blood (either whole blood, or separating plasma and red blood cells) is used to determine arrival dates and residence periods at stopovers, as this metabolically active tissue has a fast turnover rate
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thus providing information over a short time window of a few days to weeks before sampling (Hobson and Barlein, 2003; Klaassen et al., 2010; Oppel and Powell, 2010). Stable isotopes in bird toenails received comparatively rather less attention than in both feathers and blood. Toenails grow continuously and a few studies suggest that they integrate isotopic information over a time window of several weeks to a few months (Bearhop et al., 2003), making them ideal, for instance, to infer the geographic origin of migrant birds (Clark et al., 2006; Catry et al., 2012; Hopkins et al., 2013). Despite their potential as a target structure for SI work on avian migration, very few studies attempted to estimate SI turnover rates in bird toenails. The proposed time window for the information this structure integrates was estimated based on toenail growth rates (Bearhop et al., 2003; Hahn et al., 2014) and the only study we are aware of that attempted to measure the turnover rate of SI in toenails through a controlled diet switch experiment reported no shift in isotopic values even after five months (Barquete et al., 2013). Also, nail growth in higher vertebrates is a complex and not necessarily linear process (Ethier et al., 2010; Hahn et al., 2014), and some evidence suggests that toenail growth during migration may limit the use of SI data in toenails for determining breeding and wintering origins in ground foraging birds due to differences between the base and the tip of the toenail (Mazerolle and Hobson, 2005). These issues emphasize the need for more controlled experiments in order to correctly interpret SI data from bird toenails. The isotopic discrimination values, i.e. the difference between SI ratios in animals and in their diet, can also vary among tissues and structures of an animal (Hobson et al., 1996; Bearhop et al., 2002). These differences may constrain the analysis and interpretation of SI data (Bond and Diamond, 2011), but have not been described for bird toenails, limiting the use of these structures for dietary studies. In order to enhance our ability to interpret SI data derived from bird toenails, we studied the change in carbon (δ13C) and nitrogen (δ15N) stable isotope ratios of captive dunlins Calidris alpina (L.) after a controlled diet switch, aiming to 1) describe the turnover rates of δ13C and δ15N in toenails; 2) compare the turnover rates of δ13C and δ15N in toenails, plasma and red blood cells; and 3) calculate the isotopic discrimination values between diet and toenails.
2. Material and methods 2.1. Bird capture and experimental procedure We captured 40 dunlins at a high-tide roost in the southern bank of the Tejo estuary, Portugal (38°49′N, 8°57′W), using mist-nets. Birds were caught in four different events during the highest spring tides, between 20-Nov-2013 and 5-Jan-2014. Post-breeding migrant dunlins arrive in August–September in the Tejo estuary (Alves et al., 2011), where they feed mainly on the polychaete Hediste diversicolor Malmgren, the bivalve Scrobicularia plana (da Costa) and the gastropod Hydrobia ulvae (Pennant) (Santos et al., 2005; Martins et al., 2013). Therefore, we assumed that dunlins had been on this diet for ca. 3–4 months before being captured so that they were in equilibrium with the SI values of their prey. After capture, all birds were weighed (to the nearest 0.1 g), and ringed with an individual combination of one metal and four color rings. We collected the distal portion from all toenails (ca. 2 mm long) on the right foot of each bird. We also made a small incision near the base of the central toenail on the left foot, using a scalpel, and the distance between the incision and the base of the toenail was measured to the nearest 0.1 mm with a caliper, in order to estimate toenail growth rate during the experiment. For a subsample of eight birds (two groups of four individuals captured in two different dates) we also collected 150 μl of blood from the brachial vein. Blood was centrifuged for 10 min at 3400 rpm within 2 h of collection, in order to separate the
cellular and plasma components. The separated blood components were frozen at −20 °C until further analysis. Within a few hours from capture, birds were transported by car in cardboard boxes to the housing facilities of the Lisboa Wild Animal Recovery Centre (LxCRAS), located in the Monsanto Forest Park, a large forested area within the city of Lisboa, ca. 50 km from the catching area. This facility provided advice as well as logistic and veterinary support, whenever necessary. Birds were divided in three groups, each kept in an outdoor aviary (roughly l × w × h: 3 × 2 × 2 m) under natural light and temperature conditions. The ground was covered with coarse sand to mimic a natural substrate, and each aviary had a pool with sloping banks where the birds could drink and bathe. The pool was filled with fresh water and frequently flushed to minimize the risk of diseases. The walls of the cages were padded with a soft mesh to minimize the risk of injury and visits to the cages were kept to a minimum to avoid stressing the birds. During the whole captive period birds were fed ad libitum with trout pellets (T-2 Optiline-sf 1P; Skretting España S.A., Spain), which have a different isotopic value from that of the natural prey taken by birds in the estuary (see results). These pellets are manufactured from soy, fish meal, rapeseed flour, fish oil, vegetable oil, wheat, vegetable proteins, vitamins and minerals, and most of their macromolecular composition consists of protein (44%) and fat (21%). All used pellets originated from a single stock, so differences in SI values among pellets should be minimal. Food was placed in several trays per aviary and always mixed with some water to soften the pellets. During the first 2–3 days of captivity we also used live mealworms Tenebrio molitor L. to train birds to feed from the trays. Birds that were sampled for blood were only given mealworms in the first day of captivity to minimize any effect on blood isotopic values due to its fast turnover rate. To evaluate temporal variation in SI values in toenails, groups of five birds were sequentially sampled over increasing periods of exposure to the new diet. The first group was sampled after 10 days, the second after 22 days, the third after 34 days and the remaining groups each after an additional 15 days, with the last group being sampled 109 days after capture. In each case, sampling was performed as at capture, but toenails were collected from the left foot. The eight birds initially sampled for blood were subsequently resampled after 3, 6, 11, 16, 21, 26, 34, 41, 48 and 55 days of captivity. During the experiment, all birds were weighed at least once every two weeks and every time they were sampled, in order to monitor their response to artificial food. Simultaneously, to monitor toenail growth, we measured the distance between the incision made upon capture and the base of the toenail to the nearest 0.1 mm. In a subsample of birds we also measured the total length of the central toenail at the end of the captive period, just before sampling the toenail for SI. For comparison purposes, we also measured the total length of the central toenail on the left foot of 28 dunlins caught in the Tejo estuary, in April 2014. After the final toenail/blood sampling each group of birds was released back in the estuary, during low tide, in an area where dunlin flocks are frequently seen foraging near the coast. This experiment was carried out in strict accordance with the current national ethical and legal regulations. The protocol was approved by the responsible ethical and legal authority, the Portuguese Institute of Nature Conservation and Forests (ICNF) and performed under official permits 385/2013/CAPT, 386/2013/CAPT and 387/2013/ CAPT. In order to compare SI values in bird blood and toenails with that of their natural food, we collected 10–20 individuals of their three main prey, with sizes within the range known to be consumed by dunlin (H. ulvae, all available sizes; H. diversicolor, 15–50 mm total length; and S. plana, 4–8 mm shell width) in a mudflat located 1 km south of the roost where the birds were captured and thus a likely foraging location (Dias et al., 2006). The animals were kept in estuarine water for 24 h to release their gut contents and afterwards divided in 2–3 samples and frozen until further analysis. A 2 g sample of the trout pellets
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used during the experiment and 10 mealworms were also stored for SI analysis at the beginning of the experiment.
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3. Results 3.1. Stable isotope values of food samples
2.2. Stable isotope analysis Toenail samples were washed in double baths of 0.25 N sodium hydroxide solution alternated with baths of double distilled water to remove adherent contamination (Catry et al., 2012). All samples of blood, toenails and food items were dried at 50 °C for 48 h and then ground to powder and homogenized. In order to extract lipids, food samples were immersed in a 2:1 chloroform/methanol solution with a solvent volume 3–5 times larger than sample volume (Logan et al., 2008). Samples were then mixed for 30 s, left undisturbed for approximately 30 min, centrifuged for 10 min at 3400 rpm, and the supernatant containing solvent and lipids was removed. We repeated this process at least three times until the supernatant was clear and colorless after centrifugation. Samples were re-dried at 60 °C for 24 h to remove any remaining solvent. Subsamples of H. ulvae and S. plana were acidified with several drops of 10% hydrogen chloride to remove carbonates from any shell remains. After bubbling stopped, the sample was centrifuged, the acid removed and, as a final step, the sample was washed with distilled water. This process was repeated three times (Vinagre et al., 2008). A non-acidified subsample was kept for nitrogen isotope analysis. Between 0.60 and 1.20 mg of each sample was stored in tin cups and combusted at 1000 °C in a Euro EA Elemental Analyzer, at the Stable Isotopes and Instrumental Analysis Facility of the Faculty of Sciences, University of Lisbon. Resultant CO2 and N2 gases were analyzed using a continuous-flow isotope ratio mass spectrometer IsoPrime (MicroMass), with unknowns separated by laboratory standards. Results are presented conventionally as δ values in parts per thousand (‰) relative to the Vienna Pee Dee Belemnite (PDB) for δ13C, and atmospheric nitrogen (N2) for δ15N, with a precision of 0.11 to 0.25‰ for δ13C and 0.05 to 0.17‰ for δ15N (SD), calculated from standards. 2.3. Data analysis We used Generalized Linear Mixed Models (GLMM) through R package ‘lme4’ (Bates et al., 2014) to analyze toenail growth rates and the variation in body mass during the experiment, using individual as a random factor to take into account the fact that birds were sampled repeatedly while in captivity and for a different number of times. We used t-tests to compare the average length of toenails in birds at the end of captivity with that of birds captured in the wild. We compared SI values and isotopic discrimination values from different sample types (red cell, plasma and nails) and dates, using Kruskal–Wallis and Mann–Whitney tests. The change in the isotopic value of an animal following a diet switch can be described by an exponential decay curve as y = a.e− bx + c, where y is the isotopic value of the sampled body component at time x after the diet switch (in days), a is the change in isotope values associated with the change in diet, c is the isotope value at x = ∞ (the difference between isotope values at equilibrium and those at x = ∞ are likely to be very small, so this equals to the asymptotic condition), and b is the fractional turnover rate (Bearhop et al., 2002; Hobson and Barlein, 2003; Ogden et al., 2004). Turnover rates for δ13C and δ15N were estimated for toenails and both blood components by fitting the general equation using least squares nonlinear regression in the non-linear fitting module of Statistica 8.0. The half-life was calculated using −ln(0.5)/b for each case (Bearhop et al., 2002). The isotopic discrimination values (Δdt) of δ13C and δ15N for toenails and both blood fractions were calculated using the equation: Δdt = δtissue − δdiet, based on the SI signals from the last sampling dates. Unless stated otherwise, all statistical analyses were performed in R 2.14 (R Development Core Team, 2012). Throughout the text data are presented as means ± SD, unless stated otherwise.
The three invertebrate prey species collected in the estuary had similar isotopic values (H. diversicolor: δ13C − 14.6 ± 0.4‰ and δ15N 14.6 ± 0.3‰ (n = 3); S. plana: δ13C − 14.8 ± 0.3‰ and δ15N 14.8 ± 0.4‰ (n = 2); H. ulvae: δ13C − 12.9 ± 0.5‰ and δ15N 14.9 ± 0.7‰ (n = 3)), which were clearly different from the values of the trout pellets used during the experiment (δ13C − 23.4‰ and δ15N 5.3‰), with both isotopes showing more enriched values in the estuarine invertebrates. The mealworm isotopic values (δ13C −27.2‰ and δ15N 8.3‰) were also impoverished when compared to the estuarine invertebrates.
3.2. Body mass and toenail growth rates Dunlins had an average body mass of 46.6 ± 4.1 g (n = 40) at capture, and most birds lost weight in the first days after capture, reaching a minimum average body mass of 42.5 ± 6.7 g (n = 12) after three days in captivity. Afterwards they began gaining weight, eventually stabilizing about 12 days after capture at an average body mass of 51.0 ± 3.7 g (n = 14). There was no significant change in average body mass from day 12 until the end of the experiment (GLMM: t = 2.02; P N 0.1; R2 = 0.01). Toenail length, measured as the distance between the incision and the base of the toenail, increased linearly during the experiment (GLMM: t = 58.6; P b 0.01; R2 = 0.96), at a rate of 0.041 mm·day−1 (± 0.001 SE; Fig. 1). The average total length of the central toenail measured at the end of the captivity period was 4.9 ± 0.4 mm (range: 4.1–5.7; n = 29), indicating that it takes 120 days (i.e. 4.9 mm divided by a growth rate of 0.041 mm·day−1) to replace the whole toenail. However, the average total length of the central toenail in dunlins caught in the Tejo estuary in April 2014 was 4.3 ± 0.5 mm (range: 3.0–5.3; n = 28), which is significantly lower than in captivity (t = 4.4; n = 29 in captivity and 28 in the wild; P b 0.001). Assuming the same growth rate, it would take 105 days to replace the whole toenail in the wild. The two groups did not vary in terms of wing size (t = 1.74; n = 29 and 28; P N 0.05) or tarsus length (t = 1.41; n = 29 and 28; P N 0.10).
Fig. 1. Toenail length, measured as the distance between the incision and the base of the toenail over the captivity period. The black line is the linear fit between toenail length and time evidencing an average growth rate (i.e. slope) of 0.041 mm·day−1.
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3.3. Isotopic value of toenails, plasma and red blood cells of dunlins There were no significant differences among the initial SI values of birds caught at different dates in either toenails (Kruskall–Wallis test (KW) — δ13C: H(3,40) = 4.16; P N 0.10 and δ15N: H(3,40) = 4.78; P N 0.10) or red blood cells (Mann–Whitney test (MW) — δ13C: Z = 2.01; P N 0.05 and δ15N: Z = 1.73; P N 0.10). There were also no significant differences in δ15N in plasma among catching dates (MW: Z = 1.73; P N 0.10), although the difference in δ13C in plasma between catching dates was marginally significant (MW: Z = 2.30; P = 0.042). In animals sampled both for toenails and blood (Table 1), we found no significant differences in δ15N among different sample types (KW: H(2,24) = 1.99; P N 0.10) at the time of capture, but toenails tended to have higher average values. The value of δ13C varied among sample types (KW: H(2,24) = 8.02; P b 0.05), with significantly higher values in toenails in relation to plasma. After the diet switch, δ13C and δ15N values in all sampled body components declined towards the values observed in the trout pellets, showing a strong fit to the exponential decay curves (Table 2). The turnover rate in plasma was the highest, with half-lives of 1.3 days (1.0–2.2; 95% CI) for δ13C and 2.8 days (2.5–3.2; 95% CI) for δ15N (Fig. 2A). Turnover in red blood cells was intermediate, with half-lives of 8.6 days (7.2–10.8; 95% CI) for δ13C and 10.2 days (9.1–11.4; 95% CI) for δ15N (Fig. 2B). Toenails had the lowest turnover rate with half-lives of 26.7 days (19.8–40.8; 95% CI) for δ13C and 34.7 days (26.7–46.2; 95% CI) for δ15N (Fig. 2C). Turnover tended to be faster for δ13C than for δ15N (Fig. 2), but the 95% confidence intervals for each isotope overlapped in both red blood cells and toenails. The isotopic discrimination (Δdt) of captive birds showed significant differences between sample types (KW: δ13C: H(2,21) = 15.7; P b 0.001 and δ15N: H(2,21) = 17.6; P b 0.001), with higher values for toenails (Table 3). Both blood fractions had significantly lower Δ13C than toenails, but only the red blood cells had a significantly lower Δ15N than toenails. The Δ15N estimated for birds on a natural diet at capture in relation to average values for their three main prey, are very similar to those derived from the captivity experiment (plasma: 3.52 ± 1.64‰, n = 8, MW: Z = 1.34, P N 0.10; red blood cells: 3.20 ± 0.82‰, n = 8, MW: Z = 1.60, P N 0.10; toenails: 3.37 ± 1.32‰, n = 40, MW: Z = 0.52; P N 0.5). Surprisingly, Δ13C values for birds on a natural diet were negative and significantly different from those obtained during the experiment (plasma: − 3.83 ± 1.28‰, n = 8, MW: Z = 3.00, P b 0.01; red blood cells: − 2.25 ± 0.60‰, n = 8, MW: Z = 3.24, P b 0.01; toenails: −1.85 ± 1.88‰, n = 40, MW: Z = 3.27, P b 0.01). 4. Discussion In this study we provide novel information concerning estimated SI turnover rates of several body components in shorebirds under captivity, with particular emphasis on toenails, a continuously-growing structure. By sampling individuals over increasingly longer periods of exposure to an isotopic distinctive food source, we were able to establish a “time-response” curve of δ13C and δ15N, a key calibration tool to use these structures in ecological studies and particularly in ascertaining the geographic origins of migratory species. Birds caught in the wild at different dates all showed similar isotopic values, with the exception δ13C in plasma, strongly suggesting that they were in
Table 1 Average (±SD) δ13C and δ15N (‰) values in plasma, red blood cells and toenails of eight dunlins sampled at the time of capture. Different letters in superscript indicate significant differences (P b 0.05) between samples types in post-hoc Dunn tests. Sample type Plasma Red blood cells Toenails
Δ13C
Δ15N a
−18.95 ± 2.62 −17.35 ± 2.33ab −15.86 ± 1.08b
17.66 ± 2.32a 16.73 ± 2.38a 18.16 ± 1.32a
Table 2 Turnover rates of δ13C and δ15N in plasma, red blood cells and toenails of captive dunlins after the diet switch, obtained from the fitted exponential decay models. We present the turnover rate (day−1), 95% confidence intervals (95% CI), model fit and the percentage of variation in the data explained by the model (Expl. variance). (***P b 0.001). Sample type
Isotope
Turnover rate
95% CI
Model fit
Expl. variance
Plasma
δ13C δ15N δ13C δ15N δ13C δ15N
0.522 0.244 0.081 0.068 0.020 0.026
0.315–0.730 0.215–0.273 0.064–0.097 0.061–0.076 0.017–0.035 0.015–0.026
T84 = 5.0*** T84 = 1.7*** T84 = 9.7*** T84 = 16.7*** T80 = 5.9*** T80 = 7.5***
68% 92% 72% 89% 60% 67%
Red blood cells Toenails
equilibrium with their natural diet in the Tejo estuary. As expected, both toenails and the two blood components exhibited a decline in δ13C and δ15N values towards the isotopic value of the artificial food, but the turnover rates were different among sample types. The two blood components showed a much higher turnover rate than toenails, in line with previous studies (Bearhop et al., 2003; Ogden et al., 2004; Oppel and Powell, 2010), and we now have reliable estimates for the half-lives of δ13C and δ15N in dunlin toenails: 27 and 35 days, respectively, which represents the time-window to which isotopic information obtained from toenails refers to. Our data suggest that the isotopic value of dunlin toenails reach equilibrium with the new diet after 100–120 days, which fits very well with our observations of toenail growth rates. Toenail growth in captive dunlins (0.041 ± 0.001 mm·day− 1) is very similar to that measured in several European passerines (0.04 ± 0.01 mm·day−1 in Bearhop et al., 2003; 0.034 ± 0.001 mm·day−1 in Hahn et al., 2014). Although smaller than dunlins, these passerines have similar sized toenails (range 3.8–5.3 mm in Bearhop et al., 2003) indicating that the time required to replace a whole toenail should be similar. The much larger African penguin Spheniscus demersus has also been recorded to require 126 days to replace much larger toenails (16.1 ± 1.6 mm, Barquete et al., 2013) hinting for some consistency across avian taxa in the time required to replace toenails and, potentially, in the time required for toenail isotopic values to reach equilibrium with their diet. The fact that all these studies show a linear growth of the toenail while the isotope depletion curves are better modeled as negative exponentials, and that we can detect change in isotopic values just a couple of weeks after the dietary shift (even though we only sample the distal half of the nail), both indicate that not all parts of the toenail grow equally. Actually, avian toenails show conical growth (i.e. both longitudinal and lateral) which causes any part of the toenail to contain a blend of materials formed at different times (Hahn et al., 2014). Also, our toenail growth estimate only refers to the larger toenail. Since the smaller toenails are likely to have similar growth rates (Bearhop et al., 2003), this means they will be replaced faster influencing the incorporation of isotopic signals. However, due to their very small size, several nails had to be sampled from each individual to achieve the mass required for the isotopic determination, thus precluding any analysis of how the nails from different toes incorporate isotopic information. The length of the central toenail in wild caught dunlins was on average lower than that observed in our captive birds at the end of the experiment. Since this disparity is not likely to be due to allometric differences (as the two groups had similar wing length and tarsus length), it seems likely that captive dunlins suffer less wear to their toenails because they have easier access to food and spend much less time walking about. This suggests that it is likely we slightly overestimated the time dunlins require to replace their toenails. There are no published data on the half-lives of stable isotopes in each of the two blood components in dunlins. One study showed that the half-lives of δ13C and δ15N in whole blood samples of dunlins were similar to what we found for the red blood cell fraction (Ogden et al., 2004; Table 4). The half-lives of δ13C in captive red knots Calidris canutus
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Fig. 2. Temporal variation of δ13C and δ15N values in the plasma (A), red blood cells (B) and toenails (C) of captive dunlins sampled at increasing periods after the diet switch. In each sampling date black squares represent the average and whiskers show the standard deviation. The black curves are the fitted exponential decay models used to estimate turnover rates (see Table 3 for more details). The gray horizontal lines indicate the isotopic value of the artificial food pellets.
were found to be higher than our estimates for dunlin (Klaassen et al., 2010) and the same is true for the half-lives of δ15N in African penguins (Barquete et al., 2013) and of both isotopes on whole blood samples of great skua Catharacta skua (Bearhop et al., 2002; Table 4). In fact, the short half-lives recorded in our study are more similar to what is described for passerines such as the yellow-rumped warbler Dendroica coronata (Pearson et al., 2003; Podlesak et al., 2005) and the garden warbler Sylvia borin (Hobson and Barlein, 2003; Table 4).
Table 3 Isotopic discrimination values (Δdt) of δ13C and δ15N (‰) in plasma, red blood cells and toenails of captive dunlins in relation to the artificial diet used during the experiment. For both blood components n = 8 birds, while for toenail n = 4 birds. Different letters in superscript indicate significant differences (P b 0.05) between sample types in post hoc Dunn tests. Sample type Plasma Red blood cells Toenails
Δ13C
Δ15N a
0.32 ± 0.16 0.94 ± 0.24a 2.74 ± 0.68b
3.30 ± 0.20ab 2.30 ± 0.28a 4.06 ± 0.27b
Dunlins are small shorebirds, roughly 1/3 the mass of a red knot, and consequently have a higher metabolic rate (Kersten and Piersma, 1987). Several studies have suggested that isotopic turnover rates are correlated with metabolic rate and body size (Bearhop et al., 2002; MacAvoy et al., 2006), an indirect relationship probably mediated by protein turnover (Carleton and Martínez del Rio, 2005), which may explain why the isotopic turnover rate in dunlins is more similar to a passerine than to its larger congener or other larger birds. In our study, the turnover rate of δ13C tended to be faster than that of δ15N, a result consistent with some studies (Hobson and Barlein, 2003; Pearson et al., 2003), but not with others (Bearhop et al., 2003; Ogden et al., 2004). Such differences in the incorporation of the isotopic ratios of each element into animal tissues can be related with diet quality and the C:N ratio in food (Mirón et al., 2006), so any comparison between the turnover rates of different isotopes in different experiments would only be valid if the same food was used in all cases. The observed discrimination values for δ15N in our dunlin samples lay within the range 2–4‰ that have been described in most SI studies (Bearhop et al., 2002; Inger and Bearhop, 2008). Toenails tended to have higher Δ15N than blood, although this difference was only
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Table 4 Half-lives of δ13C and δ15N (in days) in blood samples of a range of bird species with different body masses. Body mass ranges (in g) were obtained from (del Hoyo et al., 2013). Species are ordered by increasing body mass. Species
Body mass
Analyzed tissue
Half-life of δ13C
Half-life of δ15N
References
D. coronata
10–17
Pearson et al. (2003)
10–17 16–22 33–85
C. alpina C. canutus
33–85 85–220
C. skua S. demersus
1100–1700 2100–3700
0.4–0.7 3.9–6.1 1.0 5.0–5.7 1.0–2.2 7.2–10.8 11.2 ± 0.8 6.03 15.07 14.4
0.5–0.7 7.5–27.7
D. coronata S. borin C. alpina
Plasma Whole blood Plasma Whole blood Plasma Red blood cells Whole blood Plasma Red blood cells Whole blood Plasma Red blood cells
significant when compared with red blood cells. The only study that compared discrimination values between blood and toenails actually found a lower Δ15N in toenails (Barquete et al., 2013), which could indicate that our values were overestimated, possibly because our last measurements were yet to reach asymptotic values. However, the fact that δ15N values show virtually no change between the two last sampling dates suggest this is not the case (after 94 days: 9.69 ± 1.14‰, n = 4; after 109 days: 9.48 ± 0.27‰, n = 4). Also, the Δ15N at the end of the experiment was similar to what was estimated for birds on a natural diet, supporting the idea that our estimates for Δ15N in toenails are not biased. Stable carbon isotope ratios usually undergo fairly limited increase between trophic levels, in most cases with Δ13C falling in the range 0–1‰ (Inger and Bearhop, 2008), which is consistent with what we observed for the two blood fraction in dunlins. However, the observed value for toenails was again significantly higher. Although in the case of δ13C the estimated values for birds on a natural diet were not consistent with what was observed during the experiment, as dunlin samples at capture had lower δ13C than their main prey, Δ13C was also higher (in this case less negative) for toenails at capture. These unexpected negative discrimination values possible derive from the fact that we cannot measure discrimination in the wild as precisely as in captivity, because the natural diet may suffer variations and natural prey may have spatial–temporal patterns of variation in their signatures that may scramble the SI values. To our knowledge, no studies have compared Δ13C in toenails and blood, but work in great skuas (Bearhop et al., 2002) and garden warblers (Hobson and Barlein, 2003) have reported higher Δ13C and Δ15N in feathers than in blood, with values for feathers (Δ13C: 2.1–2.7‰, Δ15N: 4.0–5.0‰.) that are similar to what we describe for toenails in dunlins. Studies in king Aptenodytes patagonicus and rockhopper Eudyptes chrysocome penguins (Cherel et al., 2005) and in California condors Gymnogyps californianus (Kurle et al., 2013) have also reported higher discrimination values in feathers than in blood, although not as high as we found in dunlin toenails. On the other hand, no difference was observed in Japanese quails Coturnix japonica and ring-billed gulls Larus delawarensis (Hobson and Clark, 1992b). Avian feathers and toenails are structures that become metabolically inert after being synthesized and are both mainly composed of keratin (Gill, 2007), so it seems reasonable to assume that they will have similar isotopic discrimination values, which in many cases seem to be higher than that of blood. 4.1. Implications for the use of isotopic data from avian toenails in ecological studies Feathers and blood can be collected without excessive intrusion to birds, and to a large extent this explains the extensive use of these body components in studies of avian ecology. However, toenails can
11.0 2.5–3.2 9.1–11.4 10.0 ± 0.6
15.7 ± 2.1 7.6 ± 0.7 14.3 ± 1.6
Podlesak et al. (2005) Hobson and Barlein (2003) This study Ogden et al. (2004) Klaassen et al., 2010 Bearhop et al. (2002) Barquete et al. (2013)
also be easily collected and may have a greater potential for the study of avian migration, particularly for determining the geographic origin of migratory birds. Toenails integrate isotopic information over a longer period than blood and, unlike feathers, their constant growth can deliver information during the whole yearly cycle. This means that isotopic values of the distal half of toenails from birds at a staging area are likely to be related with those found at the wintering or breeding areas from where they originate. Mapping the isotopic values at several potential breeding and wintering areas can help us identify their most likely geographic origin (Catry et al., 2012). Of course, such data needs to be interpreted carefully, as populations with uncommonly short breeding or wintering periods may not stay in an area long enough to fully incorporate the local isotopic values, while populations with very long intermediate staging periods prior to capture may mask the original SI values. Still, due to the asymptotic nature of the incorporation of isotopic information into body tissues, this should only be a problem in rather extreme cases. In order to apply toenail SI data in ecological studies, we require an in-depth knowledge on the rate at which isotopic information is incorporated, as well as on how isotope ratios change between ingested food and toenails. Initial work with SI in toenails assumed that the turnover rate would follow the growth rate of the toenails (Bearhop et al., 2003), but there was no empirical data to confirm this and the only experimental work on this topic was unable to detect isotopic turnover in toenails (Barquete et al., 2013). Our data shows a turnover rate of 0.020.day− 1 for δ13C and 0.026.day− 1 for δ15N in dunlin toenails, which indicate a period of about four months to reach asymptotic equilibrium values with their diet, translating into half-lives of about one month. Since toenail growth data also indicate a period of about four months to replace the whole toenails in dunlins, this confirms that the isotopic turnover in avian toenails indeed follows the growth rate of the nails, although one process is linear and the other is not. Moreover, in species belonging to three widely distinct avian groups (passerines, shorebirds and penguins) the time required to replace toenails is remarkably similar. Even though more data would be required to support this assumption, and toenail wear possibly varies considerably among species with different behavior and habitat use, we can speculate that the turnover rates we measured and the four months window for incorporation of isotopic information into toenails can be used when interpreting SI data for other bird species. Although our study design offers no insights into isotopic variations within individual toenails, previous studies have shown that such differences can be found in continuously growing structures such as hairs, teeth, baleen, otholits and mammalian nails, with potential applications for the study of temporal variations in SI values (revision in Dalerum and Angerbjȯrn, 2005). In fact, at least one study reported isotopic differences between the tip and base of toenails in birds (Mazerolle and Hobson, 2005), which may indicate there is some potential for
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using different sections of avian toenails to study temporal variations in diet or foraging location. However, since avian toenails grow both longitudinally and laterally (Hahn et al., 2014), such data needs to be treated with great caution until further experiments determine precisely how isotopic differences within individual toenails are originated. Toenail isotopic values appear to have a higher discrimination value than that of blood, and these values may fall above the usual figures used in most dietary studies, particularly in the case of δ13C. This is an important consideration for interpreting SI data from avian toenails, particularly in dietary studies. However, looking through the literature, it is clear that discrimination values vary considerably among species, tissues and diets (Hobson and Clark, 1992b; Pearson et al., 2003; Podlesak et al., 2005). Since we only studied individuals of a single species being fed a single diet, we must recommend caution when using the discrimination values found for dunlin toenails in other species. Glossary
Half-life the amount of time required for the amount of something to fall to half its initial value. Half-life is used to describe a quantity undergoing exponential decay, and is constant over the lifetime of the decaying quantity. High-tide roost locations, such as beaches, marshes or saltpans, where shorebirds and other intertidal foraging birds rest during the periods when their foraging areas are submerged by the tide. Isotopic discrimination enrichment of one isotope relative to another in a chemical or physical process. Migratory connectivity a term used to describe the relationship between populations of animals (especially birds) and geographic locations at different time points during the year. Stable isotope An isotope (i.e. one of several nuclides having the same number of protons in their nuclei, and hence having the same atomic number but differing in the number of neutrons) of an element that shows no tendency to undergo radioactive breakdown. Acknowledgments We would like to thank LxCRAS, and particularly Nuno Ventinhas, Manuela Mira and their staff for help preparing the housing facilities for the birds, as well as helping in the daily care and providing veterinary support. Thanks are also due to the volunteers who helped catching the birds, and especially Camilo Carneiro who also helped preparing samples for stable isotope assays. We would also like to acknowledge Sr. Almiro Sousa who gave us permission to access the private shorebird roosting site in the Tagus estuary where the bird captures took place. Two anonymous reviewers provided several useful comments on an earlier version of this paper. The authors were funded by Fundação para a Ciência e Tecnologia (FCT) through Project “Invisible Links” (PTDC/MAR/119920/2010) and postdoctoral grants to PML (SFRH/BPD/84237/2012) and TC (SFRH/BPD/102255/2014).[SS] References Alves, J.A., Dias, M., Rocha, A., Barreto, B., Catry, T., Costa, H., Fernandes, P., Ginja, B., Glen, K., Jara, J., Martins, R.C., Moniz, F., Pardal, S., Pereira, T., Rodrigues, J., Rolo, M., 2011. Monitoring waterbird populations on the Tagus, Sado and Guadiana estuaries: 2010 report. Anu. Ornitol. 8, 118–133. Barquete, V., Strauss, V., Ryan, P.G., 2013. Stable isotope turnover in blood and claws: a case study in captive African penguins. J. Exp. Mar. Biol. Ecol. 448, 121–127. Bates, D., Maechler, M., Bolker, B., Walker, S., 2014. R Package Version 1.1–7. http://CRAN. R-project.org/package=lme4 (Accessed 14 June 2014). Battley, P.F., Rogers, D.I., Hassell, C.J., 2006. Prebreeding moult, plumage and evidence for a presupplemental moult in the great knot Calidris tenuirostris. Ibis 148, 27–38.
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