SPECIAL FEATURE: ISOSCAPES

A multi-isotope (d13C, d15N, d2H) feather isoscape to assign Afrotropical migrant birds to origins
K. A. HOBSON,1, S. L. VAN WILGENBURG,1 L. I. WASSENAAR,1 R. L. POWELL,2 C. J. STILL,3 AND J. M. CRAINE4
1

Environment Canada, 11 Innovation Boulevard, Saskatoon, Saskatchewan S7N 3H5 Canada 2 Department of Geography, University of Denver, Denver, Colorado 80208-0710 USA 3 Department of Geography, University of California, Santa Barbara, California 93106 USA 4 Division of Biology, Kansas State University, Manhattan, Kansas 66506 USA

Citation: Hobson, K. A., S. L. Van Wilgenburg, L. I. Wassenaar, R. L. Powell, C. J. Still, and J. M. Craine. 2012. A multiisotope (d13C, d15N, d2H) feather isoscape to assign Afrotropical migrant birds to origins. Ecosphere 3(5):44. http://dx.doi. org/10.1890/ES12-00018.1

Abstract. A universal challenge in methodology used to study the ecology, conservation and evolutionary biology of migratory species is the quantification of connectivity among breeding, wintering and stopover sites. For the avian Eurasian-Afrotropical migratory system, knowledge of geographical wintering areas used by migrants that breed in Europe remains deficient, despite the advent of satellite transmitters and geolocators. Here we explored the use of theoretical plant d13C and d15N landscape distributions coupled with d2H hydrologic models to construct multi-isotopic avian foodweb clusters for Africa. The cluster analysis identified four distinct regions of Africa based on all three isotopes (13C, 2H, 15 N), and five regions based only on 13C and 15N. We applied known isotopic diet-tissue discrimination factors to map equivalent feather isotopic clusters for Africa. The validity of these feather isotopic clusters was tested by examining how well known- and unknown-origin species were placed in regions of Africa using previously published feather isotope data. The success of this multi-isotopic cluster model depended upon the species of interest and additionally on how well potential winter molt origins in Africa were constrained by prior information. Ground-truthing data suggested this approach will be useful for firstorder approximation of overwintering regions for Afrotropical migrants and will be improved as our understanding of the nature of isoscapes for Africa is refined.
Key words: Africa; carbon-13; deuterium; feathers; isoscapes; Isoscapes Special Feature; migratory connectivity; nitrogen-15; stable isotopes. Received 27 January 2012; revised 23 February 2012; accepted 27 February 2012; final version received 27 April 2012; published 17 May 2012. Corresponding Editor: G. Bowen. Copyright: 2012 Hobson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits restricted use, distribution, and reproduction in any medium, provided the original author and sources are credited. E-mail: Keith.Hobson@ec.gc.ca

INTRODUCTION
The abundance of naturally occurring light stable isotopes (13C, 2H, 15N, 18O, 34S) in both organic and inorganic tissues, and an understanding of how these isotopes vary in organisms both spatially and temporally, provides the
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foundation for a potentially powerful means of tracking migrant organisms that move among spatially distinctive isoscapes (Bowen et al. 2005, Hobson and Wassenaar 2008, West et al. 2010, Hobson 2011). The isotopic approach to tracking origins and movements of organisms is rooted in the fact that stable isotope ratios in animal tissues 1
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are often highly correlated to diet in the geographical area where the tissue(s) was formed, particularly for tissues having a short elemental turnover rate (Hobson 2008, Mart nez del Rio et al. 2010). For tissues composed of keratins or chitins, such as feathers, insect exoskeletons and wings, hair, or claw, isotopic dietary signals are typically locked in at formation and so retain a record of origin. Since birds typically have clearly defined feather molt schedules that can be related to time of year or geographic region, the isotope approach to tracking avian origins has predominated the field to date. In North America, most migratory birds molt flight feathers on or near natal or breeding grounds prior to their autumn migration south (Pyle 1997). Thus, isotopic analysis of feathers retrieved on the wintering grounds in central and South America allows retrospective assignment to North American breeding ground regions. In particular, hydrogen isotopes are highly suited for molting area assignment because long-term hydrogen isoscapes are well established for North America (cf. Bowen et al. 2005, among others). For the Palearctic-African avian migratory system, many birds breeding in Europe molt their flight feathers on the African overwintering grounds. Thus the isotopic analysis of feathers of birds returning to Europe are used to infer approximate winter origins, or to identify populations differing in their wintering regions or habitats (e.g., Pain et al. 2004, Bensch et al. 2006, Yohannes et al. 2005, 2008a, b, Procha zka et al. 2008, Sze p et al. 2009, Wakelin et al. 2011). Unlike North America, most research in the PalearcticAfrican migratory system has thus far relied on feather d13C and d15N values. This is because the African continent contains large climatic and ecosystem variations, resulting in large differences in the relative abundance of plants using the C3 and C4 photosynthetic pathways and precipitation (d2H, d18O) isoscapes across Africa show weaker spatial gradients compared to other continents. Plant C3 and C4 photosynthetic pathways incorporate 13C to differing degrees during photosynthesis, C3 plants typically are isotopically depleted relative to C4 plants and the abundance of each group depends largely on climate (Ehleringer 1989). Plant water-use efficiency mechanisms can also contribute to variv www.esajournals.org

ance in C3 plant d13C values (Still and Powell 2010). Several climatic factors contribute to the N isotopic composition of soils and plants with xeric and/or cultivated regions typically enriched in 15N compared to mesic or uncultivated regions (Pardo and Nadelhoffer 2010). Despite considerable studies that have used stable isotope assays of feathers to infer origins or determine isotopic characteristics of habitats used by birds wintering in Africa, the inferences have largely been qualitative. Several authors referred to general expectations of biome characteristics associated with foodweb d13C and d15N values, or speculated on possible origins using d2H based on the long-term Global Network of Isotopes in Precipitation (GNIP) summarized by Bowen et al. (2005). A more formalized approach to geospatial assignment of feathers origin in Africa may now be possible because theoretical plant CN isoscapes have recently been developed for the African continent based on (1) plant d13C values related to modeled relative cover of C3 and C4 vegetation (Still and Powell 2010) and (2) plant d15N values related to climate and plant physiology proxies (Craine et al. 2009). We used a triple-isotope (C, N, H) approach related to the distribution of modeled plant d13C and d15N and precipitation d2H values to produce a model feather isoscape surface, which was in turn derived from known diet-tissue isotopic discrimination factors related to feather growth. We combined these isoscape models into two (d13C and d15N) and three (d13C, d15N, d2H) isotope feather isoscapes for potential use in the assignment of feather origins from Africa. We tested our assignment models using published and unpublished feather C, N and H isotope data from sub-Saharan Africa. The findings revealed that spatial models of expected plant and precipitation isotope values can be combined with known diet-tissue isotope discrimination factors to produce predictive tissue isotope surfaces. These surfaces can be used in a likelihood-based assignment framework to predict the molting origins of Palearctic-African migrants.

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METHODS
Isoscapes
A raster surface of the theoretical spatial d13C distribution of plants was obtained from Still and Powell (2010). This d13C isoscape was created using an algorithm that first identified potential C4 vegetation cover by identifying herbaceous vegetation located in the C4-favored climatic zones; non-herbaceous vegetation cover (principally shrubs and trees) was assumed to be composed of C3 plants. The algorithm then integrated data on fractional cover of crop types to account for managed agroecosystems that may violate natural climatic constraints. Finally, the predicted d13C value for land grid cells was based on the relative amounts of C3 and C4 vegetation in each cell by using fixed endmember d13C values for C3 (27%) and C4 (12%) plants (Still and Powell 2010). The plant d15N isoscape was based on a model developed by Craine et al. (2009). This model derived climatic covariates (mean annual precipitation, mean annual temperature) associated with a global survey of plant foliar d15N values. Other considered covariates included foliar N concentration and method of N fixation used by the plant. The amount-weighted mean growing-season precipitation d2H isoscape (hereafter d2Hp) was derived from Bowen et al. (2005; available at www.waterisotopes.org). This surface was based on a long-term dataset provided by the International Atomic Energy Agency (IAEA/WMO 2011). The interpolated d2Hp raster accounted for altitude and was weighted by long-term monthly precipitation amount. Similarly, a d2H raster was made and constrained only to those months corresponding to winter precipitation (December through January) when feathers were grown (hereafter d2HDec-Feb). The winter raster was created using the on-line tool IsoMAP (http://isomap.org), based on a derived model with minimum temperature and precipitation amount as covariates (Mitchell and Jones 2005) comprising the best model (Van Wilgenburg 2011). Using feather d2H data from known-origin songbirds reported in Bowen et al. (2005), and Reed Warblers (Acrocephalus scirpaceus; P. Procha zka, unpublished data), feather d2H was rev www.esajournals.org

gressed against d2Hp from the isoscape of Bowen et al. (2005). The resulting calibration equation (d2Hf 6.77 1.42d2Hp), was used to create a feather d2H isoscape. For d13C values, a 1% isotopic discrimination between plants and herbivorous insects was assumed (DeNiro and Epstein 1981, Peterson and Fry 1987, France and Peters 1997). An additional 1% discrimination between insects and bird feathers was further assumed (Hobson 2007). Similarly, a 5% trophic discrimination was applied to the d15N isoscape, assuming approximately 2.5% from plant to insect, and a subsequent discrimination from insect to feather of approximately 2.5% (Vanderklift and Ponsard 2003). A series of GIS operations (Spatial Analyst extract by mask) and resampling of thee isoscapes was then conducted to ensure that all three isoscapes had matching spatial extents and resolutions. As a result, the spatial extent of the feather d2H and d13C isoscapes were reduced to match the d15N isoscape; whereas the spatial resolution of the d13C isoscape (,0.018) and the d15N isoscape (0.178 ) were resampled to match the coarser resolution of the d2H isoscape (0.338). All geographic manipulations were conducted using ArcGIS version 9.1 (ESRI, Redlands, California, USA). Feather isoscapes for d13C, d15N, and d2H were exported and merged into a single data matrix, and the data were then imported into the R statistical computing environment (R Development Core Team 2011) for further manipulation and analysis. The calibrated isoscapes were examined for geographic structure in order to find natural groupings in multivariate space. This was accomplished by cluster analysis using the partitioning around medoids (PAM clustering algorithm, a form of clustering related to Kmeans that is based on medians as opposed to means (Jain 2010)). The PAM algorithm selects data points to serve as medoids, representing potential cluster centers, and attempted to assign each data point to the nearest medoid. The algorithm sought to minimize the sum of the dissimilarities of the observations to the closest representative medoid (Jain 2010). However, the algorithm is cumbersome on large spatial data sets, thus, cluster analysis was conducted using the clara algorithm which is based on the PAM algorithm, but selects an optimal set of medoids 3
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over a series of random samples of the data to reduce computational complexity. Our analyses were conducted using Euclidean distance as the dissimilarity measure. Since predetermination of the optimal number of clusters is a major challenge, a cross-validation criterion known as prediction strength was employed (Tibshirani and Walther 2005). A random sample of 15% of the 14923 rows of data derived from the isoscapes was selected for calculation of prediction strength, and these data were randomly split into equally sized prediction and validation sets. Both sets were then classified by the clustering algorithm and prediction strength was computed by estimating the proportion of observations in the test set in which the observation was placed in the same cluster by applying cluster analysis within the test set versus using the algorithm derived using training sample; here, the minimum of the proportions is treated as the prediction strength (Tibshirani and Walther 2005). Cluster analysis and the calculation of prediction strength was conducted using the fpc package within R 2.13.0 (R Development Core Team 2011). We constrained cluster analyses to a minimum of two and to a maximum of ten clusters. Each iteration included cross-validation using 50 divisions of the data into training and validation sets for determining prediction strength. Prior to cluster analysis, each data set was standardized by subtraction of a given variables mean and division by the standard deviation of that variable (i.e., z-scores) to account for different measurement ranges between variables. Multiple cluster analyses were conducted, one using three isotopes (d13C, d15N, d2Hp), and one based upon three isotopes but substituting d2HDec-Feb for d2Hp, and finally one based on d13C and d15N alone. For clarity, we have documented the workflow for the creation of a triple isotope (d13C, d15N, d2Hp) isoscape in Fig. 1. Based on the a priori assumption (and caveat) that our initial calibration of the isoscapes accurately reflected values expected for sampled feathers, each isoscape combination was used to devise feather origin assignment algorithms. Linear discriminant function analysis (DFA) was used to derive algorithms that predicted the posterior probability that a sample with a given multi-isotope (e.g., CNH) composition
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could have originated from any given cluster within Africa, given the predicted ranges for feather d13C, d15N and d2H (see above). A leaveone-out cross-validation was used to examine the accuracy to which DFA classified GIS cells from the isoscape relative to the original cluster assignment from the cluster analysis. All discriminant function analyses were conducted using the MASS library (Venables and Ripley 2002) in R 2.13.0 (R Development Core Team 2011). In addition to cross-validation, we examined sensitivity of assignments to our choice of discrimination values. To that end, we generated hypothetical birds with isotopic feather compositions that were identical to the cluster mean values (reported in Table 1), and should thus be assigned to cluster without error. We then replicated these hypothetical samples by applying a series of systematic upward and downward shifts to one isotope at a time (holding the other variables constant at the cluster mean), with the adjustments ranging from 0150% of the cluster mean. Using the aforementioned DFA, we assigned these samples to the clusters, and we graphically assess the proportion correctly assigned to their original cluster relative to the degree of adjustment made to the isotopic values. Isotopic data from feathers grown in Africa for two (d13C, and d15N) and three isotope (d13C, d15N, d2H) measurements were obtained from previously published studies (see references in Tables 4 and 5) and from our own data. The discriminant functions were used to assess the likelihood that each isotopic cluster represented a potential origin for the data from the literature. Since individual data values were not available for most of the published studies, reported means were used for feather d13C, d15N, and d2H values in assignment tests.

RESULTS
Isotopic spatial cluster analyses
Predicted feather isoscapes for Africa showed moderately strong spatial isotopic gradients. The feather d2H isoscape ranged from 76.3 to 35.8%, which contrasted with a slightly narrower isotopic range of 74.0 and 21.7% for the d2HDec-Feb derived isoscape but regional contrasts were more striking (Fig. 2A vs. 2B, respectively). For example, sub Saharan regions were generally 4
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Fig. 1. Workflow for creation of a multi-isotope (d13C, d15N, d2H) isoscape and assignment of literature review data to the resulting isoscape. Note that the depicted workflow is for the creation of a multi-isotope (d13C, d15N, d2H) isoscape; workflow was identical minus the inclusion of the d2H isoscape when creating the dual isotope (d13C, d15N) isoscape. Table 1. Summary of the feather isotopic (d2H, d13C, and d15N, in %) composition of cells within isoscapes falling within regions defined by cluster analysis of those isoscapes. Hydrogen isotope data represents a calibration of the predicted amount-weighted growing-season average isotopic values to feather equivalent values (see Methods).
d2H Cluster 3-1 3-2 3-3 3-4 mean 24.3 37.5 22.2 6.7 SD 14.9 8.4 7.5 12.7 Min 68.7 76.3 39.5 25.7 Max 14.5 7.7 6.0 35.8 Mean 22.1 14.4 12.3 11.1 SD 2.5 2.3 2.0 1.9 d13C Min 25.0 25.0 25.0 25.0 Max 13.6 10.0 10.0 10.0 mean 9.4 9.5 10.8 11.8 SD 0.8 0.5 0.8 0.9 d15N Min 5.4 5.7 8.5 9.1 Max 13.7 10.8 14.1 20.4

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Fig. 2. d2H, d13C, and d15N isoscapes for feathers grown in Africa. Portrayed are (A) Feather d2H isoscape calibrated from amount-weighted growing-season d2H in precipitation; (B) Feather d2H isoscape calibrated from amount-weighted d2H in precipitation during the three months of highest precipitation (DecFeb); (C) predicted d13C in feathers calibrated from the isoscape of Still and Powell (2010) by assuming 2% discrimination from plants to feather; and (D) predicted feather d15N isoscape constructed by applying a discrimination of 4% to the isoscape of Craine et al. (2009).

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Fig. 3. Multi-isotope isoscape for Africa, derived from cluster analysis of the d2Hp, d13C, and d15N isoscapes (see Methods).

more enriched in 2H across the region for the December to February period than for the mean annual period. The opposite was generally the case for the southern region of the continent. Feather d13C was estimated to range between 10.0 and 25.0% (Fig. 2C), and feather d15N ranged between 5.4 and 20.4% (Fig. 2D). Combined analysis of d13C, d15N, and d2H feather isoscapes (clusters depicted with the prefix 3) suggested that partitioning the isotopic data into four clusters provided the optimal configuration based upon prediction strength. Across 50 randomized clustering experiments, sample pairs were placed in the same cluster ;88% of the time (prediction strength 0.88 6 0.05 [mean 6 SD]) using a four cluster configuration. Feather Cluster 3-1 had the most isotopically depleted 13C and 15N values and was the secondv www.esajournals.org

most depleted cluster with respect to 2H (Table 1). This cluster was associated with the Congo Basin, north Africa, and pockets of west Africa, southern Africa, and Madagascar (Fig. 3). These locations are principally associated with topographic (e.g., high elevation in Ethiopia and eastern Madagascar) and climatic features (e.g., high rainfall in the Congo rain forest, Mediterranean rainfall in North Africa and southwest South Africa, and very low rainfall in western South Africa and Namibia) that primarily determine the isoscape spatial patterns that are reflected in our cluster analysis. Feather Cluster 3-2 was the most isotopically depleted in 2H and was the second-most depleted cluster for 13C and 15N (Table 1). Geographically, this cluster was primarily associated with Angola, Zambia, Zimbabwe and southern portions of the Democratic Republic of Congo, but 7
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Fig. 4. Degree of overlap in multi-isotope (d2Hp, d13C, and d15N) space between isotopic provinces derived from cluster analysis of the d2Hp, d13C, and d15N isoscapes (see Fig. 3 and Methods). Depicted are 90% concentration ellipsoids about the cluster centroid.

also to extreme west Africa, central South Africa and much of Madagascar (Fig. 3). Many of these regions experience intermediate rainfall amounts and are dominated by savanna vegetation with extensive tree cover. Feather Cluster 3-3 was associated with a broad arc spanning the Atlantic coast of Senegal, south and east toward the Congo Basin, along Africas eastern shoreline and westward into Botswana and Namibia (Fig. 3). These regions are also associated primarily with savanna vegetation. Feather Cluster 3-4 represented areas of Africa that were the most isotopically positive for all three isotopes (Table 1) and was largely
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associated with extremely arid regions with sparse vegetation like the horn of Africa and the north-eastern Sahel region (Fig. 3). In general, the isotopic clusters separated well in multivariate space, with relatively low overlap between 90% confidence ellipsoids between clusters (Fig. 4). This was supported by a crossvalidated DFA, which suggested that 94% of GIS cells could be accurately placed back into the correct multi-isotope clusters; however, the classification accuracy differed among clusters (Cluster 3-1 95%, Cluster 3-2 92%, Cluster 3-3 99% and Cluster 3-4 84% ). Sensitivity analysis suggested that each cluster 8
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Fig. 5. Sensitivity of assignments to changes in assumed discrimination values. Data were generated with values initially equivalent to the cluster mean values for d13C, d15N, and d2H, and were subsequently altered by systematic addition or subtraction of a proportion of the mean, with one variable altered while the other two were held constant (see Methods). Samples were then assigned to clusters using a discriminant function (see Methods), and the cumulative proportion of samples that were correctly assigned are shown.

varied in the sensitivity of assignments to variation in the discrimination factor (Fig. 5). In general, assignments were most sensitive to assumed discrimination factor for d15N (Fig. 5). When d15N was altered by 11%, all samples from cluster 3-2 were correctly assigned; however, changing d15N by 1163% from the cluster mean (corresponding to !1% increase in d15N)
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resulted in those samples being assigned to cluster 3-3, and departures of 63150% from the cluster mean resulted in the samples being assigned to cluster 3-4. Similarly, all samples were correctly assigned to cluster 3-3 when d15N was altered by ,13% from the cluster mean; when the values were !13% (;1.4%) lower than the cluster mean, the samples were assigned to 9
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Fig. 6. Multi-isotope isoscape for Africa, derived from cluster analysis of the d2HDec-Feb, d13C, and d15N isoscapes (see Methods).

cluster 3-2. In contrast, samples from cluster 3-3 were assigned to cluster 3-4 when d15N was !26% (;2.8%) higher than the cluster mean. Cluster 3-1 was the cluster most sensitive to changes in d13C discrimination. All samples from cluster 3-1 were correctly assigned when samples were within 21% of the cluster mean (Fig. 5), but were being assigned to cluster 3-2 when 2154% was subtracted from d13C (;4.611.9%), and departures of 55150% from the cluster mean resulted in the samples being assigned to cluster 3-3. With respect to d2Hf, cluster 3-2 was the most sensitive to a change in d2Hf (Fig. 5); however, a !47% departure from the cluster mean (.18%) was necessary before any samples were incorrectly assigned. Substituting d2HDec-Feb for predicted feather 2 d H based on d2Hp, the cluster analysis of the isoscapes (depicted with the prefix H) resulted in
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a different clustering pattern (Fig. 6), likely as a result of strongly contrasting isotopic patterns between moist forest versus savannah regions (Fig. 2B). The two-cluster solution had a mean prediction strength of 98 6 2% across 50 randomized trials. The clusters largely divided the African continent into areas of forested and woodland regions (Cluster H-1) versus open grasslands, agricultural areas and deserts represented by Cluster H-2 (Fig. 6). These broad categories were consistent with the isotopic variation represented by the cells within the isoscapes, with all three isotopes being isotopically more depleted within Cluster H-1 and more enriched in Cluster H-2 (Table 2). DFA suggested that .99% of the cells could be correctly assigned into the correct cluster based on d13C, d15N, and d2HDec-Feb. When excluding the feather d2H isoscape, 10
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Table 2. Summary of the feather isotopic (d2H, d13C, and d15N, in %) composition of cells within isoscapes falling within regions defined by cluster analysis of those isoscapes. Hydrogen isotope data represents a calibration of the predicted isotopic composition of rainfall from December through February.
d2H Cluster H-1 H-2 Mean 22.9 1.1 SD 15.0 8.1 Min 74.0 36.2 Max 13.3 21.7 Mean 17.9 11.7 SD 4.5 2.0 d13C Min 25.0 25.0 Max 10.0 10.0 Mean 9.5 11.2 SD 0.7 0.9 d15N Min 5.4 9.1 Max 14.1 20.4

cluster analysis resulted in optimal clusters based on the remaining d13C and d15N isoscapes (clusters depicted with the prefix 2, Fig. 6). Mean prediction strength across 50 randomized clustering experiments was ;89 6 5%. Feather Cluster 2-1 was the most depleted with respect to 13C and 15N (Table 3), and was associated with north Africa, high elevation areas of Ethiopia, and areas near Lake Victoria and in Angola, South Africa , Zimbabwe, and Madagascar (Fig. 7). Feather Cluster 2-2 was largely south of the Sahel and mostly surrounded the Congo Basin, but also included large areas in West Africa, South Africa, Ethiopia and Madagascar (Fig. 7). This cluster was the second-most depleted for 15 N and the third-most depleted for 13C (Table 3). By contrast, Cluster 2-3 was the second-most depleted cluster with respect to 13C, but had intermediate d15N values (Table 3), was largely limited to the Congo Basin, but also represented pockets of west Africa (Liberia and Sierra Leone), Angola, Namibia, and South Africa (Fig. 7). Feather Clusters 2-4 and 2-5 were largely limited to the Sahel and Kalahari Desert regions (Fig. 7), and these clusters were the most isotopically enriched for C and N isotopes (Table 3). Cross-validated DFA suggested that ;95% of GIS map cells could be accurately placed back into their correct two-isotope clusters. Classifica-

tion accuracy was high for all clusters, varying from 91% to 97% (cluster 2-1 96%, cluster 2-2 95%, cluster 2-3 97%, cluster 2-4 96%, and cluster 2-5 91%).

Comparison of isotopic assignments to published studies

Previously published data were obtained for five species having feather d13C and d15N isotope data for Africa, and nine species having feather d13C, d15N and d2H analyses (Table 4). The discriminant functions were applied to assign those samples to the isoscape clusters (above). For studies that included three isotopes, they were also reanalyzed using only two isotopes (d13C, d15N) to examine the predictive power of the dual isotopes compared to all isotopes combined. The DFA analysis predominantly assigned birds (i.e., .80%) to single cluster categories using the two- and three-isotope combinations (Tables 4, 5). Using three isotopes, data reported for Blue Swallow (Hirundo atrocaerulea; Wakelin et al. 2011), Aquatic Warbler (Acrocephalus paludicola; Pain et al. 2004), River Warbler (Locustella fluviatilis; Yohannes et al. 2005), and Pied Flycatcher (Ficedula hypoleuca; Hjernquist, personal communication) showed reasonable associations (i.e., .20%) for two clusters. For assignments based on feather d13C and d15N

Table 3. Summary of the feather isotopic (d13C and d15N, in %) composition of cells within isoscapes falling within regions defined by cluster analysis of those isoscapes.
d13C Cluster 2-1 2-2 2-3 2-4 2-5 Mean 24.1 14.9 21.8 12.1 10.9 SD 1.3 1.9 1.8 1.6 1.8 Min 25.0 18.6 25.0 17.9 25.0 Max 19.0 10.0 18.3 10.0 10.0 Mean 8.5 9.6 9.8 10.5 12.1 SD 0.6 0.4 0.5 0.4 0.7 d15N Min 5.4 7.7 8.5 9.5 11.1 Max 9.5 10.8 13.1 11.4 20.4

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Fig. 7. Two-isotope isoscape for Africa, derived from cluster analysis of the d13C and d15N isoscapes (see Methods).

values, more than one cluster was also supported for Willow Warbler (Phylloscopus trochilis; Bensch et al. 2006), Reed Warbler (Procha zka et al. 2008), Blue Swallow (Wakelin et al. 2011), and Great Reed Warblers (Acrocephalus arundinaceus; Yohannes et al. 2011).

Known origin African feather samples

Consideration of how well the isoscape cluster analysis agreed with known-origin feathers for Africa provided a general evaluation of our approach. This assessment was not intended to be an exhaustive analysis or proof of the wintering ecology and distribution of species in these test cases, but rather a brief evaluation of the degree of agreement or disagreement with inferences based on the scientific literature. 1. Blue Swallow.Wakelin et al. (2011) measured d13C, d15N and d2H values in feathers of
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juvenile Blue Swallows from colonies in three areas of southern Africa. This is an isotopically diverse region of Africa with many of our clusters represented by numerous small fragments. The DFA from our assignment placed birds from South Africa and Swaziland largely to Cluster 3-3, and secondarily to Cluster 3-2. Birds known to molt in eastern Zimbabwe were correctly placed in Cluster 3-2 and those from southwestern Tanzania were feasibly placed in Cluster 3-3. However, Malawi corresponds mostly to Cluster 3-2. Considering only d13C and d15N data (Fig. 7), the DFA analysis placed most birds from South Africa and Swaziland to Cluster 2-2 (probability of 0.84) and 2-4 (0.16), those from Zimbabwe correctly in Cluster 2-2 (0.91), and those from Malawi and Tanzania less well to Clusters 2-4 (0.75) and 2-5 (0.25). From this validation test, it appeared that the multi-isotope 12
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Table 4. Assignment of published feather isotope mean (6SD) d13C, d15N, and d2H values (%) to clusters defined by multi-isotope isoscapes. Values in the Assignment to Cluster columns represent likelihoods that the sample means for d13C, d15N and d2H could have been generated via feather growth in the given isotopic cluster (see Methods). Note: location refers to the country or region in which the feathers were sampled in the original study.
Isotope values Species/location N d13C d15N d2H Assignment to cluster 3-1 3-2 3-3 3-4

Blue Swallow (Hirundo atrocaerulea) South Africa/Swaziland 40 16.2 6 1.7 10.4 6 1.0 Zimbabwe 11 15.6 6 1.1 10.1 6 0.6 Malawi/Tanzania 9 15.9 6 1.6 11.7 6 1.3 Amur Falcon (Falco amurensis) Naboomspruit A 10 17.6 6 3.5 7.8 6 2.5 Naboomspruit J 4 17.5 6 5.6 7.2 6 0.8 Middleburg A 10 19.6 6 2.0 6.9 6 2.2 Middleburg J 7 20.0 6 2.4 6.2 6 2.4 10 15.5 6 2.2 8.3 6 1.6 Ventersdorp A Ventersdorp J 10 17.9 6 2.5 8.2 6 2.2 Heidelberg A 13 20.0 6 3.0 7.2 6 2.9 Newcastle A 10 19.6 6 3.5 7.4 6 2.9 Newcastle J 11 20.3 6 3.7 7.5 6 2.7 Graaff-Reinet J 3 21.8 6 1.5 6.6 6 1.9 Aquatic Warbler (Acrocephalus paludicola) West Africa? 133 20.5 6 3.2 10.9 6 1.5 Djoudj National Park, Senegal (Diadie ` me)} 17/17/6 24.6 6 1.5 8.9 6 0.6 Djoudj National Park, Senegal (Tiguet)} 37/38/32 24.4 6 2.2 8.9 6 0.8 Djoudj National Park, Senegal (Grand Lac)} 15 22.7 6 2.7 9.7 6 1.4 Great Reed Warblers (Acrocephalus arundinaceus) Unknown# 4 15.3 6 3.0 7.9 6 0.6 unknown|| 183 16.1 6 1.7 8.9 6 3.1 unknown 16 18.6 6 3.6 9.3 6 1.7 River Warbler (Locustella fluviatilis) unknown 20 16.2 6 3.6 8.1 6 1.8 Unknown# 28 15.0 6 3.6 7.8 6 1.7 Whitethroat (Sylvia communis) unknown 15 22.5 6 1.9 11.1 6 2.7 Unknown# 40 22.0 6 1.9 11.1 6 2.9 Marsh Warbler (Acrocephalus palustris) Unknown# 35 14.3 6 4.4 7.7 6 1.9 Collared Flycatcher (Ficedula albicolis) South-central Africa 45/26 18.7 6 0.8 7.2 6 1.4 Pied Flycatcher (Ficedula hypoleuca) Sub Saharan west Africa}} 12 19.8 6 1.4 7.0 6 1.0

25.2 6 6.7 59.9 6 7.5 43.2 6 10.8 40.4 6 12.7 46.5 6 8.5 31.0 6 15.5 62.5 6 12.5 37.2 6 16.1 62.2 6 12.4 37.2 6 6.9 41.2 6 14.2 54.3 6 19.4 63.6 6 13.3 73.2 6 8.9 72.6 6 11.2 69.4 6 5.1 68.9 6 5.2 26.7 6 0.6 65.9 6 8.9 52.6 6 10.1 8.5 6 11.6 3.3 6 10.4 40 6 26.3 32.7 6 25.1 0.1 6 15.9 51.0 6 15.5 31.7 6 9.9

0.05 0.00 0.01 0.01 0.00 0.32 0.00 0.00 0.00 0.35 0.16 0.09 0.10 0.38 0.99 0.99 0.93 0.00 0.00 0.06 0.12 0.02 1.00 1.00 0.01 0.01 0.41

0.25 0.98 0.15 0.99 1.00 0.68 1.00 0.99 1.00 0.65 0.84 0.91 0.90 0.61 0.01 0.01 0.07 0.99 1.00 0.94 0.77 0.84 0.00 0.00 0.81 0.99 0.59

0.70 0.02 0.85 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.01 0.00 0.01 0.11 0.14 0.00 0.00 0.18 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Note: Sources are: Wakelin et al. 2011; Symes and Woodborne 2010; Pain et al. 2004; }Oppel et al. 2011; #Yohannes et al. (2007); jjYohannes et al. 2008a; Yohannes et al. 2011; Yohannes et al. 2005; Hjernquist et al. 2009; }}Hjernquist, personal communication.

isoscape provided a more reliable assignment. 2. Amur Falcon.Symes and Woodborne (2010) collected feathers of adults and juveniles across a latitudinal gradient in South Africa. These sampling sites fell along an isotopic divide between clusters 3-1 and 3-2. Cluster analysis overwhelmingly placed both the adults and
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juveniles into Cluster 3-2, with a lesser association with Cluster 3-1 (e.g., Adults at Middleburg (0.32), Heidelburg (0.35) and Newcastle (0.16) all of which were located in cluster 3-1). Although caution is warranted in applying the d2Hf approach to some raptor species (reviewed in Greenwood and Dawson 2011), the assignment 13
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Table 5. Assignment of previously published feather isotope means (6SD; d13C, d15N) to clusters defined by our feather isoscape. Values in the Assignment to Cluster columns represent likelihoods that the sample means for d13C and d15N could have been generated via feather growth in the given isotopic cluster (see Methods). Note: location refers to the country or region in which the feathers were sampled in the original study.
Isotope values Species/location Willow Warbler (Phylloscopus trochilis) West Liberia te dIvoire Co Nigeria Cameroon Central East Uganda Kenya Tanzania South Zimbabwe Botswana South Africa Reed Warbler (Acrocephalus scirpaceus) W. Africa? E. Africa? Barn Swallow (Hirundo rustica) SE Africa? Nigeria? South Africa} N d13C d15N 2-1 Assignment to cluster 2-2 2-3 2-4 2-5

47 7 15 13 8 26 12 5 8 27 4 14 9 83 54 46 18 98

18.9 20.9 20.9 15.5 20.3 16.4 12.9 19.5 19.6 19.7 18.7 19.1 21.0

6 6 6 6 6 6 6 6 6 6 6 6 6

3.5 0.8 1.1 4.1 1.9 3.8 2.4 1.2 1.5 1.7 0.7 1.8 1.0

8.7 6 1.9 7.7 6 1.0 9.6 6 1.3 7.7 6 1.6 9.9 6 2.1 8.5 6 1.5 8.0 6 1.0 9.1 6 0.5 8.8 6 2.2 10.2 6 2.4 11.3 6 1.4 9.2 6 1.9 11.2 6 2.8 9.8 6 1.7 10.5 6 2.0 11.6 6 0.8 11.6 6 0.8 11.7 6 1.4

0.07 1.00 0.00 0.00 0.00 0.00 0.00 0.02 0.10 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00

0.20 0.00 0.00 1.00 0.02 0.99 1.00 0.08 0.05 0.10 0.49 0.19 0.01 0.93 0.28 0.01 0.01 0.04

0.73 0.00 0.99 0.00 0.98 0.01 0.00 0.90 0.85 0.90 0.20 0.80 0.99 0.00 0.00 0.00 0.99 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.31 0.00 0.00 0.07 0.72 0.78 0.00 0.86

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.21 0.00 0.10

14.7 6 4.1 14.4 6 3.9 15.3 6 2.0 21.4 6 2.6 17.2 6 1.8

Note: Sources are: Bensch et al. 2006 ; Procha zka et al. 2008; Evans et al. 2003; }Sze p et al. 2009.

results using a triple isotope isoscape appeared largely successful. 3. Aquatic Warbler.This species was the focus of two studies (Pain et al. 2004, Oppel et al. 2011) aimed at evaluating its enigmatic winter distribution using isotopic measurement of Africangrown feathers that were sampled in Europe (Pain et al. 2004) and in Africa (Oppel et al. 2011). In both papers the evaluation appeared to be largely discouraging, with Pain et al. (2004) unable to assign origins beyond West Africa, and Oppel et al. (2011) finding large isotopic variability among individuals wintering at three wetland sites in Djoudj National Park, Senegal. The close association of this species with wetlands that are subjected to numerous processes that might cause isotopic variability (e.g., intense evaporation, flood, drought cycles) undoubtedly makes for a difficult assignment case. Our own DFA assignment to clusters placed the species overwhelmingly in Cluster 3-1 for the Oppel et al. (2011) and in Clusters 3-1 and 3-2 for the Pain et al. (2004) samples of unknown origin. Senegal, in general, was classified as Cluster 3-3. Clusters 3-1 and 3-2 in West Africa corresponded to
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Guinea, Sierra Leone and Liberia with small pockets along the coast of the Gulf of Guinea and a larger extent through Cameroon and the western Congo. Considering only the d13C and d15N isoscape (Fig. 7), DFA placed most of the Pain et al. (2004) West Africa sample to Cluster 23 (0.97) which was consistent with the triple isotope assignment. However, the Djoudji National Park samples of Oppel et al. (2011) were placed in Cluster 2-1 for the Dadieme (0.94) and Tiguet (0.93) samples, but in Cluster 2-3 for the Grand Lac sample (0.98), none of which agreed with Senegal that is split into Clusters 2-4 and 25. Thus, while our assignment approach provided some improvement over Pain et al. (2004), the isotopic assignment of this species remains problematic, primarily driven by high variance in d13C values. Further research on potential wintering habitats associated with Clusters 3-1 and 3-2 in west Africa, determination of molt phenology, and whether this species is itinerant when molting would useful in the ongoing search for the primary wintering grounds of Aquatic Warblers. 4. Willow Warbler.Bensch et al. (2006) exam14
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ined d13C and d15N in new feathers from Willow Warblers from several sites in west, central, and south Africa (Table 5). Summary data for west Africa showed birds distributed across Clusters 2-1, 2-2 and 2-3, but closer examination of each site showed variance among these countries. In West Africa, our assignment for the Cameroon samples was to Cluster 2-3 (0.73), which was in agreement with our prediction for that country. The same was true for Nigeria where Cluster 2-2 was correctly predicted for the sample and the portions of that country sampled by Bensch et al. te dIvoire samples were predicted to (2006). Co originate from Cluster 2-3 (Table 5) which was consistent with the western portion of the country (Table 5). Liberian samples were predicted to be associated with Cluster 2-1 which was inconsistent with that region, which is principally comprised of Cluster 2-3. For central-east Africa, assignment was problematic because many of these countries included more than one cluster. Uganda is largely comprised of Cluster 2-2 and to a lesser extent Cluster 2-3. Samples collected in Uganda were assigned the highest likelihood of association with Cluster 2-2, thus corresponding well to their origin (Table 5). Kenyan samples corresponded primarily to Cluster 2-3 (0.90) and to a lesser extent Clusters 2-2 and 2-1 though this region was dominated by Cluster 2-1. Tanzanian samples were assigned mostly to Cluster 2-3 and to a lesser extent Clusters 2-1 and 2-2 (Table 5), and the country falls mostly into Cluster 2-2 and to a lesser extent Clusters 2-3 and 2-4. For southern samples, a variety of assignments to various clusters likely reflected the diverse nature of isotopic provinces in this region. DFA assigned the highest likelihood of origin to Cluster 2-3 (0.49) and to a lesser extent Cluster 2-2 (0.31) for samples collected in Zimbabwe, whereas the country is dominated by Cluster 2-2. Samples from Botswana were predicted to come from Cluster 2-3 but the country is dominated by Cluster 2-4. In South Africa, samples were predicted to have originated from Cluster 2-3, however South Africa is relatively evenly split into Clusters 2-1, 2-2, 2-3 and 2-4 (Fig. 7). Overall, feather isotope and cluster analysis of these Willow Warblers indicated only moderate agreement with known sampling location, possibly indicating higher confidence in west Africa than in the south.
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Unknown origin African samples

1. Great Reed Warbler, River Warbler, Marsh Warbler and Whitethroat.In a series of stable isotope studies of feathers for a group of species captured during migration stopover, Yohannes et al. (2005, 2007, 2008a, b, 2011) attempted to gain insight into winter origins and habitat use by these species in Africa, but were restricted to gross classifications of C3 and C4 habitat use based on d13C values. Our DFA analysis of these data showed that Great Reed Warblers intercepted at African stopover sites or on Swedish breeding grounds had feathers highly associated with Cluster 3-2 (Table 4). This species is known to winter across west Africa, and our results placed birds to Guinea and southern Cameroon. Using the dual isotope isoscape, a broader range of origins corresponded equally to these regions and also the Democratic Republic of Congo (Table 5 and Fig. 7), which was in keeping with band recoveries and known winter locations (Yohannes et al. 2008a, b). Using a triple isotope isoscape, River Warblers captured in Kenya after growing feathers in sub Saharan Africa (Yohannes et al. (2007) placed primarily in Cluster 3-2 (0.84) and to a lesser extent to Cluster 3-3 (0.14). Considering areas north of Kenya, possible origins corresponded more with Cluster 3-2 (Table 4), which is a broad band across the western sub Saharan region (Fig. 3). Considering only dual isotope clusters (Fig. 7), Cluster 2-2, which represented a broad band of the southern Sahel, was well supported (1.00). Marsh Warblers captured in Kenya also showed a very similar clustering assignment to Great Reed Warblers using the triple isotope isoscape (Table 4). Whitethroats (Sylvia communis), however, differed from these other species, placing in Cluster 3-1 (Table 4), which was consistent with the Democratic Republic of Congo, western Ethiopia and also Kenya. Assignment of Whitethroat using a dual isotope isoscape placed birds in Cluster 2-3 (1.00), suggesting the Congo as the most likely origin (Fig. 7). 2. Collared Flycatcher and Pied Flycatcher. Hjernquist et al. (2009) provided published (Collared Flycatcher, Ficedula albicolis) and unpublished (Pied Flycatcher) d2H, d13C and d15N data of African grown feathers from known breeding populations in Europe. They reported strong between-year consistency in isotope val15
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ues for individuals, and argued this indicates strong migratory connectivity and philopatry to the wintering sites. Our cluster analysis placed the Collared Flycatcher in Cluster 3-2 (Table 4). This species is known to winter primarily in south-central Africa. Thus, assignments to the triple-isotope isoscape were consistent with Angola, Zambia and the southeastern Democratic Republic of the Congo (Fig. 3). By contrast, the Pied Flycatcher assigned individuals primarily to Cluster 3-2, but also to Cluster 3-1 (posterior probabilities of 0.59 and 0.41 respectively, see Table 4). Since this species is known to be distributed primarily in sub-Saharan West Africa, our analyses suggested that most birds overwintered in the region of Sierra Leone, Guinea and te dIvoire and/or Cameroon. Overall, our Co assignment of these species was in very good agreement with prior information (Hjernquist et al. 2009). 3. Reed Warbler.Procha zka et al. (2008) measured d13C and d15N in African-grown feathers of Reed Warblers from western and eastern breeding populations that were suspected of overwintering in West and East Africa, respectively. We found that the two groups indeed clustered differently with the putative western African group associated strongly with Cluster 2-2, and the eastern African sample divided between Cluster 2-2 (0.28) and Cluster 2-4 (0.72). West Africa samples were consistent with Cluster 2-2 (Table 5), and is consistent with the region of te dIvoire and also Guinea, Sierra Leone and Co southern Nigeria (Fig. 7). Thus, West African samples were consistent with the numerous band recoveries as summarized in Procha zka et al. (2008). Less is known about wintering grounds in east Africa, but Clusters 2-2 and 2-4 in east Africa were consistent with the Lake Chad basin (Fig. 7) where some band recoveries are reported (Procha zka et al. 2008). Again, our isotopic cluster assignment approach supported the available data. 4. Barn Swallow.In a study similar to Procha zka et al. (2008), Evans et al. 2003 examined feather d13C and d15N values of barn swallows breeding in England and Switzerland, and concluded that these two populations wintered in different parts of Africa. Band recoveries from Swiss birds suggested Nigeria as a primary wintering region, whereas those from the UK
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corresponded more to the south and eastern regions of South Africa. DFA assignment to the d13C and d15N clusters (Table 5), placed Swiss birds in Cluster 2-3 (0.99), which corresponded with regions southeast of Nigeria in the Democratic Republic of the Congo and Cameroon and te also with Guinea, Sierra Leone and the Co dIvoire (Fig. 7). Birds from the UK were consistent with Clusters 2-4 and 2-5 (0.78 and 0.21 respectively, see Table 5), which were consistent with more southerly wintering areas in Botswana, Namibia and western South Africa (particularly cluster 2-4, which has the much higher posterior probability). Thus, our assignment to the clusters agreed with the prevailing knowledge regarding migratory connectivity in this species.

DISCUSSION
Based on theoretical plant-based predictions (d13C, d15N) and the climatically driven longterm precipitation d2Hp patterns, we defined distinctive isotopic clusters in Africa that may be useful for assigning origins to migratory birds and other migratory species that grow their tissues there. Strong continental patterns of climatic forcing and the expected covariation of isotopes due to moisture and plant responses to climate resulted in distinct isotopic regions that conformed to general and theoretical expectations. In general, our reconsideration of some of the published feather isotope data for Africa suggests that the isotopic clustering approach should be an extremely powerful tool in the establishment of migratory connectivity. This multi-isotope clustering and assignment approach represents a clear advance over previous studies that attempted to gain insight into winter location or habitat use by Palearctic-African migrants, but which were based on less spatially explicit approaches. However, it must be recognized that the multiisotopic isotopic structure of Africa presents both opportunities and challenges for assignment of migrant organisms to regions primarily due to potential confounding ambiguities for broadly distributed species, especially where any strong prior information to constrain assignments are lacking. So, while multi-isotope assays may well place individuals, species or populations into 16
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isotopic/geographic clusters of high probability, it can be a challenge to distinguish among the number of geographical possibilities where that cluster occurs. Nor is this approach ever to likely result in pinpoint accuracy of determining location, and we should therefore be realistic with regard to methodological limitations. In many cases, combining isotopes with ancillary information on known species distribution within Africa using other data (e.g., geolocators, band recoveries) is extremely useful to help constrain assignment options. Both conventional and new Bayesian approaches to assignment are now able to make use of informative priors, such as band recovery data and genetic structure (Van Wilgenburg and Hobson 2011; Chabot et al., in review). A more fundamental challenge to using isoscape cluster model approaches to track migrant individuals in Africa is the current lack of information in determining which periods of rainfall matter to the foodwebs supporting wintering birds during their molt, particularly for hydrogen isotopes. Rainfall patterns and extremes (e.g., flood, drought) in Africa vary in such a way that the period of rainfall for contributions to feather growth for wintering birds can change both spatially and temporally. Rainfall in many regions of Africa is highly seasonal, with the northern part of the continent experiencing a wet season from April-July, and eastern North Africa a second wet season from September to October. However, in southern Africa the rainy season is essentially reversed, with the wet occurring from November to March. The strong positive shift we showed for d2HDecFeb in the Sahel was driven by low precipitation levels during the dry season period, and is likely a poor indicator of the kinds of assignment we can expect for feathers grown during that period. However, this period of isotopic integration may be more appropriate for the southern part of the continent, as December to February overlaps with much of its wet season. Until more groundtruthed plant and feather d13C, d15N and d2H datasets become available for Africa, we suggest that the amount-weighted mean growing season period of integration for d2Hp be used corresponding to the depictions that were presented here. We note that some researchers have been successful in establishing migratory connections for populations of migrants breeding in Europe
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by identifying those regions of the African continent whose long-term Normalized Difference Vegetation Index (NDVI) patterns best matched population trajectories and survivorship in Europe (Sze p and Mller 2005, Sze p et al. 2006, Ambrosini et al. 2011). Since NDVI is clearly linked to amount and timing of rainfall, these analyses may also be useful to both corroborate isotope assignments and may also ultimately help inform the future development or refinement of isotopic clusters. Another complication in deriving useful feather isotopes for Africa or other continents is of course the issue of identifying which scale is appropriate when averaging isotopic patterns. Many bird species are habitat specialists and if such habitats are rare or highly localized, then generalized isotopic clusters may simply not capture such habitat representations. Similarly, better quantification of cover by trees and shrubs (C3) vs. grasses (C4) or the distribution of agricultural crops may provide an overall better representation of the d13C isoscape regionally, but again, if birds are seeking out specific habitats, such increased resolution may not necessarily increase the accuracy of assignment. Much of the scale issue could be better understood if details of the composition and origins of diets were established for species. For example, are birds feeding locally on insects derived from specific habitats or are they consuming insects derived from many different biomes and over large spatial scales? Variance in isoscapes due to year-specific departures for all three isotopes relative to long-term averages clearly remains a challenge, as is also the relatively sparse d2Hp coverage for Africa in general. In addition, for predicted d13C and d15N isoscapes, the occurrence of anthropogenic activities, such as land clearing and agriculture can influence assignment of birds to clusters, especially for species that are able to make use of these modified landscapes (Still and Powell 2010, Craine et al. 2009). Better maps of the distribution of woody vs. herbaceous layers (rather than tree vs. non-tree) would be an improvement for modeling the d13C isoscape as would be the incorporation of detailed crop use for predicted d13C and d15N isoscapes for Africa. An additional improvement will be to assign more realistic and dynamic variations to grid cell 17
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d13C values based on variations in the tree:grass ratio and observed distributions of d13C values in leaves and soil organic matter (Powell and Still, unpublished manuscript). While we did not directly propagate errors associated with the determination of diet to feather discrimination factors and endmember values associated with the d13C and d15N isoscapes, our sensitivity analysis provided insight into which isotope and cluster combinations are potentially problematic. In particular, Clusters 32, 3-3 and 3-4 were clearly sensitive to variance in feather d15N, whereas Cluster 3-1 was most sensitive to variation in d13C. The shifts in assumed discrimination factor necessary to influence our assignments were fairly large (1 2.2% for d15N), thus assignments using our approach should generally be fairly robust to our choice of discrimination value for most species. Nonetheless, further assessment of sources of error and how they propagate to assignment are now encouraged. In particular, an analysis of how variation in d13C and d15N endmembers used to generate isoscapes influences both clustering and subsequent assignments are now needed. While we used very specific diet-tissue isotopic discrimination factors for feathers, the spatial configuration of the isotopic clusters identified will not change with tissue types because these delineations were based on patterns of rainfall and primary productivity and are thereby independent of tissue type. Further, the data standardization approach used in our cluster approach removed this source of variance. Only the absolute mean isotope values of clusters for various tissues will differ. Ideally, one would eventually obtain known-origin samples from each of these predefined clusters to determine isotopic means, variance and estimate covariance between multiple-isotopes, thus allowing assignment tests by assessing multivariate normal (mvn) probability density functions (Royle and Rubenstein 2004), or by applying DFA, as we have done. While analysis of known-origin African tissues based upon an ambitious sampling scheme may yield slightly different spatial structure than was found here, we suggest that the isoscape clustering approach provided a good first-order approximation of the minimum spatial resolution and geographic structure to
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which assignments can be made. In addition, it is fairly straightforward to convert these isotopic cluster patterns to other tissue isocapes (e.g., bone collagen, hair, muscle) by applying the appropriate discrimination factors, provided they are known. Similarly, as our understanding of isotopic discrimination factors linking feathers to foodweb or precipitation baselines improves over time with new research, then predicted mean tissue values associated with our isotopic clusters can be readily revised.

ACKNOWLEDGMENTS
We thank M.B. Hjernquist and C. Symes for providing unpublished data. Funding to KAH, SVW and LIW was provided by operating grants from Environment Canada. Gabe Bowen and three anonymous reviewers provided constructive comments on an earlier draft of the manuscript.

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