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Increasingly, stable isotope measurements are being used to assign individuals to broad geographic origins based on established relationships between animal tissues and tissue-specific isoscapes. In particular, the eastern North American population of the monarch butterfly (Danaus plexippus) has been the subject of several studies using established δ2H and δ13C wing-tissue isoscapes to infer natal origins of migrating and overwintering individuals. However, there has been no study investigating potential variance that can derive from sub-sampling different regions of the wings, especially those regions differing in pigmentation (orange versus black). Within-wing isotopic (δ2H, δ13C, δ15N) variance of 40 monarch butterflies collected from natural overwinter mortality on Mexican roost sites were split evenly into two groups: unwashed samples and those washed in a 2:1 chloroform:methanol solvent. Isotopic variance in δ2H and δ13C was related to pigment (within-wing range 5‰ and 0.5‰, respectively), but not region of subsampling. This variance was reduced 3 to 4 fold through solvent washing that removed pigmented surface scales and any adhered oils. Wing δ15N was similarly influenced by pigment (range 0.3‰), but this effect was not reduced through washing. We recommend future isotopic studies of monarchs and other butterflies for migration research to use the same region for sub-sampling consistently and to wash samples with solvent to reduce isotopic variance related to uncontrolled variance in discrimination (δ2H, δ13C, δ15N) and/or adsorbed water vapor (δ2H). These data also need to be included in description of methods.