Deuterium concentrations (δ2H) have a latitudinal structure across some continents that are linked to their concentrations in precipitation (23). Bird feathers grown at a given place have δ2H values (δ2Hf) reflecting amount-weighted mean growing season precipitation at sites where feathers were grown. Hence, by measuring δ2Hf, it is possible to infer a probabilistic geographical space where this feather might have grown. We used a spatially explicit likelihood assignment method to delineate probable origins for ortolan buntings by converting an amount-weighted growing season precipitation surface (23) to a feather isoscape using a calibration equation developed for Eurasian reed warblers (Acrocephalus scirpaceus; δ2Hf = −10.29 + 1.28* δ2Hp) (24). The residual SD [SD = 10.36 per mil (‰)] from the linear regression model used to calibrate the precipitation surface for Eurasian reed warbler feathers was included in the assignments as an estimate of error. As the ortolan bunting also forages in agricultural habitats, we did not consider feather carbon (δ13C) or nitrogen (δ15N) isotopes because their concentrations may be linked to local agricultural inputs.

Feathers were cleaned in 2:1 chloroform:methanol solvent rinse and prepared for δ2H analysis at the Stable Isotope Laboratory of Environment Canada, Saskatoon, Canada. The δ2H of the nonexchangeable hydrogen of feathers was determined using a method based on two calibrated keratin hydrogen isotope reference materials [CBS (Caribou Hoof Standard) and KHS (Kudu Horn Standard)]. We performed hydrogen isotopic measurements on H2 gas derived from high-temperature (1350°C) flash pyrolysis (EuroVector 3000; Milan, Italy) of feather subsamples (350 ± 10 μg) and keratin standards loaded into silver capsules. We analyzed resultant separated H2 on an interfaced Isoprime (Crewe, UK) continuous-flow isotope-ratio mass spectrometer. Measurement of the two-keratin laboratory reference materials corrected for linear instrumental drift were both accurate and precise with typical within-run mean δ2H ± SD values of −197 ± 0.79‰ (n = 5) for CBS and −54.1 ± 0.33‰ (n = 5) for KHS.We report all results for nonexchangeable H expressed in the typical delta notation, in units of per mil, and normalized on the Vienna Standard Mean Ocean Water–Standard Light Antarctic Precipitation standard scale.

We limited assignments to origin to the known range of the species, where feathers could have grown or molted. We obtained the digital distribution map of ortolan buntings from the BirdLife International and NatureServe (25) and modified it to include known breeding areas in Kazakhstan (see map at www.birds.kz/v2taxon.php?s=577&l=en). Ortolan buntings molt their feathers either on the breeding or wintering grounds; therefore, assignment to origin analyses only included the species’ breeding or wintering range, respectively.

To determine the wintering grounds of ortolan buntings from various breeding populations, we analyzed δ2Hf values from birds captured on their breeding territories. The ortolan bunting molts body coverts twice each year: before the autumn migration on the breeding grounds (August) and before the spring migration on the wintering grounds. Hence, breeding birds in May to June have body coverts molted on their African wintering grounds.

We analyzed samples (scapulars or body coverts) obtained from territorial males captured on their breeding grounds in May to June of 2013 to 2015 in various countries (sample sizes given in brackets): Belarus (n = 43), Finland (n = 139), France (n = 34), Lithuania (n = 43), Poland (n = 31), Serbia (n = 7), Spain (n = 19), and Russia (n = 192). We also analyzed body feathers of migrants captured in spring (April 2015) in Kuwait (n = 45) and Israel (n = 10) to similarly assign individuals to potential wintering grounds. Deuterium concentrations in these scapulars/body coverts (molted in Africa during the winter) revealed two distinct groups of countries representing an eastern flyway: (i) Belarus, Russia, and Serbia, together with spring migrants captured in Israel and Kuwait, and a western flyway: (ii) breeding birds of Finland, France, Lithuania, Poland, and Spain (fig. S1). Each group corresponds to individuals having molted in similar isotopic environments, probably in the same geographical areas.

To determine the potential geographical origins of ortolan buntings captured across France during autumn migration, we measured δ2Hf from live birds captured during migration and released in the wild. In this species, autumn birds either have tail feathers grown in the nest (for juveniles) or molted on the breeding grounds before the fall migration (for adults). We therefore used samples collected from migrating individuals in France in August to September (in 4 years, 2012–2015; n = 40) and samples collected from seized first-calendar-year individuals (n = 34), while we also did the same analysis with samples collected along the eastern flyway in Kuwait in September 2014 (n = 78).

In separate assignments to origin using δ2Hf, potential origins for birds caught along the western (n = 74) and eastern (n = 45) flyways were restricted to their respective breeding areas on either side of the longitudinal migratory divide following political borders, based on results from geolocation data and genetic structure of breeding populations. We used an odds ratio of 2:1 to assign each feather sample to potential origins, where cells in the isoscape in the upper 67% of probabilities were considered as likely (1) origins and all others were considered unlikely (0). We first conducted assignments by country in which birds were captured, and then, we grouped samples based on statistical analyses, grouping national populations with similar δ2Hf values [country effect in General Linear Models (GLMs); see, e.g., fig. S1]. Assignments resulted in a spatially explicit binary surface for individual birds, which we subsequently summed across assignments for all individuals within a group to represent potential origins for that group. For each pixel in the assignment raster, the presented value is the proportion of all sampled individuals, which could potentially have grown the analyzed feathers within the pixel, rescaled to range from 0 to 1.

To determine the latitudinal origin of migrants along the eastern flyway, we performed an assignment to origin analysis on samples collected in September 2015 in Kuwait from wild birds captured with mist nets while migrating. A preliminary assignment was conducted using the 78 individuals captured in Kuwait to the whole breeding range of the species. When restricting the assignment to countries with populations using the eastern flyway (i.e., wintering in East Africa, as defined by the genetics and light logger studies; Fig. 1D), the possible geographical origin of these migrants appears as being mainly southern Russia, southern Ukraine, and northern Romania, as well as Turkey. Given the respective population sizes breeding in these countries, we can consider that most migrants captured in Kuwait likely have origins in Russia, as almost all Russian ortolans (2 million to 4.3 million pairs) breed within the pale to dark blue areas depicted in Fig. 1D. From the assignment surface analysis, Turkish (0.5 million to 1 million pairs) and Romanian (225,000 to 550,000 pairs) populations probably also use this flyway, while Ukraine holds only 58,000 to 67,000 pairs (9).

To determine the latitudinal origin of migrant ortolan buntings captured in August to September of 2012 to 2015 along the western Atlantic flyway (in France), we performed an assignment to origin analysis on samples collected by ringers on wild birds captured in France. We derived a preliminary assignment depiction from the 74 individuals captured by ringers or seized by the police from caged birds and aged as first-calendar-year birds. When restricting the assignment to countries with populations using the western flyway (i.e., wintering in West Africa; Fig. 1C), the possible geographical origin of these migrants appears as being mainly Poland and Germany and, to a lesser extent, France, the Baltic States, and southern Sweden. Very few individuals appear to come from Norway (which has a restricted breeding population), Finland (which may use a more continental route), or northern Russia (which uses the eastern flyway and has a very low population density). Again, it is possible that such a map—restricted to the countries with populations using the western flyway—mirrors local population densities, as the largest populations in the area of concern are located in Poland. The only exceptions here are Finland, which holds 7000 to 19,000 pairs, and Sweden, where population sizes are mostly smaller than in Poland, with a very low probabilistic contribution to migrants captured in France.

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