Ethics Statement

Samples from the Schwäbische Alb (Münsingen) and Freiburg have been collected under field work and animal experiment permits granted by the responsible state environmental offices of Baden-Württemberg (RPT Tierversuch-Nr. 1056 and 55-8853.17/0, respectively). Samples collected in Kefermarkt, Austria were approved from the district Freistadt permit Nr. N10-75-2010. Sampling in Rybachy (Russia) was performed under the license from Kaliningrad Regional Agency for Protection, Reproduction and Use of Animal World and Forests. Trapping and sampling permits in Spain were granted by the Consejería de Medio Ambiente of the government of Andalucía and in Bulgaria under permit N243/1.03.2010 from Bulgarian Ministry of Environment and Waters.

Sample collection and allocation

Blood samples from 502 Blackcaps were collected at 12 sample sites across Europe from 2001 - 2011 (Figure 1 and Table 1). Individuals captured in Sevilla, Spain (Sv) during the non-breeding season (January) were determined to be overwintering, short-distance migrants based on two lines of evidence: 1) Sevilla birds had relatively short wings compared to other breeding populations in central Europe, thus travelled a shorter distance during migration, and 2) they were found to be genetically differentiated (at microsatellite loci) from resident Spanish Blackcaps identified in a previous study [20]. The remaining 11 sites were sampled during the breeding season (Table 1). While we assume most to be breeding individuals (supported by our ringing data (data not shown)), we cannot rule out that some spring migrants may have been caught as well. All birds captured at a single sampling site were considered as populations in the following analyses.

Stable isotope values from avian tissues can be used to discriminate between possible wintering grounds of migratory birds in Europe [32]. In particular, stable hydrogen isotope values (δ²H) in keratins (claws and feathers) vary by latitude in Europe and have been previously used to distinguish which individuals winter at either northern or southern latitudes among sympatrically breeding Blackcaps in southern Germany [17,18,20]. To distinguish among NW and SW migrants breeding sympatrically in the Freiburg, Germany (Fr) population in this study, approximately 2 mm of claw material was collected from Blackcaps in the spring of 2011 and analyzed for δ²H at the Environment Canada stable isotope facility in Saskatoon, Canada, using the comparative equilibrium method [33]. Claw δ²H from eight Blackcaps caught in Sevilla, Spain (in Jan. 2011) for this study was used as a southern reference to identify SW migrants (mean δ²H: -51.6‰, SD: 5.8). Due to the observed interannual variation in claw δ²H and the absence of Blackcap reference claws from the British Isles during the 2010-2011 winter, δ²H was analyzed from overwintering Blackcaps and other species (Cyanistes caeruleus, Parus major, Fringilla coelebs, Prunella modularis, Carduelis spinus) captured in Britain, Ireland and Scotland between 2002-2010 as part of this study (unpublished) and a previous study of Bearhop et al. [17]. We used the Bearhop et al. [17] estimate of overwintering C. caeruleus and P. major (2002-2003) as a northern reference (mean δ²H: -94.3, SD: 4.9) and best approximation of the wintering ground signature of NW migrants in this study. Blackcaps caught in Freiburg in the spring of 2011 were assigned to wintering grounds using a 75% probability criterion based on reference claw δ²H ( Figure S1), as previously described in Rolshausen et al. [20].

In addition to Freiburg, Blackcaps from three other collecting sites were analyzed for variation in claw δ²H (Ub, Kf, and Bw – see Figure S1). The distributions of δ²H were found to differ significantly among these four populations (Kruskal-Wallis P < 0.05). Apart from the Freiburg population (see above), only one individual caught in Uebersyren, LU was marginally categorized (78.4% probability) with a NW claw δ²H value among the other populations tested; therefore, we found little indication of NW migrants present at these three additional locations during spring sampling. Due to the lack of longitudinal spatial resolution in claw δ²H for birds in Europe, Blackcap populations (excluding Fr) were therefore categorized as SW or SE migrants using the approximate location of the SW/SE migratory divide in central Europe found in other studies [26–28] and published ring recoveries for this species [25,34–36] as classification criteria. The autumn migratory direction of Blackcaps breeding on the southern coast of Norway (Os) likely encompasses a broad front spanning SW – SE orientations [36,37] and so, while we initially analyzed this population as SW migrants, we also repeated the analyses by considering this population as SE migrants.

PCR and genotyping

Genomic DNA was extracted from blood samples using DNeasy Blood and Tissue kits (Qiagen) following manufacturer’s protocol. A total of 14 microsatellite markers were utilized (Table S1), comprising nine markers developed for this study (Syla1, Syla2, Syla3, Syla12, Syla14, Syla15, Syla18, Syla19, Syla20 [38]) and five markers previously developed for this species (Syl1, Syl2, Syl6, Syl9, and Syl10 [39]). Microsatellite loci were amplified with polymerase chain reaction (PCR) for all individuals in 20µl reactions including: 1x Qiagen CoralLoad Concentrate, 1x Qiagen PCR Buffer (containing KCl and (NH₄)₂SO₄), 0.25 µM labeled forward primer, 0.25 µM reverse primer, 200 µM of each dNTP, 1.25 U Qiagen Top Taq DNA polymerase, and 1.0µl DNA template. Mastercycler Gradient Thermocyclers (Eppendorf GmbH, Hamburg Germany) were used to run PCR under the following conditions: initial denaturing at 94 °C for 5 min, followed by 20 cycles of 94°C for 30 s, 60°C for 30 s (decreased by 0.5°C per cycle), and 72°C for 45 s, followed by 15 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 45 s with a final extension of 72°C for 3 min. Forward primers were labeled with FAM or HEX florescent dyes (Applied Biosystems), PCR products were genotyped on an ABI3130xl Genetic Analyzer (Applied Biosystems), and genotypes were analyzed with PeakScanner v1.0 software (Applied Biosystems). Raw genotype sizes were rounded and binned according to repeat size using TANDEM [40]. The mean number of effective alleles (A_E) and observed and expected heterozygosities (H_O and H_E) were estimated for each population with GenAlEx v6.41[41]. Deviations from Hardy Weinberg Equilibrium (HWE), genotypic linkage disequilibrium (LD) between loci, and the frequency of null alleles were estimated with Genepop v4.1.2 [42]. Estimated P-values from HWE and LD tests were corrected for false discovery rate using the Qvalue program [43].

Genetic structure

Pairwise genetic differentiation among Blackcap populations was estimated with the fixation index D_est (the unbiased estimator of D, [44]) using the R package “DEMEtics” [44,45]; complementary F_ST values estimated with Arlequin v3.5.1.3 [46] were found to be highly correlated with D_est values (Kendall’s tau = 0.637, P < 0.0001) and so, we report only D_est values hereafter. Locus Syl10 has been shown to deviate from HWE in a previous study [20]. We included this marker in our dataset as it has been proven to be highly variable. However, we ran D_est analyses including and excluding this marker for all comparisons and found that D_est values were highly correlated between analyses (Kendall’s tau = 0.842, P < 0.0001). Wilcoxon Sum Rank tests were implemented to investigate the independence of the distributions of D_est values within and among migratory orientations; the Uebersyren NW migrant identified via claw δ²H was excluded from these analyzes.

Isolation-by-distance among Blackcap populations was explored with Mantel tests using Isolation by Distance Web Service v3.23 [47] to investigate the relationship between geographic distance and genetic distance: Euclidean geographic distance among all 12 populations was calculated with the R package “fossil” [48], pairwise genetic distance between populations was calculated as D_est /(1- D_est) following Slatkin [49], and 10,000 randomizations were implemented. Additional Mantel tests were run for only those populations west of the SW/SE migratory divide and those populations east of the divide to investigate differential effects of isolation-by-distance on either side of the SW/SE divide.

The relationship between genetic distance and longitudinal geographic distance was investigated between Blackcap populations located on opposing sides of the SW/SE migratory divide, which was approximated at 13°E (Figure 1); we acknowledge that the contact zone between SW and SE migrants is potentially a broad, diffuse zone where migrants come into contact and here use this precise position as an approximate zone center (consistent with Blackcap ring recovery data [25–27,50]) for analytical purposes only. The latitude of each population was set to zero and the Euclidean (longitudinal) distance between populations was calculated with the R package “fossil” [48]. Spearman-Rank correlations were used to explore the relationship between genetic distance and longitudinal geographic distance between populations. To determine if the relationship between genetic distance and geographic distance was significantly different from random, measures of geographic distance were resampled 10,000x and tested against pairwise genetic distances with Spearman rank correlations. True rho values determined for genetic distance vs. geographic distance were then compared to confidence intervals constructed from random distributions to determine significance of true values. Statistical analyses were conducted with R statistical computing environment [51].

Genetic structure was further investigated from genotype data using a MCMC model-based clustering method implemented with STRUCTURE software v2.3.4 [52]. The most likely number of genetic clusters (K) was assessed assuming admixture and locprior of the individual populations and five iterations were run per K (Burn-in: 100,000; Repetitions: 500,000).

Hierarchical structuring of genetic variance

Populations were grouped by autumn migratory orientation (i.e. NW, SW, SE) and migratory distance (i.e. short-, intermediate-, long-distance) inferred from ring-recoveries (Table 1 and Figure 1; [25,35]). We investigated the effect of hierarchical structuring on the partitioning of genetic differentiation by categorizing individuals by migratory orientation, migratory distance, and the population level (in decreasing hierarchical order) using the R package “HierFstat” [53]. Here, the population level was nested within the level of migratory distance, and migratory distance was nested within the migratory direction level (population/distance/direction); the effect of each level was tested with 10,000 permutations to determine the level of significance of this structuring. Hierarchy A categorizes the Norwegian population (Os) with a SW migratory direction, while Hierarchy B categorizes the same population with a SE migratory direction. The Uebersyren NW migrant identified via claw δ²H was excluded from these analyses.

Genetic diversity

Observed multilocus population heterozygosities were high and ranged from 0.60 - 0.73, with a mean observed heterozygosity of 0.69 and mean number of effective alleles of 4.26 (Table 1). Only 18 of 168 total HWE tests indicated a deficiency of heterozygotes (Q < 0.01), although patterns were not consistent across populations (Table S1). The Syl10 locus deviated from HWE at a high frequency (i.e. in nine populations). In a global analysis including all populations, one comparison was in linkage disequilibrium (Q < 0.05: Syl2/Syl6), but found to be significant only in the Kefermarkt population when populations were analyzed separately. Overall we thus can assume independence between loci used in this study. Allele frequencies at each population per locus are listed in Table S2.

Genetic structure

All birds could be assigned to one single population as STRUCTURE analysis identified the highest posterior probability for the number of genetic clusters to be K=1 (Table S3). Despite this lack of resolution in genetic structure, significant neutral genetic differentiation was evident among some population comparisons (Table 2). Most notably, genetic differentiation was significant between sympatric NW and SW migrants in Freiburg, with D_est = 0.029 (F_ST = 0.007). NW migrants breeding in Freiburg were also found to be significantly differentiated from allopatric populations in Uebersyren, Kalimok, and Sevilla, while SW migrants in Freiburg were undifferentiated from all allopatric populations. Freiburg NW migrants were most genetically similar to populations within/near the contact zone at the SW/SE migratory divide (Table 2).

Within the migrant groups, comparisons among SE migrants had higher D_est values than SW migrants, although distributions did not differ significantly between the groups (Wilcoxon test P > 0.05; Figure 2). Among the migrant groups, comparisons among NW vs. SW migrants had the highest D_est values and significantly differed from lower D_est values estimated from SW vs. SE migrant comparisons (Wilcoxon test P = 0.013; Figure 2). Levels of genetic differentiation among NW vs. SW comparisons did not significantly differ from NW vs. SE comparisons; similarly, NW vs. SE comparisons did not significantly differ from SW vs. SE comparisons (Wilcoxon tests P > 0.05; Figure 2).

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Figure 2. Distribution of D_est values among population comparisons.

Comparisons within NW, SW, SE migratory orientations are shown as open bars and comparisons among NW, SW, SE migratory orientations are shown as shaded bars. Within SW orientations, D_est comparisons did not differ from comparisons within SE orientations (Wilcoxon test P > 0.05). NW vs. SW comparisons were found to have significantly higher D_est values than SW vs. SE comparisons (Wilcoxon test P = 0.013). Remaining comparisons among groups were non-significant (P > 0.05). Width of bar is proportional to sample size.

https://doi.org/10.1371/journal.pone.0081365.g002

Isolation-by-distance

Mantel tests revealed significant isolation-by-distance among all 12 Blackcap populations (Figure 3A: r = 0.424, P = 0.045), although the relationship no longer occurred when the non-breeding, short-distance migrant population in Sevilla is excluded (r = 0.285, P = 0.117). Among the five eastern populations (i.e. those east of the SW/SE divide), isolation-by-distance was found significant (Figure 3B: r = 0.744, P = 0.008). Conversely, isolation-by-distance was non-significant among the seven western populations (Figure 3C: r = 0.364, P = 0.174), even when excluding the non-breeding Sevilla population (r = -0.275, P = 0.754).

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Figure 3. Isolation-by-distance results among Blackcap populations: the relationship between geographic distance (km) vs. D_est genetic distance.

(A) All populations included: r = 0.424, P = 0.045; (B) Eastern populations only: r = 0.744 P = 0.008; (C) Western populations only: r = 0.364, P = 0.174. Os population considered as SW migrants.

https://doi.org/10.1371/journal.pone.0081365.g003

By exploring the relationship between genetic distance and the longitudinal geographic distance of population pairs located on opposing sides of the SW/SE divide, a strong, positive correlation was evident (Figure 4, rho = 0.467 P = 0.005). The least genetic distance was found between populations near the center of the SW/SE divide (approximated at 13°E) and genetic distance increased significantly with increased geographic distance between populations. For example, all pairwise D_est comparisons between Münsingen (Mn), Kefermarkt (Kf), and Vienna (Vn) were found to be non-significant and for the majority of these estimates (5/6 comparisons) D_est = 0, indicating a lack of genetic differentiation between these populations near the center of the zone (Table 2). Permuting the geographic distances between populations determined that the true rho values for genetic distance vs. geographic distance lie outside the confidence interval generated from randomly-sampled rho values (95% CI: -0.337 - 0.333), affirming the observed relationship between genetic and geographic distance is not a random effect. A positive relationship between pairwise genetic distance vs. geographic distance was also apparent after excluding the non-breeding Sevilla population (rho = 0.377, P = 0.040).

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Figure 4. Relationship between geographic distance (km) and D_est genetic distance.

Pairwise measurements estimated between population pairs located on opposing sides of the SW/SE migratory divide revealed a positive relationship (rho = 0.467, P = 0.005). All 12 populations included with the Os population considered as a ‘western’ population.

https://doi.org/10.1371/journal.pone.0081365.g004

Hierarchical structuring of genetic variance

No evidence of hierarchical structuring of genetic variation was found by grouping the populations by three migratory directions and three migratory distances (all F-statistics P > 0.05; Table S4). These results indicate the underlying genetic structure among populations is more complex than simply classifying populations into three migratory orientations and three migratory distances. HierFstat analysis was additionally repeated by categorizing the Norwegian population (Os) as SE migrants (as opposed to SW migrants), but similarly resulted in weak genetic structure; marginally better support for underlying genetic structure was found by categorizing this population as SW migrants (i.e. slightly higher F-statistics, albeit still non-significant).

Genetic structure among Blackcap populations

Overall, genetic differentiation across all the sampled populations was low and Bayesian clustering, pairwise comparisons and hierarchical analyses all failed to show consistent, significant genetic structure among populations corresponding to different migratory behaviors. A positive relationship was found between genetic distance and (longitudinal) geographic distance of population pairs of Blackcaps located on opposing sides of the SW/SE migratory divide. This result is consistent with the historical isolation of western and eastern breeding populations in Europe previously restricted to potential glacial refugia on the Iberian and Balkan peninsulas, respectively [2,7,54]. Despite this observed relationship between genetic and geographic distance, the only SW/SE pairwise D_est comparison found to be significantly differentiated was between Ub and Km (over 2000km apart). Such results indicate a lack of neutral genetic divergence between populations of SW and SE migrants and, in fact, many populations within/near the approximated SW/SE contact zone cannot be differentiated with our marker set. We suggest therefore that the SW/SE migratory divide is an ineffective barrier to neutral gene flow across this zone of contact.

Although isolation-by-distance appears to be maintained among eastern populations, this pattern is not found among western populations and is potentially confounded by the occurrence of sympatrically-breeding populations of NW and SW migrants (as observed in Freiburg, DE). Indeed, NW vs. SW D_est comparisons were found to significantly differ from SW vs. SE comparisons, confirming beliefs that different dynamics of genetic divergence occur with the NW/SW migratory polymorphism and across the SW/SE migratory divide. In southern Germany (i.e. Freiburg), weak, albeit significant, genetic differentiation was found between NW and SW migrants breeding sympatrically, corroborating results of a previous study by Rolshausen et al. [20], who suggest that only low levels of genetic divergence may be needed to maintain differences in migratory behavior.

The low nuclear genetic differentiation found across the SW/SE migratory divide in the Blackcap is consistent with similar patterns found across migratory divides in Eurasian Reed Warblers (Acrocephalus scirpaceus) in central Europe [31] and Willow Warblers (Phylloscopus trochilus) in Scandinavia [12]. A lack of neutral genetic differentiation was similarly found across the Red-billed Quelea (Quelea quelea) migratory divide in Africa [10] and between populations of Black-throated Blue Warblers (Setophaga caerulescens) with different migratory routes in North America [55]. In addition, complete introgression has recently occurred between populations of Savi’s Warblers (Locustella luscinioides) in Iberia despite Pleistocene divergence and long periods of geographic isolation between populations [56]. Without sufficient contemporary reproductive barriers it thus seems that interbreeding often occurs across migratory divides, regardless of historical geographic divergence between populations.

Potentially, NW migrant Blackcaps may also contribute to breeding populations in other areas in central Europe (e.g. in upper Austria, see 27,57 and Czech/Slovak Republics, see 35). Our pairwise D_est comparisons suggest the Fr_NW population is most genetically similar to the Kf population (i.e. the least genetic distance was found among Fr_NW comparisons). This would support the hypothesized origination of NW migrants from within the SW/SE contact zone, first proposed by Helbig [27] from observations of hand-raised Blackcaps from Linz, AU (~25km SW of Kefermarkt) orienting SW-NW directions (mean 268°) during the autumn migratory period. Alternatively, since our δ²H assignment method did not indicate a presence of NW migrants in the Kefermarkt population, this result may indicate that the location of the Blackcap migratory divide has shifted eastward; the low genetic divergence found between Fr_NW vs. Vn supports this hypothesis, potentially being driven by the expansion of SW migrants into eastern European populations, as proposed previously by Wesołowski [58].

Isolation-by-distance (IBD) was found to be non-significant among the 11 breeding populations (after excluding Sevilla). However, patterns of IBD differed on either side of the SW/SE migratory divide: significant IBD was evident among eastern populations (with greater levels pairwise genetic divergence), but not among western populations (with lower levels of pairwise genetic divergence); this result is consistent with the genetic structure of Blackcap mtDNA by Pérez-Tris et al. [28], who found no relationship between genetic and geographic distance among western continental populations (although results may be influenced by the different sets of populations sampled in each study). The relationships between geographic and genetic distance were similarly found to contrast among populations of Eurasian Reed Warblers (Acrocephalus scirpaceus) located on opposing sides of their migratory divide in central Europe [31]. Such contradictory patterns geographically spanning migratory divides may be indicative of different ecological and/or evolutionary dynamics acting on opposing sides of migratory divides in central Europe. Unlike that found for eastern Blackcap populations, the lack of IBD demonstrated among western Blackcap populations (i.e. both SW and NW migrants) suggests a mechanism other than geographic isolation may be promoting genetic divergence among western Blackcap populations in the face of gene flow.

Mechanisms promoting / preventing genetic divergence

Our results provide little evidence that the historical migratory divide in central Europe provides sufficient reproductive isolation to influence mating preferences based on migratory orientation in the Blackcap. Rather, it appears that interbreeding between SW and SE migrants (and potentially also NW migrants) results in high levels of gene flow across the migratory divide where populations come into contact. Asymmetrical gene flow across the SW/SE migratory divide (i.e. SW migrant Blackcaps outcompeting SE migrants) has been argued to be responsible for recent demographic changes at Polish breeding grounds (e.g. advanced arrival time and increased population size)[58]; interbreeding between Blackcaps with different migratory strategies could therefore potentially occur across an even larger area, resulting not in a divide per se, but a gradual transition between migratory orientations across central Europe. Our results indicate an absence of NW migrants in the Białowieża population (from isotope analysis), but cannot exclude the occurrence of SW migrants occurring at this location. In fact, low levels of genetic divergence are found between Białowieża Blackcaps and several SW migrant populations (Ub, Fr_SW, Mn) as well as SE migrant populations (Kf, Vn, Ry), suggesting Białowieża birds are genetically similar to many other European population due to introgression. Additionally, the distribution of claw δ²H from Białowieża birds is roughly consistent with the δ²H distribution of Freiburg SW migrants (although we are unable to distinguish SW from SE migrants based solely on claw δ²H). Our results, together with those of Wesołowski [58], may provide evidence for either an eastward shift in the geographic position of the divide/contact zone (i.e. asymmetrical hybridization) or a widening of the zone in both east and west directions (i.e. symmetrical hybridization) [59,60]. While further studies are needed to better characterize the dynamics of this zone, movement of the SW/SE contact zone remains plausible for Blackcaps.

Recent studies are now utilizing geolocators to record the annual cycle of long-distance passerine migrants, including migratory routes, durations, and strategies [61,62], as for example in the Swainson’s Thrush (Catharus ustulatus) where the existence of a migratory divide in North America has been found [63]. At this narrow migratory divide, low frequencies of Swainson’s Thrush hybrids may be suggestive of post-mating isolation, while evidence for pre-mating isolation (e.g. ecological selection and difference in arrival time) indicates an important role for reinforcement in the maintenance of this hybrid zone which spans a migratory divide [14,19]. Similarly, in Blackcaps both pre- and post-mating isolating mechanisms may likely influence genetic divergence among populations. Crosses between SW and SE migrant Blackcaps produced viable F1 hybrids with intermediate (southerly) orientations [64], but wild-caught Blackcaps from the center of the contact zone (Linz, AT) were found to orient SW-NW (mean 268°) suggesting selection against S-SE orientations (and thus selection against hybrid or SE x SE pairings) at this location [27]. Still, 58% of Blackcaps ringed in the Czech Republic and Slovakia were recovered at SE locations during the non-breeding period [35]. Nevertheless, within the SW/SE contact zone, female preference for males with SE orientations could be responsible for eastward introgression of SW migrant Blackcaps into eastern breeding populations (e.g. Poland) [58].

Rapid genetic divergence between sympatric NW and SW migrant Blackcaps may be promoted by pre-mating isolation. Temporal isolation in spring arrival to breeding grounds in southern Germany has been documented between NW and SW migrants [17,18,30] and presents also an effective isolating mechanism among Swainson’s Thrushes breeding in North America [19]. Such phenological changes can be further affected by global climate change which is impacting the phenology of migratory birds and influencing the advancement of spring migration and arrival to the breeding grounds [58,65,66]. It remains unclear however, if such temporal divergence is locally adaptive in sympatry only, or if NW and SW migrants reflect significant divergence also in allopatry.

We not only find evidence of significant genetic differentiation between NW and SW migrant Blackcaps in sympatry, but NW migrants were significantly differentiated from other populations of SW migrants in western Europe. The observed divergence between NW and SW migrants may be the consequence of a founder effect resulting from a small number of genetically distinct individuals initially establishing the NW migratory route. Allopatric genetic differentiation demonstrated between Fr_NW and SW migrants (i.e. Sv and Ub) may indicate that such divergence is not locally adaptive, but that this polymorphism has evolved repeatedly, independent of adaptive pressures on the breeding ground. The behavioral, morphological, and genetic traits observed to be diverging in the Blackcap may be part of a ‘migratory syndrome’ or ‘migratory gene package’ in this species whereby a suite of correlated traits that evolve together are targeted by correlational selection [67–69]. Whether such syndromes are common and generalized among migratory birds may be debatable [70], but provide an exciting frontier to explore.

Although our Bayesian clustering and hierarchical analyses failed to find significant genetic structure among populations corresponding to the different migratory behaviors investigated, significant (but weak) genetic structure in mtDNA was found among eastern (including SE migrants) and western Blackcap populations (including SW and NW migrants) in a previous study (although only two eastern populations were considered)[28]. Maternally-inherited mtDNA is less likely to introgress across a contact zone than autosomal DNA [71], and reflects only uni-parental gene flow which limits interpretation among populations with highly dynamic migratory behaviors. Migratory orientation may instead be controlled by very few genes [22,24], and the possibility that one or few of microsatellite loci used in our study are linked to functional loci cannot be ruled out, though we did not find any evidence using outlier detection methods (data not shown). Although microsatellites were found to be undifferentiated across the Willow Warbler migratory divide, two AFLPs were shown to exhibit clinal variation corresponding to phenotypic differences [12]. Developing large sets of genetic markers spanning the entire genome and searching for informative markers associated with migratory behavior will provide better resolution in the genetic variation and structure of recently divergent populations, such as the Blackcap [69]. Furthermore, combining molecular and stable isotope markers (e.g. see 72) with data obtained from ring recoveries and geolocators may greatly improve the spatial resolution of species’ movements and thus provide a better understanding of the ecology and evolution of migratory divides.