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Hybrids originating from migratory divides are hypothesized to migrate intermediately relative to the parental populations. Few studies have tested this hypothesis in wild birds, and only in hybrids that have completed the migration back to the breeding grounds. Here, we make use of the well-established migration routes of willow warblers (<jats:italic>Phylloscopus trochilus<\/jats:italic>), for which the subspecies <jats:italic>trochilus<\/jats:italic> and <jats:italic>acredula<\/jats:italic> have migration-associated genetic markers on chromosomes 1 and 5. The genetic approach enabled us to analyze the geographic distribution of juveniles during their first autumn migration, predicting that hybrids should be more frequent in the central flyway over Italy than along the typical SW routes of <jats:italic>trochilus<\/jats:italic> and SE routes of <jats:italic>acredula<\/jats:italic>.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>Blood and feather samples were collected from wintering birds in Africa (<jats:italic>n<\/jats:italic>\u00a0=\u200969), and from juveniles during autumn migration in Portugal (<jats:italic>n<\/jats:italic>\u00a0=\u200933), Italy (<jats:italic>n<\/jats:italic>\u00a0=\u200938) and Bulgaria (<jats:italic>n<\/jats:italic>\u00a0=\u200932). Genotyping was carried out by qPCR SNP assays, on one SNP each on chromosome 1 (SNP 65) and chromosome 5 (SNP 285). Both these SNPs have alternative alleles that are highly fixed (&gt;\u200997%) in each of the subspecies.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>The observed combined genotypes of the two SNPs were associated with the known migration routes and wintering distributions of <jats:italic>trochilus<\/jats:italic> and <jats:italic>acredula<\/jats:italic>, respectively. We found hybrids (HH) among the juveniles in Italy (5\/38) and in Portugal (2\/33). The proportion of hybrids in Italy was significantly higher than expected from a background rate of hybrid genotypes (1.5%) in allopatric populations of the subspecies.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>Our genetic approach to assign individuals to subspecies and hybrids allowed us to investigate migration direction in juvenile birds on their first migration, which should better reflect the innate migratory direction than studies restricted to successful migrants. The excess of hybrids in Italy, suggests that they employ an intermediate route relative to the parental populations. Our qPCR SNP genotyping method is efficient for processing large sample sizes, and will therefore be useful in migration research of species with known population genetic structure.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s40462-020-00209-7","type":"journal-article","created":{"date-parts":[[2020,5,29]],"date-time":"2020-05-29T09:02:43Z","timestamp":1590742963000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Autumn migration direction of juvenile willow warblers (Phylloscopus t. trochilus and P. t. acredula) and their hybrids assessed by qPCR SNP genotyping"],"prefix":"10.1186","volume":"8","author":[{"given":"Tianhao","family":"Zhao","sequence":"first","affiliation":[]},{"given":"Mihaela","family":"Ilieva","sequence":"additional","affiliation":[]},{"given":"Keith","family":"Larson","sequence":"additional","affiliation":[]},{"given":"Max","family":"Lundberg","sequence":"additional","affiliation":[]},{"given":"J\u00falio M.","family":"Neto","sequence":"additional","affiliation":[]},{"given":"Kristaps","family":"Sokolovskis","sequence":"additional","affiliation":[]},{"given":"Susanne","family":"\u00c5kesson","sequence":"additional","affiliation":[]},{"given":"Staffan","family":"Bensch","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2020,5,29]]},"reference":[{"issue":"8","key":"209_CR1","doi-asserted-by":"publisher","first-page":"1744","DOI":"10.1111\/j.1420-9101.2011.02302.x","volume":"24","author":"A M\u00f8ller","year":"2011","unstructured":"M\u00f8ller A, Garamszegi L, Peralta-S\u00e1nchez J, Soler J. 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An ethical permission from Lund-Malm\u00f6 Djurf\u00f6rs\u00f6ksetiska N\u00e4mnd (M45:14) was given to SB for collecting blood and feather samples in Sweden in association with mist-netting and ringing (SB Swedish ringing license nr. 555). Feather samples from Portugal were collected under the licenses 86\/2009, 122\/2009\/CAPT and 92\/2010 issued by CEMPA\/ICNF. Blood samples from Bulgaria were collected under permission 213\/26.6.2009 issued by Ministry of Environment and Water of Bulgaria.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval"}},{"value":"Not applicable.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"22"}}