Amplified fragment length polymorphism analysis identifies hybrids between two subspecies of warblers

In the western Pyrenees (Southwest France and Northwest Spain), a narrow hybrid zone exists between the common chiffchaff Phylloscopus (collybita) collybita and the Iberian chiffchaff Phylloscopus (c.) brehmii. In this zone, which is approximately 20 km wide, mixed matings and individuals singing the songs of both taxa occur at substantial frequencies (24 and 8.6%, respectively), suggesting frequent hybridization. Previous studies have shown very weak mitochondrial gene flow (Nm = 0.065), whereas four microsatellites suggested much higher nuclear gene flow (Nm = 4.9). In this study we used the amplified fragment length polymorphism (AFLP) method in order to identify hybrids and early backcrosses. We typed 91 birds from both allopatric and sympatric areas for 12 informative AFLP markers (of > 141 polymorphic fragments), obtained by screening 13 AFLP primer combinations. These individuals were previously typed for song (brehmii, collybita or mixed singers), mitochondrial DNA (mtDNA) haplotype and allelic genotypes at four microsatellite loci. Assignment tests demonstrated that in the zone of sympatry, a substantial number of intermediate genotypes existed among the birds previously believed to be pure collybita and brehmii, based on song and mtDNA haplotype. The majority of the mixed singers had intermediate genotypes. Our data suggest that the fraction of the adult population having a hybrid origin (hybrids or backcrosses) is in the order of 10%. With such a frequency of genetic hybrids, there would have been much more mtDNA introgression than observed, had female hybrids been perfectly fertile/viable. This result is consistent with male-biased gene flow and Haldane's rule.