Genetically, the four Western Palearctic black-and-white Ficedula species are all quite distinct from each other, and the feathers from the Flamborough bird provided a potentially foolproof way of deciding whether it was an Atlas Flycatcher. At the mitochondrial DNA level, the Pied, Atlas, Semi-collared and Collared Flycatchers show about 2.5-3.5% divergence in their sequences. On the first round of analysis we successfully sequenced 3000 base pairs of mitochondrial DNA from 4 separate genes and the Flamborough bird was identical, or almost identical (0 to about ~0.1% divergence) to nominate Pied Flycatchers. While we can occasionally make small mistakes in reading long stretches of DNA sequence, there is no way to make enough mistakes on that scale to turn Atlas Flycatcher DNA into another species. For example for 991 bases of the cytb gene, the Flamborough bird was identical to, or only 1 base pair different from, nominate Pied Flycatchers but 29-35 bases different from Atlas, Collared and Semi-collared Flycatchers. Whatever the Flamborough bird was, it wasn’t an Atlas Fly.
Another initial possibility was that the bird was a Collared x Pied Fly hybrid. The mitochondrial DNA comes down the female line, so to that point we only knew that its mum was a Pied Fly. To find out what its dad was we needed to sequence nuclear DNA, which would be a mixture of genes from both parents. Fixed differences between Collared and Pied Flycatcher genes have been defined previously by other labs – e.g. at position 315 of the RHO-1 gene, Collared Flycatchers always have a ‘G’ and Pied Flycatchers always have an ‘A’. If the bird was a hybrid, at that point the sequencing machine would get confused as an ‘A’ and a ‘G’ went through the reader together. For the Flamborough bird, the sequence was only ‘A’ – its dad was a Pied Flycatcher too.
Iberian Pied Flycatcher was the final possibility. Genetically, the iberiae subspecies is very close to nominate birds but appears to be, just about, reliably distinguishable. At the cytb gene, the Flamborough Flycatcher was 4 bases different from Iberian Pied Fly sequence, and similarly different at other genes. There wasn’t a lot in it, but it seems as though it was not an Iberian bird either.
Conclusion? Pied Fly. More cautiously, either the bird really was ‘just’ an aberrant, or extreme, Pied Flycatcher showing plumage features more consistent with Atlas or Iberian Pied Flycatcher, or there is something even more weird going on – maybe a second or third generation hybrid, or with some Iberian ancestry that we have not picked up on the first round of analysis. We will continue to work on this bird. It seems to have made the certain field identification of vagrant black-and-white Ficedulas a more daunting task.”